ISEV2025 Резюме книги.

About ISEV

With a membership of nearly 2000 individuals spanning the globe, the International Society for Extracellular Vesicles (ISEV) stands as the premier professional organization for scientists and researchers engaged in the exploration of extracellular vesicles (EVs). Established in 2012 in Sweden, ISEV subsequently relocated its headquarters to New Jersey, USA. ISEV is dedicated to fostering global consistency and robustness in EV research, as underscored by the MISEV guidelines of 2014 and 2018 and the update in 2023. The society facilitates this mission through an array of initiatives, including educational offerings, task forces, special interest groups, workshops, and summer schools, while also managing two peer‐reviewed, gold open access journals—the Journal of Extracellular Vesicles and the Journal of Extracellular Biology. A cornerstone of ISEV's activities is its flagship annual gathering, a focal point that provides a crucial avenue for knowledge exchange. By means of its comprehensive programmes and services, ISEV plays an indispensable role in delivering vital training and research prospects for those immersed in the realm of EV research.

Our Mission

To provide global leadership in the extracellular vesicle field through research, education, translation, and fostering partnerships.

Our Vision

Advancing EV research and applications worldwide.

ISEV2025 Annual Meeting

The International Society for Extracellular Vesicles is the premier international conference of extracellular vesicle research, covering the latest in exosomes, microvesicles and more. With more than 1500 attendees, the meeting has achieved a new level of recognition in the global community and features presentations from the top researchers in the field, as well as providing opportunities for talks from students and early‐career researchers.

ISEV2025 International Organizing Committee

IOC Chairs: Eva Rohde (Austria), Eva‐Maria Krämer‐Albers (Germany)

IOC Members: An Hendrix (Belgium), Andreas Moeller (China), Bo Li (China), Edit Buzas (Hungary), Johannes Grillari (Austria), Lucia Languino (USA), Mario Gimona (Austria), Sun Young Lee (USA), Wolf Holnthoner (Austria), Cristóbal Cerda‐Troncoso (Belgium), Madhusudhan Bobbili (Austria).

ISEV2025 Oral Presentations

Thursday, 24th April

24 April 9.00–10.00 Plenary 1 and featured abstract

Converging EV, LNP and virus biology: towards oral RNA drugs

Plenary Presenter: Nicole Meisner‐Kober

Ludwig Boltzmann Institute for Nanovesicular Precision Medicine, Vienna, Austria

Development of membrane fusion engineered extracellular vesicles as antigen presentation carriers to improve the efficacy of HER2 antibody‐drug conjugates

Presenter: Shili Yao

Tianjin University, Tianjin, China

Introduction : The advent of HER2‐targeted antibody drug conjugates (HER2‐ADCs) has marked a revolutionary advancement in targeted breast cancer therapy. However, 60%–70% of patients who are completely HER2 negative and do not meet the HER2‐low criterion are unable to benefit from these treatments. Engineered extracellular vesicles (EVs), which are naturally occurring nanocarriers, possess the distinctive attributes of high biocompatibility and low immunogenicity. This study aims to utilize engineered EVs to facilitate the continuous and safe presentation of the HER2 antigen to breast tumour cell membranes via membrane fusion, thereby exploring the potential for enhancing HER2‐ADCs' efficacy.

Methods : Firstly, magnetic bead‐assisted high‐speed dispersion technology was employed to generate large‐scale engineered EVs, EV‐eHER2, derived from genetically engineered 293T‐eH cells overexpressing HER2 antigens without the intracellular kinase domain (eHER2). Then, the TEM and NTA were used to verify the structure and size of EV‐eHER2, while the WB was deployed to validate the distinctive protein markers of these EVs. Furthermore, the WB, flow cytometry, and nanoflow were employed to confirm that the EV‐eHER2 membrane carries the antigen eHER2. Secondly, DSPE‐PEG2000 lipids were loaded into purified EV‐eHER2 to enhance the membrane fusion function of engineered EVs (PEG‐EV‐eHER2). Subsequently, the antigen presentation ability of PEG‐EV‐eHER2 was verified by flow cytometry, confocal microscopy, and IHC. Finally, in vitro IC50 trials and in vivo HER2‐negative tumour models were employed to determine the synergistic efficacy and safety of HER2 antigen presentation by PEG‐EV‐eHER2 combining HER2‐ADC.

Results : First, our WB results show that the genetically modified 293T‐eHER2 cells don't activate the oncogenic HER2 function–PI3K/Akt signalling pathway, while maintaining high avidity and specificity to HER2 antibodies. Second, 293T‐eHER2 cell‐derived PEG‐EV‐eHER2 displays the typical characteristics of EVs and is capable of fusing with HER2‐negative tumour cell membranes, facilitating the transfer of eHER2 antigens to the surface of these tumour cells. In vivo and in vitro results further demonstrated that PEG‐EV‐eHER2 effectively and safely enhanced the efficacy of HER2‐ADC.

Summary/Conclusion : This study develops a novel method for engineering antigen presentation EVs, employing membrane fusion to load HER2‐negative tumours with antigens, which could expand the suitable patient population benefiting from the transformative HER2‐ADC therapy.

24 April 10.30–12.00 Disease and therapy

Session title OT01 EV biomarkers

Liquid biopsy for CNS tumours using tumour‐secreted EVs

Invited Speaker: Franz Ricklefs

University Medical Center Hamburg, Hamburg, Germany

Longitudinal monitoring of glioblastoma sEV evolution using a nanodiagnostic to detect the emergence of glioma stem cells driving recurrent disease

Presenter: Zhen Zhang

University of Queensland, Brisbane, Australia

Introduction : Assessing therapeutic response in glioblastoma (GBM) is limited by the inability to perform serial biopsies due to the tumour's intracranial location. This challenge restricts longitudinal monitoring of the tumour's molecular profile and hinders the development of effective treatment strategies. Liquid biopsy techniques, particularly small extracellular vesicle (sEV) analysis, offer promising opportunities by providing real‐time insights into tumour molecular profiles using peripheral blood. To address this, we developed the GBM Extracellular Vesicle Monitoring Phenotypic Analyzer Chip (GEMPAC), which profiles a novel central nervous system (CNS) and glioma stem cell (GSC) biomarker panel (ATP1B2, EAAT2, CD24, CD44, CD133, and EGFR) on circulating sEVs.

Methods : GEMPAC identifies a unique GBM signature in sEVs by targeting CNS‐specific markers ATP1B2 and EAAT2. Using a nanoshearing multiplex surface‐enhanced Raman spectroscopy (SERS) approach, the GEMPAC platform simultaneously tracks GSC subpopulations (CD24 in neural progenitor cell‐like, CD44 in mesenchymal cell‐like, CD133 in oligodendrocyte progenitor cell‐like, and EGFR in astrocyte‐like) to monitor tumour progression and therapeutic resistance. We applied this platform to 36 GBM clinical samples, including 12 longitudinal samples with five time points, to detect tumour recurrence and track therapeutic response.

Results : GEMPAC successfully captured and analysed GBM‐specific sEVs, enabling real‐time monitoring of GSC dynamics on sEVs during treatment. Key findings included elevated CD44 levels, indicating mesenchymal‐like (MES‐like) GSCs associated with disease progression and recurrence. Additionally, CD133+ GSCs emerged in a subset of patients, suggesting resistance to treatment. While EGFR and CD24 markers did not show a strong link to recurrence, a shift to a MES‐like state was observed with downregulated EGFR and elevated CD44 during therapy. These findings highlight the platform's potential to provide real‐time insights into tumour heterogeneity and therapeutic resistance through sEV analysis.

Summary/Conclusion : The GEMPAC platform offers a non‐invasive, real‐time method for monitoring tumour evolution in GBM patients, with significant potential to improve treatment outcomes. It provides valuable insights into tumour heterogeneity and can detect therapy‐resistant GSC subpopulations, creating opportunities for targeted interventions. With further validation in larger clinical cohorts, GEMPAC could become a tool in personalized GBM management, enhancing therapeutic strategies and improving patient survival.

From challenge to opportunity: A methodology to measure reproducible EV concentrations

Britta A. Bettin 1,2,3 , Ton G. van Leeuwen 2,3 , Edwin van der Pol 1,2,3 , Rienk Nieuwland 1,3

1 Laboratory of Experimental Clinical Chemistry, Amsterdam UMC, Location University of Amsterdam, Amsterdam, The Netherlands; 2 Biomedical Engineering & Physics, Amsterdam UMC, Location University of Amsterdam, Amsterdam, Amsterdam, The Netherlands; 3 Amsterdam Vesicle Center, Amsterdam UMC, Location University of Amsterdam, Amsterdam, Amsterdam, The Netherlands

Introduction : Concentrations of extracellular vesicles (EVs) in human blood plasma are potential disease biomarkers and have been measured by flow cytometry for over 25 years. However, despite numerous interLaboratory comparison studies, measured EV concentrations still differ between instruments and labouratories. To advance EV‐based diagnostics, a robust methodology is needed to standardize EV concentration measurements. We validated such a calibration methodology to measure reliable and comparable EV concentrations across different FCMs and by different labouratories.

Methods : The methodology was evaluated in a global interLaboratory comparison study involving 19 labouratories and 25 flow cytometers (FCMs) of various brands and models. Erythrocyte‐ and platelet‐derived EV concentrations were measured in a ready‐to‐measure human plasma EV test sample. The flow rate, fluorescence, and light scattering signals of the FCMs were calibrated using reference materials. Variations in EV concentrations are expressed as the median absolute deviation.

Results : The calibration methodology allows erythrocyte‐ and platelet‐derived EV concentrations to be measured with a median absolute deviation of 16% to 33% across various FCMs and labouratories.

Summary/Conclusion : The calibration methodology enables reliable and comparable EV concentration measurements within well‐defined fluorescence and size ranges. Importantly, the obtained data are from different FCMs and from different labouratories, demonstrating that reproducible EV concentration measurements are achievable. To further demonstrate the clinical utility of this methodology, we are currently establishing reference ranges of blood cell‐derived EV concentrations in blood plasma from a cohort of 224 healthy individuals.

Funding : Funding for project 18HLT01: METVES II, which has received funding from the EMPIR program, cofinanced by the participating states and from the European Union's Horizon 2020 Research and Innovation Program.

Plasma extracellular vesicle‐associated protease activity as a technically robust functional biomarker for uveal melanoma treatment response

Jan Van Deun, Bita Karimi, Giuseppe Di Fonzo, Bettina Wachter, Michael Erdmann, Carola Berking, Andreas S. Baur

Department of Dermatology, Universitätsklinikum Erlangen, Friedrich‐Alexander‐Universität Erlangen‐Nürnberg, Erlangen, Germany

Introduction : Extracellular vesicles (EVs) can be used for disease diagnosis, prognosis, treatment selection and evaluating treatment response. Previously, we found that blood plasma EVs contain active proteases in a range of pathological conditions. Physiological EV‐associated protease activity is therefore emerging as a promising functional biomarker. A main bottleneck for EV biomarkers is establishing robust isolation/analysis workflows that are stable across common pre‐analytical variations such as transport/storage conditions. Here, we show that the combination of dual‐mode chromatography‐based EV enrichment and FRET‐based protease activity assay (DMC‐PAA) fulfils all conditions for a technically robust liquid biopsy assay. As a clinical use case, we show that DMC‐PAA correlates with treatment response in uveal melanoma patients treated with tebentafusp.

Methods : Blood plasma EVs were purified by dual‐mode chromatography. EV‐associated protease activity was measured by adding FRET substrates and performing a kinetic fluorescence measurement. Assay robustness was tested by comparing DMC‐PAA performed on the same sample by different operators. To verify test stability across common pre‐analytical conditions, DMC‐PAA was measured fresh versus (1) 48–72 h whole blood shipping at ambient temperature and (2) 28‐day plasma storage at −80°C. For uveal melanoma patients, a baseline sample was collected at treatment initiation, followed by regular sequential sampling. All blood donors provided informed consent.

Results : Screening of FRET substrates for several proteases revealed that melanoma patient plasma EVs contain a broad spectrum of activity. When comparing different operators, inter‐assay coefficients of variation for the DMC‐PAA were below 10% on average. Plasma storage at −80°C was not found to significantly affect DMC‐PAA, with deviations generally below 20% compared to freshly processed plasma. Interestingly, for several proteases we found that multiple‐day shipping of whole blood also did not significantly affect the DMC‐PAA results. Sequential sampling of healthy volunteers revealed stable measurements over time. However, in uveal melanoma patients treated with tebentafusp, changes in DMC‐PAA correlated with treatment response and cancer relapse.

Summary/Conclusion : The DMC‐PAA is a highly repeatable and precise EV biomarker assay that is not significantly affected by external sources of variation. These characteristics highlight its clinical potential for evaluating treatment response in cancer.

Funding : This work was funded by Bundesministerium für Bildung und Forschung (grant number: 16LW0338K).

Unveiling neuroblastoma: Characterizing the proteome and surface markers of extracellular vesicles to discover new biomarkers and elucidate premetastatic niche formation

Martin Auber 1 , Angelique Nejaime 1 , Maddalena Grimaldi 1 , René Maier 2 , Jennifer Schwarzkopf 4 , Marieluise Kirchner 4 , Erwin Lankes 1 , Peter Kühnen 1 , Johannes Hubertus Schulte 3 , Holger Gerhardt 4 , Marco Londrini 1 , Hyun Dong Chang 2 , Philipp Mertins 4 , Annette Künkele‐Langer 1 , Angelika Eggert 1 , and Hedwig Elisabeth Deubzer 1

1 Charité—University Hospital, Berlin, Germany; 2 German Rheumatology Research Center Berlin, Berlin, Germany; 3 University Hospital Tübingen, Tübingen, Germany; 4 Max‐Delbrück‐Center at Charité—University Hospital Berlin, Berlin, Germany

Introduction : Neuroblastoma (NB), a tumour derived from neuroectodermal embryonic crest cells, is responsible for 11% of cancer‐related deaths among children. We are investigating NB‐secreted extracellular vesicles (EVs) regarding their crucial impact on the tumour microenvironment, their role in premetastatic niche formation and their potential as biomarkers and/or therapeutic targets.

Methods : First, we isolated EVs from different NB cell lines and from plasma of high‐risk NB patients. After standard EV characterization (nanoparticle tracking analysis, electron microscopy, visualization of EV‐markers), the proteome of the EVs was analysed. Flow cytometry methods allowed us to display the surface marker profiles of NB‐EVs by using different MACSPlex Kits (Miltenyi Biotec). To evaluate the impact of NB‐EVs on angiogenesis, endothelial cells were treated with NB‐EVs in an in vivo model designed to mimic angiogenesis.

Results : Analysing the vesicle's proteome revealed EV‐protein profiles crucial for distinguishing between three prominent genetic subgroups of neuroblastoma donor cell types. Additionally, we were able to indicate the cell state (adrenergic vs. mesenchymal) of the donor cells by analysing their EV‐proteome. Flow cytometry data of EVs isolated from plasma indicates a different profile of EV surface markers for NB‐high‐risk patients compared to healthy controls. Isolated EVs from different NB‐cell lines indicate distinct EV‐marker profiles. NB‐EV‐treated endothelial cells during angiogenesis show a modified cell growth.

Summary/Conclusion : Our findings will provide clinicians with a novel, non‐invasive tool to obtain important tumour information about genotype and cell states. Both pieces of information, cell state and genotype, are crucial for finding the best therapy for patients. Particularly for underage patients, non‐invasive methods for diagnosing and monitoring cancer diseases are important, as these tools are less traumatizing for body and mind. The extensive characterization of surface markers and protein cargos of EVs from different NB origins will help to understand the EV uptake of potential recipient cells for pre‐metastatic niche formation and shed light on possible tumour‐microenvironment processes. Both processes are potential targets for new drugs.

Funding : Deutsche Forschungsgemeinschaft (DFG).

24 April 10.30–12.00 Basic biology

Session title OT02 mechanism of EV biogenesis

The winding road of CD63

Invited Speaker: Guillaume van Niel 1,2

1 Institut de Psychiatrie et Neurosciences de Paris, Paris, France; 2 Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers (CRCI 2 NA), Angers, France

RalA/B cooperate with EHD4 to shape multivesicular bodies for tumour EV secretion

Kuang‐Jing Huang 1,2 , Louis Bochler 1,2,3,4,5 , Katerina Jerabkova‐Roda 1,2,3,4,5 , Valérie Demais 6 , Victor Hanss 2,7 , Danièle Spehner 8 , Annabel Larnicol 1,2,3,4,5 , Olivier Lefebvre 1,2,3,4,5 , Laetitia Paulen 1,2,3,4,5 , Jean‐Yves Rinckel 2.3,9 , Avais M. Daulat 10 , Jean‐Paul Borg 10 , Etienne Coyaud 11 , Florent Colin 1,2,3,4,5 , Patrick Schultz 2,7 , Jacky G Goetz 1,2,3,4,5* , Vincent Hyenne 1,2,3,4,5,6*

1 Tumor Biomechanics, INSERM UMR_S1109, Strasbourg, France; 2 Université de Strasbourg, Strasbourg, France; 3 Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France; 4 Equipe Labellisée Ligue Contre le Cancer, Nantes, France; 5 Centre de Recherche de Biomédecine de Strasbourg (CRBS), Université de Strasbourg, Strasbourg, France; 6 Plateforme Imagerie In Vitro, CNRS UAR 3156, Neuropôle, University of Strasbourg, Strasbourg, France; 7 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France; 8 CNRS, SNC5055, Strasbourg, France; 9 Etablissement francais du sang (EFS), INSERM UMR_S1255, Strasbourg, France; 10 Aix‐Marseille Université, CNRS, INSERM, Institut Paoli‐Calmettes, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille Protéomique, Marseille, France; 11 Department of Biology, INSERM UMR_S1192, Protéomique Réponse Inflammatoire Spectrométrie de Masse‐PRISM, Université de Lille, Lille, France

Introduction : Extracellular vesicles (EVs) are membrane‐bound particles naturally released by cells and become crucial mediators of intercellular communication. There are two primary sources of EVs based on their biogenesis pathways: exosomes and ectosomes. Exosome secretion is a multi‐step process including endosome maturation into multi‐vesicular bodies (MVBs), intra‐luminal vesicle (ILV) budding, cargo loading, MVB transport, and fusion with the plasma membrane. Our previous studies revealed that Ral family GTPases regulate MVB maturation and exosome secretion (Hyenne et al. JCB 2015), influencing the protein and RNA composition of extracellular vesicles (EVs) from a mouse mammary cancer cell line, thus enhancing their pro‐metastatic function (Ghoroghi et al. eLife 2021). Here, we investigate the molecular mechanisms through which Ral proteins drive exosome secretion.

Methods : Using BioID, we analysed the interactome of RalA/B, identifying 400 interacting proteins, including EHD4, a dynamin‐like protein that promotes membrane fission. We used CRISPR to knock out EHD4, examining its impact on MVB maturation and EV secretion with total internal reflection fluorescence (TIRF) microscopy to quantify MVB secretion, spinning‐disc confocal microscopy to observe MVB motility, and electron microscopy to study endosome ultrastructure. Live imaging with confocal microscopy captured Ral‐EHD4 interactions.

Results : EHD4 is critical for EV secretion, a function restored by re‐expressing WT EHD4 or its paralog EHD1. Using high‐pressure freezing sample preparation and volume‐EM with FIB‐SEM, we revealed MVB morphology and found that EHD4 can shape the size of MVB, similarly to Ral GTPases. Live‐cell imaging showed that EHD4 KO impairs MVB mobility, highlighting EHD4's role in secretory MVB maturation. Rescue experiments using EHD4 mutants reveal its role in different steps of MVB maturation and identify essential EHD4 domains. In parallel, to assess the impact of EHD4 on tumour EVs function, we performed an orthotopic mammary tumour growth analysis in a syngeneic mouse model and observed that EHD4 knock‐out significantly delays primary tumour growth.

Summary/Conclusion : EHD4 modulates EV secretion by interacting with Ral GTPases to regulate MVB maturation. Our study uncovers a novel ‘MVBs‐shaping’ mechanism that affects the biogenesis of tumour EVs and their oncogenic function.

Phase separation induces the generation and cargo sorting of exosomes in multivesicular bodies

Kaixiang Zhang, Gaoge Sun, Hang Hubert Yin, Ying Zhang

Tsinghua University, China

Introduction : Exosomes are EVs derived from multivesicular bodies. Multiple mechanisms are involved in the generation of exosomes and make contributions to the sorting and transportation of exosomal cargos, including mRNAs. Here we have identified that the phase separation of MVB‐related proteins, such as ESCRT family proteins, can induce the bubbling of exosomes. The RNA‐binding protein cargo NPM1 can be involved in this condensate and further transport its binding RNAs into exosomes. This work reveals a new mechanism for exosome generation and cargo transportation.

Methods : EVs from cultured cells were collected with ultracentrifugation. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot (WB) were carried out for the characterization of EVs according to MISEV2023 guidelines. Immunofluorescence microscopy was used to observe the proteins' subcellular locolization and the formation of phase separation condensate. Fluorescence recovery after photobleaching (FRAP) was used for the validation of condensate. In vitro experiments were used to simulate the relationship between phase separation and exosome generation. RNA‐binding protein cargos in EVs were identified by proteomics. Next‐generation sequencing was employed to identify total mRNAs in EVs.

Results : In this work, we found that some ESCRT family proteins can form phase separation condensate in cells and co‐localize with CD63‐marked MVBs. We have also identified NPM1 as an RNA‐binding protein that accounts for the sorting of mRNA into EVs. NPM1 can be involved in the phase separation condensate with the binding of RNAs in MVB, which can be further covered and released by EVs into the extracellular environment. Utilizing computational analysis, we identified motifs in mRNA that are specifically recognized by the NPM1 protein and facilitate the sorting into EVs.

Summary/Conclusion : In conclusion, our findings reveal that the phase separation process is important in the generation and cargo sorting process of exosomes. The RNA‐binding protein NPM1 is a cargo involved in this phase separation condensate. This mechanism can contribute to the sorting of protein and RNA cargos into exosomes, which can have further functions in intercellular communications.

Funding : This work was funded by the National Key R&D Program of China (no. 2023YFC3605400, 2021YFE0109300), the National Natural Science Foundation of China (no. 22137004, 21977061), and the Beijing Natural Science Foundation (IS23100, IS23107).

Amphiectosome release and ‘torn bag’ secretion: a general exocytosis‐independent mechanism for small extracellular vesicle release

Tamás Visnovitz 1 , Dorina Lenzinger 1 , Anna Koncz 1,2 , Lilla Lankovics 1 , István Dudás 1 , Tünde Bárkai 1 , Krisztina V Vukman 1 , Kelsey Fletcher 1 , Csaba Cserép 3 , Ádám Dénes 3 , Péter Lőrincz 4 , Martin Krátký 5 , Szilvia Bősze 1,6 , Gábor Valcz 1 , Edit I Buzás 1,2,7

1 Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary; 2 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Budapest, Hungary; 3 Laboratory of Neuroimmunology, HUN‐REN Institute of Experimental Medicine, Budapest, Hungary; 4 ELTE Eötvös Loránd University, Department of Anatomy, Cell and Developmental Biology, Budapest, Hungary; 5 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, Czech Republic; 6 HUN‐REN–ELTE Research Group of Peptide Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 7 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary

Introduction : Small extracellular vesicle (sEV) biogenesis remains partially understood. Our recent findings show that all tested cell types, including cell lines and cells in their tissue environment, release multivesicular large extracellular vesicles (MV‐lEVs) that enclose intraluminal vesicles (ILVs). When the external membrane of these MV‐lEVs ruptures spontaneously, ILVs are released into the extracellular space through a ‘torn bag’ mechanism, a process distinct from multivesicular body (MVB) exocytosis and plasma membrane shedding of small ectosomes.

Methods : Here we investigated this unique MV‐lEV‐mediated sEV release pathway. We employed high‐resolution confocal microscopy, super‐resolution nanoscopy (Lattice SIM2 and STED) on both fixed and live cells, and transmission electron microscopy (TEM) with or without immunogold labelling. ATG5 and Rab27a genes were silenced using siRNAs, and protein analysis was conducted by Western blotting. We modulated various cellular pathways with chemicals (e.g., chloroquine, bafilomycin, rapamycin, cytochalasin B and colchicine) as well as drug candidates (e.g., niclosamide and salicylanilide). Additionally, we examined the effects of the A23187 Ca 2+ ionophore on sEV release using STED nanoscopy, TEM and high‐resolution flow cytometry. The subcellular origin of the external membrane of MV‐lEVs was tracked with PalmGFP and fluorescently labelled wheat germ agglutinin.

Results : We identified the observed MV‐lEVs as secreted amphisomes containing ILVs from both MVBs and autophagosomes. The external membrane of the budding MV‐lEVs is clearly derived from the plasma membrane; therefore, we termed the released MV‐lEVs ‘amphiectosomes.’ By super‐resolution nanoscopy, we captured consecutive stages of the intracellular amphisome formation, including fragmentation of the LC3‐positive autophagosome‐derived membrane and ILV formation. Amphiectosome secretion was found to be an active process, distinct from any other known EV release pathways. Surprisingly, under steady‐state conditions, exocytosis‐dependent exosome release was not observed by TEM; however, it was significantly induced by A23187.

Summary/Conclusion : Our findings suggest that sEV release by the ‘torn bag’ mechanism of amphiectosomes is a general, essential sEV secretion pathway in non‐stressed, steady‐state mammalian cells. It is a novel EV secretion mechanism which is substantially distinct from the known exocytosis of exosomes.

Funding : This study was supported by the NVKP_16‐1‐2016‐0004; OTKA K120237; FK138851 ); EKA2022/045‐P101; VEKOP‐2.3.2‐162016‐00002; VEKOP‐2.3.3‐15‐2017‐00016; TKP2021‐EGA‐23; RRF‐2.3.121‐2022‐00003; 2019‐2.1.7‐ERA‐NET‐2021‐00015; EU's Horizon 2020 No.739593; NAP2022‐I‐1/2022; János Bolyai Research Scholarship; LP2022‐13/2022; LP2022‐5/2022.

Systemic propagation of STING signalling via generation of large extracellular vesicles

Jiae Lee 1 , Hyung Joon Park 2 , Annabel Vernon 2 , Young Kwon 2

1 California State University Long Beach, Long Beach, CA, USA 2 University of Washington, Seattle, Washington, USA

Introduction : Cells produce diverse extracellular vesicles (EVs) for intercellular communication, transferring bioactive cargo and impacting processes such as development and tumour‐host communication. Tumour‐derived EVs, such as large oncosomes, carry oncogenic molecules and interact with immune cells, affecting tumour progression. However, the mechanism by which tumour cells produce large EVs and their functions in vivo remain unclear.

Methods : Given Drosophila's transparency and genetic tools, it serves as an ideal model for studying EV‐mediated tumour‐immune interactions, potentially unveiling conserved mechanisms in cancer biology. We employ a well‐characterized Drosophila imaginal disc epithelial tumour model and demonstrate that Drosophila tumours produce large EVs, which are readily traceable in vivo. Using live imaging techniques, we were able to track tumour‐derived large EVs in living animals and observe their formation directly from tumours. Additionally, we enriched these large EVs biochemically, enabling us to perform injections into either wild‐type or genetically modified larvae to study their physiological functions.

Results : We found that the production of large EVs from tumours is a conserved process in Drosophila. Our genetic analyses elucidate the essential role of STING signalling, which mediates the cytosolic DNA response associated with chromosomal instability (CIN), in controlling the production of large EVs and inducing a systemic immune response to tumours. Notably, injection of large EV‐enriched fractions recapitulates the systemic immune response to tumours by activating STING signalling in macrophage‐like immune cell haemocytes.

Summary/Conclusion : Thus, our study establishes an animal model that allows us to investigate large EVs in action in a native context and provides insights into how the cytosolic nucleic acid response mediated by STING signalling in tumours propagates to the immune system to induce a systemic immune response to tumours.

24 April 10.30–12.00 Physiology/pathobiology

Session Title OT03 Host−pathogen interaction

Molecular insights on EV‐mediated host‐pathogen interactions

Invited Speaker: Hernando A. del Portillo

Barcelona Institute for Global Health, ISGlobal, Barcelona, Spain

Proteomic and lipidomic insights into toxoplasma gondii extracellular vesicles

Teresa Cruz‐Bustos 1 , Anna Sophia Feix 2 , Anja Joachim 1

1 Department of Biological Sciences and Pathobiology, Institute of Parasitology, Center of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria; 2 Department of Animal and Veterinary Sciences, Aarhus University, Tjele, Denmark

Introduction : Toxoplasma gondii is a widespread intracellular parasite capable of infecting nucleated cells of a broad range of warm‐blooded animals and poses a significant public health threat. Despite its widespread impact, critical aspects of T. gondii pathogenesis, host‐parasite interactions, and immune response mechanisms are yet to be fully understood. This study aimed to elucidate the strategies and virulence mechanisms of T. gondii by analysing the EVs it releases during infection of different host cell lines, focusing on the proteomic and lipidomic profiles of these EVs.

Methods : T. gondii tachyzoites were cultured in four host cell lines: human fibroblasts, green monkey Vero epithelial cells, murine myoblasts, and porcine intestinal epithelial cells (IPEC). EVs were isolated using ultracentrifugation and characterized through nanoparticle tracking analysis, Fourier‐transform infrared spectroscopy, transmission electron microscopy, and proteomic and lipidomic analysis via liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Differentially expressed proteins were analysed through Gene Ontology (GO) and KEGG pathway enrichment.

Results : Our analysis revealed host cell line‐specific differences in the EVs shed by T. gondii . NTA revealed that T. gondii EVs from host cell lines differed significantly in size and concentration. FT‐IR spectroscopy revealed distinct lipid and protein profiles. TEM confirmed vesicle integrity and revealed heterogeneous EV populations across cell types. Proteomic analysis revealed a conserved core of 1244 proteins in all T. gondii host cell‐derived EVs, including the characteristic EV marker. However, significant differences in EV protein abundance were observed depending on the host cell line. We found distinct abundance patterns for invasion‐related proteins such as micronemal proteins (MICs), rhoptry proteins (ROPs and RONs) and dense granule proteins (GRAs). Complementary lipidomic analysis revealed distinct lipid abundance in EVs, with significant differences in lysophosphatidylcholine, sphingomyelin and ceramide species.

Summary/Conclusion : Understanding the host‐specific variations in T. gondii EV composition provides crucial insights into the parasite survival strategies across diverse host environments. This study demonstrates that T. gondii adjusts its EV cargo in response to the cellular environment of the host. These findings emphasize the pivotal role of EVs in modulating host‐pathogen interactions and identify host‐specific EV profiles as potential targets for therapeutic interventions to combat toxoplasmosis.

Endothelial colony‐forming cell‐derived extracellular vesicles mitigate immune and cardiac dysfunction in a sepsis model

Luigi Menna 1 , Nóra Fekete 1 , Boglárka Nagy 1 , Kristóf Csont 1 , Eszter Lajkó 1 , Zsófia Szász 1 , Csenger Kovácsházi 2 , Zoltán Giricz 2 , Edit I. Buzás 1,3,4 , Hargita Hegyesi 1

1 Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary; 2 Department of Pharmacology, Semmelweis University, Budapest, Hungary; 3 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary; 4 HUN‐REN‐SU Translational EV Group, Budapest, Hungary

Introduction : Sepsis represents a severe systemic condition with profound impacts on immune regulation and cardiac function. This underscores the critical need for innovative therapies that can target both immune cell dysfunction and cardiac recovery. Our study explores the therapeutic potential of small extracellular vesicles (sEVs) derived from stem cells like endothelial colony‐forming cells (ECFCs), known for their anti‐inflammatory and regenerative capabilities.

Methods : ECFCs were derived from bone marrow of bi‐transgenic (CDH5 Cre/mTmG) mice and cultured to confluence. LEVs and sEVs were separated from conditioned media by differential (ultra)centrifugation. Characterization of ECFCs and ECFC‐EVs was performed using flow cytometry, while sEVs were further analysed by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and mass spectrometry. In vitro assays included exposure of neonatal cardiomyocytes (NCMCs) to lipopolysaccharide (LPS) and subsequently to ECFC‐sEVs. After 24 h, cells were harvested for qPCR analysis of inflammation‐associated genes. Furthermore, the conditioned medium was assayed for TNFα and troponin I levels using ELISA. For in vivo experiments, LPS was administered to CDH5/mTmG mice, followed by ECFC‐sEV treatment 4 h post‐injection. Peripheral blood samples were taken after 24 h for WBC, T cell subsets, and B cell counts, and endothelial EVs were analysed by flow cytometry. Cardiac function was evaluated via echocardiography before and after sepsis induction.

Results : ECFCs exhibited endothelial progenitor markers (CD31, CD34, VCAM), and ECFC‐EVs expressed both CD31 and the vesicular marker CD63. In vitro exposure to LPS significantly upregulated inflammatory markers as measured by qPCR and ELISA. Notably, treatment with ECFC‐sEVs reduced the expression of pro‐inflammatory molecules, particularly CCL‐2, and troponin I levels, relative to the LPS‐treated controls. In vivo, LPS markedly reduced circulating immune cell counts across all analysed subsets. ECFC‐sEV administration, however, restored immune cell counts to near‐baseline levels for most cell types, except for B cells.

Summary/Conclusion : These findings underscore the potential of ECFC‐derived sEVs as anti‐inflammatory agents capable of mitigating sepsis‐induced immune and cardiac dysfunctions. Their application may offer a promising avenue to enhance both immune recovery and cardiac resilience in septic patients.

Funding : NVKP_16‐1‐2016‐0004 NKFIH, VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, the TTP TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003 (NCLP), 2019‐2.1.7‐ERA‐NET‐2021‐00015, and the EU's Horizon 2020 R&IP No. 739593.

Translocation and implication for disease pathogenesis of bacterial extracellular vesicles from gut commensal pathobiont Klebsiella pneumoniae in distant organs

Shogo Tsubaki 1 , Juntaro Matsuzaki 2 , Rika Tanaka 3 , Yusuke Yoshioka 4 , Hitoshi Tsugawa 1,5

1 Transkingdom Signaling Research Unit, Division of Host Defense Mechanism, Tokai University School of Medicine, Isehara, Kanagawa, Japan; 2 Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Tokyo, Japan; 3 Division of Host Defense Mechanism, Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, Japan; 4 Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; 5 Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, Japan

Introduction : Klebsiella pneumoniae, a gut commensal bacterium, primarily causes pneumonia and liver abscesses in elderly individuals. However, the mechanisms behind the development of diseases in distant organs due to K. pneumoniae are not well understood. Recently, we identified bacterial extracellular vesicles produced by K. pneumoniae (Kp‐bEVs) using scanning electron microscopy. This study examined whether Kp‐bEVs are involved in diseases occurring in distant organs far from the K. pneumoniae ‐colonized gastrointestinal tract.

Methods : Kp‐bEVs were collected using ultracentrifugation and characterized via transmission electron microscopy. DiR‐stained Kp‐bEVs were administered to mice, and their distribution was traced using NEWTON imaging analysis. Bone marrow‐derived macrophages (BMDMs) were cultured in DMEM with M‐CSF for 7 days, while osteoclasts were induced in DMEM with M‐CSF and RANKL for 7 days. Osteoblasts were induced by Osteoblast‐Inducer Reagent (TaKaRa Bio Co.) for 21 days.

Results : To assess localization within BMDMs, GFP‐labelled Kp‐bEVs were constructed by fusing GFP to TufA, a protein localized in Kp‐bEVs. Kp‐bEVs did not co‐localize with organelles such as the Golgi, endoplasmic reticulum, or early endosome; instead, they distributed throughout the cytoplasm. These findings suggest that Kp‐bEVs exert biological activity on macrophages. Therefore, we next examined the polarization behaviour of Kp‐bEV‐treated BMDMs. Treatment with Kp‐bEVs to BMDMs significantly increased the population of CD206‐positive cells, an M2 phenotype marker. Moreover, Kp‐bEVs significantly inhibited nitric oxide (NO) production during K. pneumoniae infection. In vivo imaging analysis using Kp‐bEVs‐administered mice revealed that Kp‐bEVs accumulate in the liver, lungs, and vertebrae. In Kp‐bEVs‐treated osteoclasts, cell viabilities were significantly reduced, and tartrate‐resistant acid phosphatase (TRAP)‐staining positive cells were scarcely detected following treatment with Kp‐bEVs. Conversely, in osteoblasts, the number of alkaline phosphatase (ALP)‐positive cells significantly increased by the 21st day in the presence of Kp‐bEVs, and the intensity of Alizarin Red staining, indicating calcium deposition, was significantly enhanced by treatment with Kp‐bEVs.

Summary/Conclusion : Kp‐bEVs attenuate the immunological activity against pathogens of macrophages. Moreover, in vertebral bone, Kp‐bEVs are thought to disrupt the balance of bone metabolism through their effects on osteoblasts and osteoclasts. Our findings suggest that Kp‐bEVs may contribute to disease development in the organs where they translocate and accumulate.

Single‐cell RNA‐Seq revealed that Legionella pneumophila‐derived extracellular vesicles reprogram host macrophages to facilitate pathogen survival and spread

Ayesha, Franklin Wang‐Ngai Chow, Polly Hang‐Mei Leung

The Hong Kong Polytechnic University, Hong Kong SAR, Hong Kong, China

Introduction : Legionella pneumophila is a pathogen associated with the respiratory disease Legionellosis . Over the past two decades, our understanding of bacterial EVs has evolved from mere metabolic by‐products to active agents in bacterial communication and survival. While it's known that bacterial EVs trigger a pro‐inflammatory response upon entering host cells, few studies have investigated the precise functions they carry out in manipulating the host cell pathways. This led us to conduct the single‐cell RNA sequencing to investigate the intricate cross‐talk between host and pathogen.

Methods : Pure EVs were obtained from L. pneumophila by combining ultracentrifuge and size exclusion chromatography techniques. TEM and nanosight were used to validate EVs purity and size. THP‐1‐derived macrophages were treated with Vybrant‐labelled pure EVs and flow cytometry was performed to sort EV‐positive THP‐1 derived macrophages. The 10x Genomics platform was utilized to profile host cell responses to EVs at single‐cell resolution.

Results : NTA and TEM analysis confirmed that the diameter of the L. pneumophila EVs ranged from 100 to 250 nm. Single‐cell RNA‐Seq revealed that, compared to mock‐treated controls, a high number of differentially expressed genes were present across all the THP‐1‐derived macrophage clusters. Significantly up‐regulated genes were enriched in inflammatory responses, including the TNF signalling pathway, Chemokine signalling pathway, NFKB signalling pathway, Legionellosis, and IL‐17 signalling pathway. On the contrary, pathways critical for bacterial clearance, including lysosome function, focal adhesion, and vesicle trafficking, were downregulated. Other down‐regulated pathways included amino acid metabolism, like glycine, serine, and threonine; aspartate, alanine, and glutamate metabolism; and aminoacyl‐tRNA biosynthesis. Further, the replication of L. pneumophila was also found to be higher in EVs‐treated THP‐1‐derived macrophages than in the untreated control, evidenced by the higher numbers of CFU.

Summary/Conclusion : The current study highlights an underestimated but critical role of bacterial EVs in the host‐pathogen dynamics. Bacterial EVs may create a more permissive environment to improve pathogen survival and replication by pre‐activating bystander cells before direct pathogen invasion.

24 April 10.30–12.00 Technology and methods

Session Title OT04 EV enrichment and capture

Integrating CRISPR into extracellular vesicle‐focused assays for precision oncology

Invited Speaker: Cesar M. Castro, MD

Massachusetts General Hospital Cancer Centre, Boston, Massachusetts, USA

Extracellular vesicle online knowledge explorer (EVOKE)—a guiding tool for the selection of extracellular vesicles preparation methods

Presenter: Claudio A. Pinheiro

Ghent University, Ghent, Belgium

Introduction : EVs exert multiple biological functions, which has spurred their development as disease biomarkers and drug delivery vehicles. However, the wide range of EV preparation methods and the complexity of biofluids pose significant challenges for rigorous and reproducible research essential for EV discovery and clinical application. To address this difficulty, we developed the Extracellular Vesicle Online Knowledge Explorer (EVOKE)—a pioneering tool designed to assist researchers in evaluating and comparing commonly used EV preparation methods.

Methods : To establish EVOKE, the international Extracellular Vesicle Quality Control (EVQC) consortium was formed to systematically compare 13 widely used EV preparation methods across three biofluids: cell culture supernatant, urine, and blood plasma. We utilized complementary characterization methods, including LC‐MS/MS, RNA‐seq, high‐resolution flow cytometry, RT‐qPCR, NTA, and TEM, to assess key performance metrics—efficiency, specificity, and repeatability. Additionally, we explored the impact of each method on the biological applicability of EVs across multiple downstream dimensions, including biomolecular component analysis (proteins and nucleic acids), diagnostic use (healthy vs. cancer), and therapeutic implementation (EV uptake by cellular models).

Results : We established robust quality metrics to objectively assess the performance of EV preparation methods. Utilizing traceable spiked reference material, we identified method‐dependent variations, demonstrating an inverse correlation between efficiency and specificity. Repeatability, assessed via proteome and transcriptome profiling, revealed high consistency across technical replicates (max fold change of 1.1). Investigation of the EV biomolecular landscape portrayed method‐dependent variations in composition, including protein corona, and in EV‐enriched or depleted markers. These variations define downstream diagnostic use and therapeutic implementation. Notably, certain methods captured cancer‐specific signatures related to chronic inflammation, immune dysregulation, and neutrophil activation, while other methods did not reveal these disease‐associated profiles. Functional assays uncovered distinct EV uptake levels, with some methods indicating non‐specific surface binding rather tahn active internalization, impacting therapeutic applications.

Summary/Conclusion : EVOKE is the first tool to guide optimal EV preparation method selection, currently powered by over 2.4 million data points. It offers crucial insights into efficiency, specificity, and method‐dependent biomolecular variations, enhancing reproducibility and clinical applications while remaining adaptable to future developments.

Selective enrichment of circulating cardiac‐derived extracellular vesicles for disease monitoring

Presenter: Monique S. Bagwell

Mayo Clinic, Rochester, Minnesota, USA

Introduction : Endomyocardial biopsies (EMB) are routinely used in the diagnosis of inflammatory or infiltrative cardiac conditions. However, invasive biopsies are burdensome, imposing morbidity and rarely mortality risk to patients. In this study, a reproducible and efficient method to isolate cardiac‐specific extracellular vesicles (EVs) from peripheral blood is here described.

Methods : Utilizing previously reported selective surface markers of cardiomyocytes, we developed an immunocapture ELISA and magnetic immunoprecipitation assay. We utilized plasma from heart transplant patients to determine the enrichment capabilities of our immunoprecipitation targets. Due to the difference in HLA type between the donor heart and the recipient, we can compare the EV populations from the donor heart and circulating EVs by measuring the level of cardiac donor HLA compared to recipient HLA.

Results : Immunocapture targets demonstrated a high degree of cardiac EV selectivity, as indicated by the presence of cardiac donor HLA type and the absence of recipient HLA type. Furthermore, the immunocapture methodology was successfully leveraged for western blot, ELISA, and qPCR analysis, making it broadly applicable for downstream EV analyses.

Summary/Conclusion : An immunocapture approach efficiently enriches cardiac EVs, providing a non‐invasive diagnostic alternative to EMB for cardiac disease surveillance. Funding: Van Cleve Cardiac Regenerative Medicine Program.

NANOSPACER: nanofluidic sizing of extracellular vesicles and biomacromolecules in solution

Presenter: Oliver Vanderpoorten

UiT The Arctic University of Norway, Tromsø, Norway

Introduction : Nanofluidic devices provide great application potential ranging from human health to single molecule metrology. However, their broad application remains largely untouched as microfluidic equipment with pumps, setups and complex chip handling protocols limit their usability—while the research community remains in urgent need of a standardized way of sizing biomacromolecules and vesicles in an easy manner. A method for the detection of nanoscopic specimens and rapid size‐exclusion of larger particles such as blood cells/platelets/cells without time‐consuming steps (e.g., ultracentrifugation) would be of great benefit to the field.

Methods : Here we demonstrate the usage of disposable nanofluidic capillaries (NANOSPACERS) for rapid nanoparticle tracking analysis (NTA) of body fluid‐derived EVs and other biomolecules in solution using fluorescence and label‐free microscopy methods (Raman, phase‐contrast, and dark‐field).

Results : Within seconds we measure the size of up to thousands of exosomes in parallel using conventional microscopy equipment within a single pipetting step. EVs can be classified into individual subpopulations upon hydrodynamic size and scattering intensity using open‐source software (FIJI, ImageJ). The method is demonstrated on various biomolecules in the micro‐ to nano‐regime: from highly motile bacteria (∼1 µm) to nanocolloids (44–500 nm), extracellular vesicles (40–120 nm), viruses (40–45 nm), DNA (100 bp–1.5 kbp), and down to proteins (1–3 nm) at the single‐molecule level.

Summary/Conclusion : We showcase with this approach a versatile method for EV researchers to use their existing lab equipment for rapid EV size analysis, spectroscopy, and classification. Nanospacers allow biological labouratories with limited funds to implement cutting‐edge single‐molecule research in their facilities—enhancing various sectors of life science and biotechnology.

Funding : HORIZON‐MSCA‐2021‐PF‐01‐01, 101064246; UiT Talent Innovation Grant 2024‐2026.

Targeted isolation of prostate‐derived EVs enhances the detection of tumour‐specific markers for cancer diagnosis

1 Yohan Kim, 3 Ishwor Thapa, 1, 4 Khaled Elshaer, 3 Hesham Ali, 1, 2 Fabrice Lucien

1 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 2 Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA; 3 School of Interdisciplinary Informatics, College of Information Science and Technology, University of Nebraska at Omaha, Omaha, Nebraska, USA; 4 College of Medicine, Alfaisal University, Riyadh, Kingdom of Saudi Arabia

Introduction : Tumour‐derived extracellular vesicles (tdEVs) are promising non‐invasive biomarkers because they carry tumour‐specific molecular features in biofluids. However, tdEVs represent a minor fraction of circulating EVs, posing challenges for the sensitive and specific detection of tumour markers. Immunocapture strategies have gained interest for tdEV isolation, yet the standardization and rigorous evaluation of capture efficiency, sensitivity, and specificity of these methods are required. In this study, we optimized an immunoaffinity‐based strategy to capture prostate‐derived EVs (ProstEVs) from blood and urine and characterize their molecular cargo in patients with prostate cancer.

Methods : We optimized an immunoaffinity‐based ProstEV capture method using magnetic beads conjugated with anti‐PSMA antibody. This method was validated with PSMA+EV spiked plasma and urine samples ( n = 3/each sample type). Bulk EV isolation via ultracentrifugation was used as a reference. The performance of the ProstEV immunocapture method was evaluated through nanoscale flow cytometry, mass spectrometry, immunoblotting, and RT‐qPCR. Additionally, proteomics and transcriptomics on urine ProstEVs isolated from patients with benign prostatic disease ( n = 10) and localized prostate cancer ( n = 12) were performed.

Results : ProstEV capture efficiency was 91% ( p < 0.01), 75% ( p < 0.01), 95% ( p < 0.01) and 90% ( p < 0.01) for PSMA+ EV spiked plasma, localized prostate cancer patient plasma, PSMA+ EV spiked‐urine and localized prostate cancer patient urine, respectively. Compared to bulk EV isolation by the ultracentrifugation method, ProstEV capture from localized prostate cancer patient urine led to the enrichment of prostate‐ and tumour‐specific markers, while kidney‐ and bladder‐derived markers were undetectable. In contrast, ProstEV capture from patient blood showed co‐isolation of blood‐borne soluble proteins and EVs (e.g., immunoglobulins, lipoproteins, and platelet‐derived EVs). Lastly, multi‐omic profiling of urinary ProstEVs of benign prostatic disease and localized prostate cancer patients revealed 169 RNA transcripts and 291 proteins that were upregulated in tumour tissue of localized prostate cancer patients.

Summary/Conclusion : This study optimized a reproducible isolation method to capture tumour‐derived EVs in patient blood and urine samples. Additionally, we identified tumour‐specific features in urine ProstEVs that may serve as novel diagnostic biomarkers for prostate cancer.

24 April 14.30–16.00 Disease and therapy

Session title OT05 EV therapeutics development

MSC‐EVs: unlocking clinical potential and overcoming translational challenges

Invited Speaker: Bernd Giebel

Institute for Transfusion Medicine, University Hospital Essen, University Duisburg‐Essen, Essen, Germany

Dual‐function extracellular vesicles for mRNA and enzyme co‐delivery in Fabry disease therapy

Sin‐Yu Chen 1# , Hsin‐Tung Chen 1 , Tai‐Shan Cheng 1 , Guan‐Wan Liu 1 , Ly James Lee 1 , Chi‐Ling Chiang 1 *, Dau‐Ming Niu 2 *, Chi‐Ying F. Huang 1,2 *

1 Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan; 2 Institute of Clinical Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan

Abstract unavailable

Enhanced delivery of RNA therapeutics via extracellular vesicles for targeted treatment of peritoneal and lung metastases

Tram T. T. Nguyen, Phuong H. D. Nguyen, Xuan T. T. Dang, Cao Dai Phung, Nhut M. Tran, Eric Y. M. Yeo, Nguyen T. P. Do, Minh T. N. Le

National University of Singapore, Singapore, Singapore

Introduction : Extracellular vesicles (EVs) have great therapeutic potential; however, their rapid clearance from the circulation and limited bioavailability in the target organs restrict their applications. Addressing this pharmacokinetic challenge could significantly maximize the therapeutic efficacy of EV‐based therapies. We have developed a new RNA‐EVs therapeutic formulation for cancer immunotherapy by using red blood cell‐derived extracellular vesicles (RBCEVs). RBCEVs can deliver customized antisense oligonucleotides (ASOs) against the KRAS gene (G12D and G12V), in combination with immunomodulatory RNA (immRNA), a 5′‐triphosphate hairpin RNA molecule designed to activate the retinoic acid‐inducible gene I (RIG‐I) and stimulate antitumour immune responses. We have previously shown that this EV formula is very effective in treating KRAS‐driven cancers by local administration. However, the systemic administration of EVs is more ideal for the treatment of metastatic cancers.

Methods : Here, we tested the efficacy, biodistribution, uptake, and action of RNA‐loaded RBCEVs via intraperitoneal and intravenous injections for the treatment of peritoneal and lung metastasis of pancreatic cancer. To enhance the circulation half‐life and tumour accumulation of EVs, we developed a novel peptide‐based method for hitchhiking EVs onto red blood cells (RBCs).

Results : We found that selective KRAS gene (G12D and G12V) inhibition and the synergistic anticancer effect of KRAS ASO and immRNA delivered by RBCEVs in mutant‐specific pancreatic cancer cell lines and patient‐derived organoids. Compared to unloaded RBCEVs, KRAS‐targeting ASOs and immRNA‐loaded RBCEVs accumulated more in the pancreas, liver, and tumours in the peritoneum, synergistically inducing RIG‐I signalling and inhibiting tumour growth in a pancreatic peritoneal metastatic model. Notably, we demonstrated that hitchhiking of RNA‐loaded RBCEVs onto RBCs enhanced drug delivery to the lungs and reduced uptake by macrophages, thus facilitating the clearance of metastatic tumours at this site. The mechanism of action and safety of engineered RBCEVs with these payloads, as well as the hitchhiking approach, were further evaluated in rodent and non‐human primate models.

Summary/Conclusion : By employing an innovative payload delivery system in conjunction with a novel hitchhiking approach of EVs onto RBCs, we present an effective and safe targeted therapeutic platform for the treatment of metastatic pancreatic cancer.

Funding : This study was funded by Singapore's Ministry of Health (NMRC/OFIRG22jul‐0029).

RNA‐based therapies by red blood cell‐derived extracellular vesicle mimetics: a new platform for the development of easily translatable and scalable therapeutics for chronic and metabolic diseases

Sara Biagiotti, Elena Perla, Debora Libetti, Barbara Canonico, Mattia Tiboni, Francesca Pierigè, Stefano Papa, Luca Casettari, Marzia Bianchi, Luigia Rossi, Michele Guescini, Mauro Magnani

University of Urbino, Urbino, Italy

Introduction : Extracellular vesicles (EVs) are promising nanocarriers for the delivery of drugs and biologics. Red blood cells (RBCs) are one of the most interesting sources for EV generation since they are easy to obtain, safe, non‐immunogenic and do not contain DNA. However, producing RBC‐derived extracellular vesicles (RBCEVs) loaded with therapeutics is still challenging for several reasons (e.g., production yield, loading efficiency, and translatability to the clinics).

Methods : RNA‐loaded RBCEV mimetics were produced by a newly developed and patented physical method starting from preloaded RBCs. Cargo preloading in RBCs was obtained by a process already established and used in clinics. Vesiculation was carried out by ‘soft extrusion,’ a non‐disruptive process that mimics what naturally happens in the spleen.

Results : Thanks to their unique biological and physicochemical features, RBCs were efficiently preloaded with several molecules and further used to generate a very high yield of cargo‐loaded RBCEV mimetics. The obtained RBCEV population was deeply characterized according to the MISEV2023 guidelines, and it showed great homogeneity in terms of size, biochemical features, surface antigens and cargo. Feasibility studies have been accomplished at the lab scale using dextran‐conjugates, while the next experiments were carried out with RNAs. In the first application, RBCEVs were used to deliver a small RNA (i.e., miR‐210‐3p) to treat endothelial dysfunctions in Type 2 Diabetes. In vitro results showed a good uptake in HUVEC, efficient delivery, and biological effect, as proved by PTP1B protein inhibition and reduction of mitochondrial metabolism. A second application for delivering a long RNA to treat a rare metabolic disease is at the preclinical level. A synthetic mRNA coding for the GAMT enzyme was efficiently loaded in RBCs and retained in the produced RBCEVs. Here, we successfully scaled up the procedure using an automated extruder, and GAMT mRNA‐loaded RBCEVs were administered to GAMT‐/‐ mice. Preliminary results showed a half‐life in circulation longer than expected. Biodistribution and efficacy studies are ongoing.

Summary/Conclusion : Our RBCEV mimetics can be efficiently used for the loading and delivery of biologics and represent a promising tool for the development of RNA‐based therapies, being easily translatable, devoid of chemicals and fully automated. Funding: PRIN 2022 PNRR under Grant P202284WZH.

An engineered siRNA backbone improves siRNA delivery via extracellular vesicles

Lieke Velthuis 1 , Puck H. de Rooij 2,3 , Olivier G. de Jong 4 , Sander A.A. Kooijmans 2,3,

1 University Medical Center Utrecht, Utrecht, The Netherlands; 2 Wilhelmina's Children's Hospital, Utrecht, The Netherlands; 3 Regenerative Medicine Center Utrecht, Utrecht, The Netherlands; 4 Utrecht University, Utrecht, The Netherlands

Introduction : Extracellular vesicles (EVs) are frequently recognized as biocompatible carriers for therapeutic RNA, such as small interfering RNAs (siRNA). However, effectively loading siRNAs into EVs at high concentrations while ensuring efficient release of siRNAs into the cytoplasm after EV uptake remains a challenge. We aimed to overcome this challenge by designing novel siRNA precursor molecules that can be actively sorted into EVs and released upon delivery to induce gene silencing.

Methods : Gene silencing of short hairpin RNAs (shRNAs) targeting enhanced green fluorescent protein (eGFP) or firefly luciferase (Fluc) was assessed in eGFP+ or Fluc+ HEK293T reporter cells using flow cytometry and luciferase assays. RNA processing was visualized via northern blot analysis. EVs were isolated from HEK293T or HeLa cells cultured in serum‐free medium using tangential flow filtration followed by Sepharose 4 Fast Flow‐based size exclusion chromatography. RNA quantification was performed using RT‐qPCR.

Results : To improve shRNA loading in EVs, we engineered shRNAs targeting eGFP with folded RNA structures (aptamers) that can bind aptamer‐binding proteins (ABPs). In eGFP‐expressing HEK293T cells transfected with various designs of these aptamer‐shRNAs, we found that shRNA‐induced eGFP silencing depended on the aptamer's position within the shRNA sequence. Silencing activity correlated with cellular processing of shRNAs into single‐stranded siRNAs. Similar outcomes were observed when aptamers were inserted into Fluc‐targeting shRNAs. Notably, silencing activity and processing of the most effective aptamer‐shRNAs were unaffected by co‐expression of cytosolic ABPs, indicating that endogenous RNA processing enzymes can release aptamer‐shRNAs bound to ABPs. Furthermore, co‐expression of aptamer‐modified shRNAs and CD9‐fused ABPs in HeLa cells significantly increased shRNA loading in EVs. These EVs reduced eGFP expression in HEK293T reporter cells in a dose‐dependent manner, which was further potentiated when EVs were engineered to express the viral fusogen VSV‐G.

Summary/Conclusion : We have developed a novel aptamer‐based engineering strategy to efficiently load shRNAs into EVs and facilitate their functional release in recipient cells. This approach is universally applicable to any shRNA, opening exciting new opportunities for the development of EV‐based siRNA therapeutics against clinically relevant target genes.

Funding : This project is funded by an Open Mind grant from the Dutch Research Council (NWO), domain Applied and Engineering Sciences (no. 21580).

24 April 14.30–16.00 Basic biology

Session Title OT06 EV release, targeting and uptake

Cell‐based glycoengineering for modulation and improvement of delivery and uptake of EV to target cells

Invited Speaker: Steffen Goletz

Technical University of Denmark, Lyngby, Denmark

Stress‐induced modifications in NPCE‐derived extracellular vesicle lipidome and microRNA content alter trabecular meshwork cell function: implications for glaucoma pathogenesis

Elie Beit‐Yannai 1 , Valeria Feinshtein 1 , Boris Knyazer 1 , Sofia Schreiber‐Avissar 1 , Padmanabhan Pattabiraman 2 , Christina Ferrara 2 , Isana Veksler‐Lublinsky 1

1 Ben‐Gurion University of the Negev, Be'er Sheva, Israel; 2 Ophthalmology Department, Indiana University, Indianapolis, Indiana, USA

Introduction : Primary open‐angle glaucoma (POAG) is a leading cause of irreversible blindness, characterized by progressive optic neuropathy often associated with elevated intraocular pressure (IOP). The non‐pigmented ciliary epithelial (NPCE) cells produce aqueous humour, while the trabecular meshwork (TM) regulates its outflow, together maintaining IOP homeostasis. Oxidative stress has been implicated in POAG pathogenesis, particularly in TM dysfunction. However, the molecular mechanisms of communication between NPCE and TM cells under stress conditions remain poorly understood. Extracellular vesicles (EVs) are emerging mediators of intercellular communication, yet their lipid composition and functional significance in ocular physiology are understudied. We investigated how oxidative stress modifies NPCE‐derived EV lipidome and microRNA content and characterized their effects on TM cells.

Methods : We employed a three‐phase experimental approach: (1) human NPCE cells were exposed to oxidative stress using AAPH under acute (15 mM, 90 min) or chronic (1.5 mM, 24 h) conditions. (2) EVs were isolated from control and stressed NPCE cells, characterized by nanoparticle tracking analysis and CD9 Western blotting, then analysed for lipid composition using Bligh‐Dyer extraction with MRM‐based untargeted lipidomics and microRNA content via microarray. (3) Human TM cells were treated with these characterized EVs, and subsequent transcriptional changes were assessed through RNA sequencing. Data analysis utilized MetaboAnalyst 6.0 for lipidomics and standard RNA‐seq pipelines.

Results : Oxidative stress significantly modified NPCE‐derived EVs, altering both their lipid and microRNA cargo. Lipidomic analysis revealed distinct changes in bioactive lipid species, while microarray identified 54 differentially expressed microRNAs in stress‐derived EVs. When TM cells were treated with these stress‐modified EVs, RNA sequencing revealed 146 differentially expressed genes (88 upregulated, 58 downregulated). Gene ontology analysis showed enrichment in pathways governing extracellular matrix organization and cell proliferation, processes crucial in glaucoma pathogenesis.

Summary/Conclusion : This study provides new insights into stress‐induced EV lipid modifications in the eye and their functional impact on recipient cells. Our findings indicate that EV lipids actively participate in intercellular communication, influencing recipient cell behaviour. This work advances our understanding of EV‐mediated cell‐cell communication in ocular health and disease, suggesting potential therapeutic strategies targeting EV lipid composition in glaucoma.

Funding : Israel Science Foundation 1545/20.

Deciphering the fate of tumour‐derived EVs within dendritic cells

Louise Merle, Clotilde Thery, Alain Joliot

INSERM U932, Institut Curie Centre de Recherche, PSL University, Paris, France

Introduction : Extracellular vesicles (EVs) are key players in mediating the communication between tumour cells and their environment. Tumour‐derived EVs promote pre‐metastatic niches in distant organs and boost tumour aggressiveness or, conversely, stimulate anti‐tumour responses. EVs can act through transfer of their bioactive content or through receptor ligand interaction with immune cells. These two distinct modes of action might contribute to their functional diversity. The aim of our project is to decipher the capacity of different types of EVs to reach and interact with target cells and ultimately deliver their content within these cells.

Methods : To investigate this interaction mechanism of EVs with their target cells, we took advantage of a bi‐molecular complementation assay based on the production of light upon spontaneous assembly of 2 complementary nanoluciferase fragments (N1 and N2). We successfully generated E0771 murine mammary cancer cell lines secreting N1‐loaded EVs upon fusion to different EV markers targeting different EV subtypes and engineered a reporter immune cell line expressing the complementary fragment N2 in the cytosol. We monitored the cytosolic delivery of EV content in a time‐ and dose‐dependent manner and investigated its sensitivity to trafficking inhibitors.

Results : Our results indicate that the respective localizations of N1 and N2 fragments influence the sensitivity of the readout, as well as the position of the N1 fragment within the fusion protein. As a proof of concept, we investigated the cytosolic delivery of several EV subtypes, and we demonstrated that EV cargo delivery is dependent on dynamin mechanisms.

Summary/Conclusion : We describe an assay based on spontaneous bimolecular complementation to decipher the capacity of EVs to interact with target cells and ultimately deliver their content within these cells. This model monitors the total binding, uptake and cytosolic delivery of EV content and its sensitivity to trafficking inhibitors. We anticipate that our findings will help to better characterize this EV's mode of action and apprehend its role in the crosstalk between tumour and immune cells.

Lipid fluidity regulates EV budding from the plasma membrane

Alissa Rumin 1 , T. Chase White 1 , Dinah Loerke 1 , Ann M. Wehman 1,2

1 University of Denver, Denver, Colourado, USA; 2 Texas A&M University, College Station, Texas, USA

Introduction : Microvesicle or ectosome budding from the plasma membrane is poorly understood. We previously discovered a conserved inhibitor of extracellular vesicle (EV) release from the plasma membrane, the C. elegans P4‐ATPase TAT‐5. TAT‐5 acts as a lipid flippase to maintain phosphatidylethanolamine (PE) asymmetry in the membrane bilayer. When TAT‐5 activity is lost, PE is exposed on the surface of cells, ESCRT proteins are recruited to the cytosolic face of the plasma membrane, and EV budding increases a 100‐fold. TAT‐5's essential flippase activity is required for embryonic development and fertility, but how PE lipids regulate EV release or other physiological processes is unclear.

Methods : PE lipids are often polyunsaturated, which can alter physical properties of the membrane, such as increasing membrane fluidity. Therefore, we asked whether disrupting lipid desaturases that form double bonds in fatty acid tails would alter EV release. We also performed temperature shift experiments to alter membrane fluidity.

Results : We found a three‐fold decrease in EV release after disrupting several lipid desaturases, suggesting that long‐chain (C20) or polyunsaturated lipids promote EV release. As these lipids promote membrane fluidity, these findings raise the possibility that PE exposure could alter membrane fluidity by decreasing the saturation of the outer leaflet of the membrane bilayer. Furthermore, worms raised at 23°C release significantly fewer EVs after a 4‐h shift to 20°C and significantly more EVs after a 4‐h shift to 26°C.

Summary/Conclusion : The temperature shift experiments and lipid desaturate knockdown experiments are consistent with changes in membrane fluidity altering plasma membrane budding. These studies also highlight the importance of maintaining consistent feeding and temperature conditions for quantitative studies of EV release.

Funding : Allen Distinguished Investigator grant, NIH R35GM152234.

Decoding de novo synthetized proteins in the polarized macrophage´s soluble and EV‐enclosed secretomes and their uptake into fibroblasts

Lena Keufgen 1,2 , Michael Saynisch 1 , Patrick Hölker 3 , Daniel Bachurski 3 , Sebastian Willenborg 4 , Marcus Krueger 1 , Ulrike Resch 1,5

1 CECAD, University of Cologne, Cologne, Germany; 2 University Hospital Cologne and University of Cologne, Department of Pediatrics, Pulmonology and Allergology, Cologne, Germany; 3 University Hospital Cologne, Klink I für Innere Medizin, Cologne, Germany; 4 University Hospital Cologne, Klink für Dermatologie und Venerologie, Cologne, Germany; 5 Medical University of Vienna, Institute of Vascular Biology and Thrombosis Research, Vienna, Austria

Introduction : Communication between cells and tissues is vital for any organism and accomplished via secreted soluble and extracellular vesicle (EV) enclosed proteins. Macrophages are key players in homeostasis, stress response, immunity and cell‐to‐cell communication. They integrate incoming and outgoing signals, which result in inflammation, antigen presentation, tissue repair and remodelling in crosstalk with other cells, including fibroblasts. Knowledge of intracellular and secreted protein dynamics following pro‐ or anti‐inflammatory polarization signals is needed to better understand macrophage plasticity.

Methods : We utilized BMDMs expressing a mutant methionyl‐tRNA synthetase (MetRS*) to facilitate the detection of newly synthetized proteins (NSPs) through bio‐orthogonal non‐canonical amino acid tagging (BONCAT). BMDMs were polarized to pro‐inflammatory M1 (LPS + IFNg) or anti‐inflammatory M2 (Il‐4, Il‐13) phenotypes for 4, 20 and 40 h. The uptake of secreted soluble and EV‐enclosed proteins was investigated in immortalized dermal fibroblasts cultured in stable isotope (SILAC) medium. Proteomes and affinity‐purified NSPs from cells, crude secretomes as well as SEC‐purified EVs were analysed with mass spectrometry (LC‐MS).

Results : We identified 544 de novo proteins in whole cells, 136 de novo proteins in crude secretomes and 38 de novo proteins in SEC‐purified EVs with increased abundance during M1 and M2 polarization. A large number of EV‐ and soluble BMDM‐secreted proteins were detected in recipient dermal fibroblasts.

Summary/Conclusion : Our unbiased proteome analyses offer new insights into intra‐ and extracellular protein dynamics and communication routes. Funding: German Federal Ministry of Education and Research (‘NMJ‐on‐a‐Chip,’ 01ED2006) to M.K.

24 April 14.30–16.00 Physiology/Pathobiology

Session title OT07 Tissue homeostasis

Preserving quiescence: multidrug transporter‐driven EV release in stem cell homeostasis

Invited Speaker: Stefan Momma

Goethe University Frankfurt, Frankfurt, Germany

Role of cardiomyocyte‐derived extracellular vesicles in post‐ischaemic cardiac remodelling

Lélia Borowski 1 , Paul Alayrac 2 , Vincent Duval 3 , Alina Ramazanova 1 , Estelle Robidel 1 , Camille Knosp 1 , Jean‐Sébastien Silvestre 1 , Chantal M. Boulanger 1 , Xavier Loyer 1 , Stéphane Camus 1

1 Université Paris cité, INSERM U970, PARCC, Paris, France; 2 Institut Pasteur, Paris, France, 3 Polygon Therapeutics, Paris, France

Introduction : Cardiovascular diseases remain the leading cause of death worldwide. Following myocardial infarction (MI), inflammatory cells are mobilized to the injured myocardium from distant compartments to coordinate tissue remodelling. Although soluble mediators are well known to influence local inflammation, their short half‐life conditions their restricted range of action. The molecular mechanisms underlying the extracellular vesicles (EV)‐mediated interorgan communication and the role of cardiomyocyte‐EV (CM‐EVs) in the pathophysiology of MI remain unknown. In this work, we aimed at determining how CM‐EVs released during MI shape the inflammatory response and cardiac remodelling.

Methods : We employed mice with cardiomyocyte‐specific Cre recombinase alone (Myh6‐Cre) or in combination with ubiquitously expressed mT/mG fluorescent reporters (Myh6‐Cre/mTmG) to define CM‐EV composition and tropism on one hand, and to highlight transfer of biological information from CM‐EV to target cells on the other. Intracardiac and circulating EVs were isolated from naïve, ischaemic or sham‐operated male or female mice by differential ultracentrifugation. CM‐EV concentration was determined by flow cytometry, and their protein composition was determined by proteomic analysis. We assessed CM‐EV biodistribution in vivo by monitoring GFP fluorescence in remote organs after MI by flow cytometry and 2‐photon microscopy and identified the identity of CM‐EVs target cells. To investigate the interaction between CM‐EVs and their target cells, we incubated CM‐EVs with primary macrophages and visualized the interaction using imaging flow cytometry. We then monitored GFP expression in vitro by flow cytometry as a proxy for functional transfer of biological material from CM‐EVs to macrophages.

Results : First, we showed that our EV characterization strategy comply with MISEV recommendations. Proteomics data show that male‐ and female‐derived IM CM‐EVs are similar and that they are mainly enriched in mitochondrial proteins. Then, we demonstrate CM‐EVs are preferentially targeting spleen and lung macrophages after infarction. We validated an interaction between EV and macrophages and confirmed CRE transfer from CM‐EVs to macrophages as shown by induction of GFP expression.

Summary/Conclusion : Our data show that CM‐EVs generated upon MI, express a unique set of proteins and establish long‐range, inter‐organ communication routes by targeting innate macrophages from the spleen and lungs.

Novel insights into extracellular vesicle trafficking in cardiac injury

Presenter: Xinjie Wang

Texas A&M University, College Station, Texas, USA

Abstract unavailble

Podocyte exopher formation in membranous nephropathy

Karen Lahme 1 , Wiebke Sachs 1 , Desiree Loreth 1 , Stephanie Zielinski 1 , Johannes Brand 1 , Kristin Surmann 2 , Uwe Völker 2 , Thorsten Wiech 3 , Tobias N. Meyer 4 , Lars Fester 5 , Catherine Meyer‐Schwesinger 1

1 Institute of Cellular and Integrative Physiology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 2 Interfaculty Institute for Genetics and Functional Genomics, Greifswald, Germany; 3 Institute of Pathology, Nephropathology Section, University Medical Center Hamburg‐Eppendorf, Germany; 4 Nephrology, Asklepios Klinikum Barmbek, Hamburg, Germany; 5 Institute of Neuroanatomy, Medical Faculty, University Bonn, Bonn, Germany

Introduction : Membranous nephropathy (MN) is an autoimmune glomerulonephritis of kidney podocytes caused by circulating autoantibodies directed against podocyte foot process proteins. The morphologic hallmark of MN is glomerular antigen‐autoantibody deposition. The underlying mechanisms and clinical significance of glomerular aggregate formation and release in MN are unknown. This study analysed podocyte‐derived urinary extracellular vesicle (EV) formation in human nephrotic patients and experimental murine MN and identified exophers as the pathobiological correlate of glomerular urinary space antigen/autoantibody aggregates.

Methods : EVs were isolated from mouse and human urine by differential ultracentrifugation, followed by ultrafiltration (human urine) or by dialysis (mouse urine). EVs were quantified by nanoparticle tracking analysis, characterized by electron microscopy, ImageStream, and immunoblotting. The proteostatic content of human urinary EVs was further investigated by mass spectrometry. Immunohistology from human nephrotic patient biopsies was performed.

Results : We identified podocyte exophers as biological correlates of glomerular urinary space aggregates in MN. These large, stalked vesicles are formed by translocating THSD7A and bound autoantibodies from the subepithelial to the urinary side of podocyte plasma membranes. Exophers in MN patients contain antigen/autoantibody complexes and disease‐associated proteins like complement. Exopher‐release enhanced by proteotoxic stress reduces the glomerular aggregate burden in experimental MN. Monitoring urinary exopher abundance and autoantibodies is essential for assessing immunologic activity in MN, especially in the setting of negative serum autoantibody titres. The proteostatic content of podocyte‐derived urinary EVs changes disease‐dependently, reflecting the disease‐associated proteostatic situation of podocytes.

Summary/Conclusion : Exopher‐genesis is a protective pathomechanism in MN. Tracking of the autoantibody from the serum to the urinary exopher‐bound state assesses the podocytes’ propensity to deal with the disease initiating autoantibodies. This will provide a non‐invasive diagnostic tool with prognostic potential for clinical diagnostics of MN.

Intra‐nephron cross talk through exosomes

Janina Kern, Sindhu Thiagarajan, Nina Sopel, Janina Müller‐Deile

Uniklinikum Erlangen, Erlangen, Germany

Introduction : Insufficient cell‐cell communication of glomerular endothelial cells (GECs) and podocytes leads to a dysfunctional glomerular filtration barrier, and not much is known about the intra‐glomerular as well as the glomerular to tubular signalling. Exosomes are 30 to 160 nm small extracellular vesicles released by multivesicular bodies from various cell types. These transport vehicles encapsulate signalling molecules such as mRNAs, microRNAs or proteins. Exosomes secreted from cells facing the urinary space might be taken up by tubular cells. However, it is unclear if exosomes can serve as mediators of GEC to podocyte crosstalk in vivo, as it is unknown if they are able to pass the glomerular basement membrane (GBM).

Methods : RNA‐seq analysis and proteomics were used to characterize exosome cargo of glomerular cells. A CD63 (surface protein of exosomes) plasmid tagged with mScarlet and pHluorin (labelling intracellular exosomes in red and secreted exosomes in green) was used to investigate exosome secretion in glomerular cell culture models. By injecting zebrafish eggs in the one‐cell stadium with a pHluorin CD63 reporter plasmid, including one with a podocin promoter for podocyte‐specific exosome labelling exosomes could be visualized in zebrafish larvae with a multiphoton microscope in high resolution. Moreover, in vitro labelled exosomes were injected into the zebrafish circulation and visualized with immunofluorescence and electron microscopy to examine their ability to pass the GBM.

Results : In vitro podocytes and GECs secreted exosomes that could be live‐tracked with the help of a spinning disc microscope. Live tracking analyses in 2D and 3D glomerular co‐culture detected exosome‐transfer from one cell type to another. Incubation of GEC‐derived exosomes changed expression in podocytes, and likewise podocyte‐derived exosomes had effects on tubular cell expression. Injecting fluorescently labelled exosomes into the zebrafish circulation allowed to study the permeability of exosomes for the GBM. Using CD63 plasmids encoding podocyte‐specific labelled exosomes helped to investigate cell‐cell communication via exosomes originating from podocytes.

Summary/Conclusion : Live tracking experiments of glomerular cell‐type‐derived exosomes will help to get more insights into inter‐glomerular and glomerular‐to‐tubular cell–cell communication. Funding: DFG, TRR‐374.

24 April 14.30–16.00 Technology and methods

Session Title OT08 Tissue homeostasis

Understanding EV heterogeneity—the final frontier?

Invited Speaker: André Görgens André Görgens 1 , Samir EL Andaloussi 1

1 Karolinska Institutet, Stockholm, Sweden

Squeezable hydrogel microparticles for single extracellular vesicle protein profiling

Presenter: Jina Ko

University of Pennsylvania, Philadelphia, Pennsylvania, USA

Introduction : Extracellular vesicles (EVs) are promising for molecular diagnostics, but current analyses are limited by the rarity and compositional heterogeneity of EV protein expression. Therefore, single EV profiling methods require high sensitivity, multiplexing, and throughput to address these issues. Here a single EV analysis technique that utilizes squeezable methacrylated hyaluronic acid hydrogel microparticles (MHPs) is described as a scaffold to immobilize EVs and perform an integrated rolling circle amplification (RCA) assay for an ultra‐sensitive and multiplex analysis of single EV proteins.

Methods : To perform ultra‐sensitive and multiplex protein analysis of single EVs, uniform droplets consisting of methacrylated hyaluronic acid (MeHA) polymer precursor and bulk EVs were generated with a flow‐focusing microfluidic droplet generator. EVs are physically arrested within hydrogel microparticles by photopolymerization. Consequently, captured EVs are labelled with DNA barcoded antibodies (Ab‐DNA), and multiplex protein signals are amplified by incorporating RCA into the hydrogel microparticle assay without Poisson and steric limitations.

Results : We validated that MeHA hydrogel microparticles (MHPs) exhibit high compressibility upon physical squeezing, which can align RCA products in a single plane to uniquely enable imaging without the need for high‐magnification z‐stack imaging. As a result, image acquisition is cost‐effective, simple, and fast, and EV analysis is high throughput, as a single image contains > 1000 EVs. An automated pipeline was developed to describe the presence/absence and co‐expression of protein markers for single EVs from different cancer cell lines. For validation, we profiled the protein expression patterns of highly expressed cancer cell markers among single EVs from different cancer lines and elucidated their heterogeneity. As a result, this method bypasses the Poisson distribution and multiplexing limits of digital ELISA platforms, enhances sensitivity by enabling a larger surface area of interactions with epitopes, and provides a scaffolding for conducting RCA steps to enhance visualization of rare protein signals.

Summary/Conclusion : We present a simple, robust, and scalable platform that can enable the identification of rare EV subpopulations with diagnostic potential and advance the development of molecular assay technologies.

Single Extracellular VEsicle Nanoscopy‐Universal Protocol (SEVEN‐UP): accessible imaging platform for quantitative characterization of single extracellular vesicles

Benjamin Purnell 1 , Andras Saftics 1 , Balint Beres 1,2 , Nan Jiang 1 , Carinna Lima 1 , Ima Ghaeli 1 , Brian Armstrong 1 , Rupangi Vasavada 1 , Tijana Jovanovic‐Talisman 1

1 Beckman Research Institute, City of Hope, Duarte, California, USA; 2 Budapest University of Technology and Economics, Budapest, Hungary Benjamin Purnell and Andras Saftics contributed equally .

Introduction : Extracellular vesicles (EVs) are key orchestrators of intercellular communication. These nanoscopic particles, which are shed by all cells, have emerged as intriguing therapeutic modalities, exciting delivery vectors, and enticing biomarker sources. Despite these important features, EVs have been challenging to reproducibly characterize because they are heterogeneous in size, origin, and molecular content. Most EVs also have diameters below 200 nm—the resolution limit of conventional light microscopy. To advance EVs in biology and biomedicine, tools for biophysical analysis of individual EVs that do not require highly specialized equipment are urgently needed.

Methods : We quantified individual EVs by combining affinity isolation of EVs with super‐resolution radial fluctuations (SRRF) microscopy and new analysis algorithms supported by machine learning. This combined approach, which we called Single Extracellular VEsicle Nanoscopy‐Universal Protocol (SEVEN‐UP) is compatible with a wide range of microscopes and does not require specialized fluorophores. SEVEN‐UP can assess abundant EVs enriched in classical EV markers (e.g., tetraspanins CD9, CD63, and CD81) and rare EV subpopulations enriched in specific biomarkers (e.g., those associated with a particular cell type or disease).

Results : We first optimized the workflow and validated the data obtained on widefield and total internal reflection fluorescence microscopes by correlating SRRF and single‐molecule localization microscopy images. Using both recombinant and plasma EVs, we robustly assessed their captured density, size, and molecular content of tetraspanins. Further, we used SEVEN‐UP to assess plasma EVs enriched in different glycosylation motifs such as mannose, branched N‐glycans, N‐acetylglucosamine, chitin, sialic acid, and fucose. We show that EVs enriched in specific glycosylation patterns have unique biophysical properties.

Summary/Conclusion : We developed a new tool to characterize the biophysical properties of single EVs for the general research community. The approach is economical; the cost of supplies for one experiment is typically well below the price of a Sachertorte slice in Vienna cafes. By democratizing single EV imaging, SEVEN‐UP may help unravel basic EV biology and move EVs toward the clinic.

Funding : UG3/UH3 TR002878, Dorrance Family Research Fund, Board of Governors of the City of Hope, T32 PROMT, Fulbright US Student Program.

Mapping single extracellular vesicle landscapes to dissect tumour heterogeneity and evolution of drug resistance in glioblastoma

Presenter: Mahsa Jalali

Research Institute of McGill University Health Center, Montreal, Quebec, Canada

Introduction : High‐grade brain tumours, such as glioblastoma multiforme (GBM), present substantial diagnostic challenges due to their cellular and molecular heterogeneity coupled with rapid biological evolution of the disease, including therapy resistance. These features are difficult to track longitudinally due to the absence of reliable biomarkers reflective of tumour complexity, which evade conventional diagnostic imaging. The systemic and dynamic representation of the GBM cellular ecosystem is reflected in the repertoire of extracellular vesicles (EVs) released from the tumour into plasma and biofluids. Thus, complex landscapes of individual EVs may serve as a unique source of actionable information in GBM given the development of high‐resolution tools. Here we report the coupling of Raman spectroscopy and nanoimaging of single EVs to obtain the multidimensional EV panorama of GBM lesions progressing toward temozolomide (TMZ) resistance.

Methods : We employed patient‐derived, tumour‐initiating mesenchymal glioma stem cells (GSC1123 and GSC83) to isolate small EVs through ultracentrifugation and size exclusion chromatography, following MISEV guidelines. These EVs were analysed for their responses to TMZ‐induced stress both in vitro and in vivo (subcutaneous models). Using bulk mass spectrometry, high‐resolution single EV microscopy (dSTORM) and Raman spectroscopy (SERS), we generated integrated multimodal molecular profiles of individual EVs and their populations with diversity measured using Mahalanobis distance to classify deconvoluted EV fingerprints.

Results : We observed that the single EV SERS spectra of GSC1123 and GSC83 cells, along with their TMZ‐resistant variants, exhibited significant variations in both peak positions and intensities. Deconvoluted peak combinations specific to the presence of hypermutation or MGMT hypermethylation patterns of TMZ resistance were identified through coupled EV dSTORM nanoimaging and SERS. These findings suggest that Raman spectroscopy of evolving EV populations can be adapted as a liquid biopsy technique to detect the impending transition from therapeutic responsiveness to resistance during brain tumour progression.

Summary/Conclusion : Single EV heterogeneity offers a unique insight into dynamic repertoires of EVs and their heterogeneous parental cancer cell populations. This approach could serve as a new modality of longitudinal liquid biopsy in GBM.

Funding : Fondation Charles Bruneau and CIBC, Canadian Institutes of Health Research.

Single vesicle‐resolved lipidomics of extracellular vesicles via mass spectrometry imaging to untangle the EV interactome in ovarian cancer

Presenter: Victor Chiu

University of California, Davis, Davis, California, USA

Introduction : Ovarian cancer (OvCa) remains a significant health burden, with early detection being crucial for improved survival rates. Circulating extracellular vesicles (EVs) represent promising non‐invasive biomarkers due to their unique molecular cargo, including lipids and small metabolites reflective of their cellular origins and pathological role in metabolic reprogramming of cancer. Our study utilizes MALDI‐MSI (matrix‐assisted laser desorption/ionization mass spectrometry imaging) to perform high‐resolution lipidomic profiling of EVs, aiming to analyse tumour‐associated EVs enriched from peripheral circulation. Here, we report the first demonstration of single‐vesicle resolution in EV lipidomics, enabling previously unattainable insights into individual vesicle profiles and their relation to tumour biology.

Methods : EVs were enriched from serum samples of OvCa patients (IRB‐approved protocol with informed consent) using size exclusion chromatography (SEC), characterized in accordance with MISEV 2023 guidelines, and immobilized on ITO‐coated glass slides via simple dilution and drop drying. Matched fresh frozen tumour tissue from each patient was cryo‐sectioned and mounted. By rastering a UV laser across either the deposited EVs or tissue, MALDI spectra were collected at each position for subsequent construction of ion images, allowing for profiling of subcellular features (< 1 µm). MALDI‐MSI and EV deposition were optimized to detect lipid features down to single EVs, using synthetic liposome controls to establish sensitivity and specificity. Atomic force microscopy (AFM) verified spatial distribution, confirming single‐vesicle detection capability.

Results : MALDI‐MSI was able to resolve dozens of lipid species in individual vesicles, with several distinct EV subtypes arising from analysis of shared lipid profiles. Certain specific subpopulation profiles were detected with signatures correlating to those in matched ovarian tumour tissue. Key lipids, including PC(38:4;O) associated with tumour cells and PS(26:0) associated with adjacent areas in the tumour microenvironment, were consistently present, demonstrating the potential for tissue‐of‐origin tracing and early disease detection.

Summary/Conclusion : Our findings introduce a new approach to EV lipidomics, achieving single‐vesicle sensitivity for the first time and correlating lipid signatures with OvCa progression and mapping tissue‐ and cell‐of‐origin. This capability establishes a new benchmark for EV analysis in cancer research and paves the way for deeper insights into general EV biology that extend beyond biomarker discovery and precision diagnostics.

Friday, 25th April

25 April Plenary 2 and featured abstracts

The ESCRT machinery in biogenesis of intraluminal and extracellular vesicles

Plenary Presenter: Harald Stenmark

Oslo University Hospital, Oslo, Norway

Development of a live‐cell imaging assay to elucidate spatiotemporal dynamics of extracellular vesicle fusion with target cells

Jasper van den Ende 1 , Kyra Defourny 1 , Huib Rabouw 2 , Marvin Tanenbaum 2 , Richard Wubbolts 1 & Esther Nolte‐‘t Hoen 1

1 Utrecht University, Utrecht, The Netherlands; 2 Hubrecht Institute, Utrecht, The Netherlands

Introduction : Cells communicate via extracellular vesicles (EVs), which transfer functional RNAs and proteins. Knowledge on the fate of internalized EVs and their mechanisms for breaching cellular membranes to release luminal cargo into the cytoplasm is limited. In available functional assays, EV‐cargo delivery readouts are a cumulative result of EV‐binding/uptake, EV‐fusion and the target cell's potential to use cargo for a functional response. As these assays are unlikely to uncover molecular players and conditions that specifically boost the fusion step, new strategies are needed. Here, we developed a live‐cell imaging assay for simultaneous detection of EV uptake and fusion, both in time and space.

Methods : EV‐donor cells were engineered to tag the lumen of EVs with a red fluorophore and repeating units of the SunTag peptide. EVs from these cells, with or without transfection of fusogenic VSV‐G, were isolated by differential (ultra)centrifugation and density gradient purification, then added to recipient cells and tracked by live cell confocal microscopic imaging. Recipients expressed green fluorescent anti‐SunTag antibody (STAb), which binds SunTag upon its cytosolic exposure, or Galectin‐3 (Gal3), which clusters on carbohydrates available upon endosomal rupture. Quantity and subcellular localization of fluorescent spots was then analysed.

Results : EVs taken up by STAb recipient cells appeared as red intracellular spots which increased over time. A subset of EVs fused and became red‐green double positive if VSV‐G was present. In Gal3 cells, red spots never became double positive. Thus, our assay successfully detects EV uptake and fusion, which can occur without endosomal rupture. Prolonged imaging of the same cells allowed for analysis of fusion kinetics and efficiency, assessment of specific (chemical) inhibition of fusion, and determination of fusion localization, by co‐staining for several cellular compartments.

Summary/Conclusion : We developed a highly sensitive imaging method to detect EV fusion events in real time. This method has the power to illuminate the underexplored spatiotemporal dynamics of EV‐fusion. It can also be utilized to identify fusogenic EV subsets, cell states and molecular players facilitating EV fusion, as well as drugs influencing it. Ultimately, increased knowledge about the fusion step of the EV life cycle can aid in the engineering of vesicles carrying intraluminal therapeutic payloads.

Funding : Funded by the NWO (Dutch Research Council).

Clinical trial: first‐in‐human intracochlear application of vesicle‐enriched secretome fractions (ESCRT)

Athanasia Warnecke 1 , Odett Kaiser 1 , Susanne Sasse 1 , Jennifer Harre 1 , Max Eike Timm 1 , Jurgita Ivanauskaite 1 , Anke Lesinski‐Schiedat 1 , Andreas Büchner 1 , Lutz Gärtner 1 , Daniel Kley 1 , Daniela Auer 2 , Tanja Schally 2 , Stefan Rund 2 , Carina Kals 2 , Christina Folie 2 , Elisabeth Bayer 2 , Hinrich Staecker 3 , Mario Gimona 2,4 , Eva Rohde 2 , Nils Kristian Prenzler 1

1 Hannover Medical School, Hanover, Germany; 2 Paracelsus Medical University (PMU), Salzburg, Austria; 3 University of Kansas School of Medicine, Kansas City, Kansas, USA; 4 Paris‐Lodron University Salzburg, Salzburg, Austria

Introduction : After cochlear implantation, an immune response can impair hearing performance or even lead to loss of residual hearing. Currently, there are no approved pharmacological therapies available to compensate for this. A novel medicinal product derived from mesenchymal stromal cells, that is, vesicle‐enriched secretome fractions (VSF), is intended to attenuate the reaction of the inner ear tissue. Preclinical investigations demonstrated safety as well as immunomodulatory and/or neuroprotective effects of VSF. In a first named patient use, intra‐cochlear application of VSF during cochlear implant surgery was feasible. Consequently, a first‐in‐human Phase I/II clinical trial (ESCRT) was approved by the Central European System for Clinical Trials in October 2024.

Methods : In this open‐label monocentric Phase I/IIa clinical trial, a single dose (30 µL of 0.5–1 × 10 11 particles/mL) of VSF is applied to the inner ear during cochlear implant surgery as adjuvant treatment prior to insertion of the electrode array. Eleven patients (aged ≥ 18) with profound hearing loss receiving cochlear implantation will be included. The number and severity of adverse events (AEs) are documented, and immune response, auditory nerve function, and speech perception are evaluated. A sentinel approach is applied after the first three patients in January 2025. The study duration per subject is approximately 6 (±1) months, with the entire trial scheduled to last approximately 21 months.

Results : After approval, three patients have been enrolled and are undergoing a sentinel safety assessment based on the detection and documentation of severe and serious AEs. The report will be evaluated by the ethics committee, and the remaining eight patients will be enrolled upon ethical approval. First, data regarding the primary endpoint (safety) and secondary endpoints (efficacy) will be presented.

Summary/Conclusion : This clinical trial of the first intracochlear approach of EV application can serve as an exemplary pathway to achieve the broader goal of developing precision medicines with the potential to improve and preserve hearing after cochlear implantation trauma.

Funding : This work was funded by MED‐EL (Austria), by Deutsche Forschungsgemeinschaft (DFG; EXC 2177/1, Project ID 390895286), and Project ‘ExtraNeu’ from the State of Salzburg, Austria.

25 April 8.30–10.00 Disease and therapy

Session Title OF09 EVs in tissue injury and repair

Decoding the healing dialogues

Invited Speaker: Subhadip Ghatak

Indiana University, Indianapolis, Indiana, USA

Classical Ly6C + high monocytes play a critical role in mediating neuroprotection by mesenchymal stromal cell‐derived small extracellular vesicles after ischaemic stroke in mice

Chen Wang 1 , Tobias Tertel 2 , Yiqiao Zhang 1 , Yanis Mouloud 2 , Xiaolong Liu 1 , Matthias Mack 3 , Bernd Giebel 2 , Dirk M. Hermann 1

1 Department of Neurology, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany; 2 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany; 3 Department of Nephrology, University Hospital Regensburg, Regensburg, Germany

Introduction : An appropriate understanding of the modes and sites of action is essential for the successful clinical translation of mesenchymal stromal cell‐derived small extracellular vesicles (MSC‐sEVs). Our previous studies demonstrated that MSC‐sEVs fail to synergistically provide neuroprotection when neutrophils are depleted in a mouse ischaemic stroke model. In addition to neutrophils, monocytes/macrophages may also act as key cellular targets of MSC‐sEVs, considering the close interaction between neutrophils and monocytes/macrophages and the pronounced response of monocytes/macrophages to MSC‐sEVs. Herein, we explored the effects of MSC‐sEVs in the presence and absence of monocytes/macrophages in our mouse ischaemic stroke model to identify the specific monocyte/macrophage subpopulation responsible for MSC‐sEV‐induced neuroprotection.

Methods : Male C57BL/6J mice were subjected to transient middle cerebral artery occlusion (MCAO). Following reperfusion, either vehicle or MSC‐sEVs (equivalent to 2 × 10 6 MSCs), were administered intravenously. Prior to MCAO and sEV treatment, peripheral monocytes/macrophages—or, more specifically, classical Ly6C + high monocytes, were depleted using clodronate liposomes (50 mg/kg) or anti‐CCR2 antibodies (MC‐21; 20 µg), respectively. Neurological deficits and ischaemic brain injury were evaluated over 3 days post‐MCAO.

Results : MSC‐sEVs consistently induced neuroprotection, reducing neurological deficits, brain infarct size, and brain oedema in MCAO mice treated with PBS‐loaded control liposomes or isotype antibodies in both study sets. Administration of clodronate liposomes induced total peripheral monocyte/macrophage depletion, which ensued with neuroprotection similar to that of MSC‐sEV treatment. Yet, when MSC‐sEVs were administered to monocyte/macrophage‐depleted MCAO mice, the neuroprotection provided by MSC‐sEVs was abolished, resulting in worsened stroke outcomes. MC‐21 antibody‐induced selective Ly6C + high monocyte depletion provided milder neuroprotection than total monocyte/macrophage depletion, reducing neurological deficits only. When MSC‐sEVs were administered to Ly6C + high monocyte‐depleted MCAO mice, neurological deficits were again worsened, and infarct size increased.

Summary/Conclusion : Classical Ly6C + high monocytes are key mediators of MSC‐sEV‐induced neuroprotection in ischaemic stroke.

The effect of trimeric superoxide dismutase 1 on EVs in ALS

Presenter: Brianna Hnath

Penn State University College of Medicine, Hershey, Pennsylvania, USA

Introduction : Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease resulting in paralysis and death within three to 5 years. Mutations in fifty different proteins have been linked to ALS, leading to controversy over whether ALS is one disease or many diseases with a similar phenotype. Mutations in Superoxide Dismutase 1 (SOD1) are only found in 2%–3% of amyotrophic lateral sclerosis (ALS) cases, yet misfolded SOD1 is found in both sporadic ALS patients as well as patients with other ALS mutations. The toxic trimeric form of misfolded SOD1 is difficult to identify due to structural differences, perpetuating the controversy. Yet, expressing mutations in TDP‐43 or FUS increases the level of misfolded SOD1 on extracellular vesicles (EVs). Additionally, small EVs isolated from ALS patient samples caused cell death of wild‐type motor neuronees and myotubules. We hypothesize that toxic trimeric SOD1 is spreading on EVs in ALS and altering the spread of other ALS‐related proteins, linking them to a common mechanism.

Methods : We isolated EVs (ultracentrifugation) from motor neuronee‐like cells expressing trimer stabilizing mutations and performed a sandwich enzyme‐linked immunoassay (ELISA) (CD9 capture antibody) to quantify whether misfolded SOD1 and twenty other ALS‐related proteins increase or decrease on EVs with trimer stabilization. Next, we investigated which EV release pathway is being affected by trimeric SOD1 utilizing endocytosis and exocytosis inhibitors. We determined the timeline of protein increases on EVs using cell lines stably expressing SOD1 WT or mutants (as opposed to an overexpression model used for the earlier assays). Finally, we differentiated human‐induced pluripotent stem cell lines into motor neuronees or myofibrils and determined any cell‐specific differences in ALS‐related proteins on EVs between wild type and an SOD1 mutant.

Results : Misfolded SOD1 and multiple other ALS‐related proteins increased on EVs with trimer stabilization. Inhibiting common endocytosis and exocytosis pathways increased the level of the altered proteins on EVs when the trimer was stabilized. Two different release timelines were observed in the stable cell line assay.

Summary/Conclusion : Stabilizing trimeric SOD1 alters the concentration of ALS‐related proteins on EVs. Investigating the mechanism of spread on EVs gives us targets for new biomarkers and therapeutics to treat ALS.

Mechanically activated extracellular vesicles regulate angiogenesis for bone repair at different stages of the osteogenic lineage

Carolina S. Martins 1,2 , Mimma Maggio 1,2, , Mathieu Y. Brunet 1,2 , Cansu Gorgun 1,2,3 , Rawan Almasri 4 , Lorraine O'Driscoll 4 , David A. Hoey 1,2,5

1 Trinity Centre for Biomedical Engineering, Ireland, 2 Dept. of Mechanical, Manufacturing, and Biomedical Engineering, School of Engineering, Trinity College Dublin, Ireland, 3 School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Ireland, 4 School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute & Trinity St James's Cancer Institute, Trinity College Dublin, Ireland, 5 Advanced Materials and Bioengineering Research Centre, Trinity College Dublin & RCSI, Ireland

Introduction : The formation of new blood vessels, angiogenesis, plays a critical role in bone repair. We have previously highlighted the significance of bone cell‐derived extracellular vesicles (EVs) in mediating angiogenesis. However, the impact of mechanical stimulation, a potent regulator of bone regeneration, on bone cell‐derived EVs remains uncertain. Therefore, this study aimed to determine the role of EVs derived from cells at different stages of the osteogenic lineage in regulating angiogenesis, and how this is influenced by mechanics.

Methods : Fluid shear was applied to human MSCs, human osteoblasts (OBs), or osteocyte‐like (OCY) cells at 1 Pa, 1 Hz for 2 h. Following 24 h in culture, the conditioned media was collected. EVs were collected by ultracentrifugation at 110,000 × g for 75 min at 4°C, using a 70Ti fixed‐angle rotor. NTA, flow cytometry and TEM were used for characterization. Human umbilical vein endothelial cells (HUVECs) were used to assess tube formation or fixed for immunofluorescent imaging (CD31 + ). Proliferation was assessed through BrDU incorporation and migration was evaluated by the Transwell assay.

Results : The treatment of HUVECs with EVs collected from statically cultured MSCs (st‐EV) inhibited junction formation, although this affect was rescued following mechanical stimulation of the parent cell. EVs collected from mechanically activated OBs (maEVs) further enhanced the number of junctions compared to the negative control, which was further augmented with OCY‐derived EVs. In particular, OCY‐derived maEVs led to a significant increase in tube formation with thicker vessels present, comparable to the +VEGF control, and also increased HUVEC migration and proliferation. Remarkably, treatment with OCY‐derived maEVs leads to a substantial increase in CD31 + expression.

Summary/Conclusion : We demonstrate the importance of the stage of differentiation, as well as mechanical stimulation on the angiogenic effect of EVs released by bone cells, highlighting OCY maEVs as a possible therapy for bone repair.

Recurrent intranasal administrations of neural stem cell‐derived EVs restrain neuroinflammation and maintain better cognitive function for extended periods in a model of Alzheimer's disease

Presenter: Ashok K. Shetty

Texas A&M University College of Medicine, USA

Introduction : Alzheimer's disease (AD), a progressive neurodegenerative condition, results in significant cognitive and mood dysfunction. Neuroinflammation is one of the vital factors contributing to AD progression. Intranasal (IN) administrations of extracellular vesicles produced by human induced pluripotent stem cell‐derived neural stem cells (i.e., hiPSC‐NSC‐EVs) have been shown to reduce neuroinflammation by inducing transcriptomic changes in activated microglia and reactive astrocytes and curtailing neuroinflammatory signalling cascades in 5xFAD mice, a model of early‐onset AD (Madhu et al., Journal of Extracellular Vesicles, 2024).

Methods : Using the 5xFAD mice, this study investigated the efficacy of hiPSC‐NSC‐EV treatments at 3 months of age (two 30 billion intranasal doses separated by a week) versus monthly intranasal treatments at 3–7 months of age (30 billion EVs/month) for maintaining better cognitive and mood function at 8 months of age. Treatments at 3 months represent intervention at an early stage of AD, whereas treatments at 3–7 months reflect regular interventions in the early and mid‐phases of AD.

Results : 5xFAD mice receiving two intranasal doses of hiPSC‐NSC‐EVs at 3 months of age displayed cognitive and mood impairments at 8 months. In contrast, 5xFAD mice receiving monthly doses of hiPSC‐NSC‐EVs at 3–7 months of age maintained better cognitive and mood function, evidenced by their ability to (1) discern minor changes in their environment in an object location test, (2) encode similar but not identical experiences in a non‐overlapping fashion in a pattern separation test, and (3) prefer sweet water in a sucrose preference test. 5xFAD mice receiving monthly doses of vehicle treatment were impaired in such cognitive tasks and displayed anhedonia. Analyses of brain tissues revealed that AD mice receiving monthly doses of EVs exhibited reductions in astrocyte hypertrophy, microglial clusters, microglia presenting inflammasomes, amyloid‐beta plaques, and the concentrations of oxidative stress markers and proinflammatory cytokines in the hippocampus.

Summary/Conclusion : Intranasal hiPSC‐NSC‐EV treatments only during the early stage of AD are insufficient for maintaining better brain function in the advanced AD stage. However, intermittent intranasal hiPSC‐NSC‐EV administrations are efficacious for restraining neuroinflammation and maintaining better cognitive and mood function for extended periods.

Funding : Supported by a grant from the National Institutes for Aging (1RF1AG074256‐01A1 to A.K.S.)

25 April 8.30–10.00 EV Sources and Basic Biology

Session Title OF10 EV Corona

Modulating protein corona on EVs when they are born for effortless cell targeting

Invited Speaker: Khuloud Al‐Jamal

The University of Hong Kong, Hong Kong

Single‐particle analysis reveals the presence of tRNA fragments on the surface of extracellular vesicles

Hannah Weissinger 1 , Madhusudhan Reddy Bobbili 3,4 , Mette Malle 1 , Yan Yan 2 , Johannes Grillari 3,4 , Jørgen Kjems 1,5

1 Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark. 2 Omiics ApS, Aarhus, Denmark. 3 Institute of Molecular Biotechnology, Department of Biotechnology, BOKU University, Vienna, Austria. 4 Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation with AUVA, Vienna, Austria. 5 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Introduction : tRNA fragments are highly modified RNA species found to be involved in the regulation of various cellular processes, including different types of cancers. Furthermore, they can be associated with extracellular vesicles (EVs), but like other EV cargo they were expected to be in the vesicle lumen. We were able to show for the first time that several tRNA fragments are present on the surface of EVs.

Methods : EVs from bovine milk and immortalized human mesenchymal stem cells (MSCs) were isolated by size exclusion chromatography (SEC) or tangential flow filtration (TFF), respectively. We performed RNA sequencing of Proteinase K and RNase‐treated EVs and compared the data of treated and untreated EVs. Single‐particle total internal reflectance (TIRF) microscopy enabled us to analyse the fraction of EVs with tRNA on the surface. Moreover, bead‐based capturing of the surface‐bound tRNA fragments allowed for the visualization of surface‐bound tRNA fragments on denaturing gels.

Results : In comparison with untreated EVs, tRNA fragments were significantly less abundant on EVs that were treated with proteinase K and RNase. This effect was not significant for miRNA or other types of small RNA. TIRF microscopy confirmed the presence of tRNA fragments on the surface of EVs. EVs could be immobilized with locked nucleic acid (LNA) complementary to the identified tRNA fragments, while LNAs with a random sequence or LNAs annealed to a competitor strand immobilized significantly fewer EVs, confirming the tRNA‐driven capturing. The directionality of the capture strand (5’‐end coupling vs 3’‐end coupling) further impacted the number of bound EVs. Moreover, the specific isolation of surface‐bound tRNA on beads resulted in fragments of the expected size.

Summary/Conclusion : The presence of surface‐bound tRNA fragments is conserved across cell types and species. We anticipate that the knowledge of nucleic acids on the surface of EVs will improve our understanding of the formation and release of EVs, as well as their uptake into cells and their potential clinical applications.

Funding : Novo Nordisk Foundation

Chitosan oligosaccharides inhibit protein corona formation on extracellular vesicles to facilitate sepsis treatment

Mengyun Wang, Jin Wang, Shuangshuang Liu, Lingting Kong, Linli Lv, Bicheng Liu, Taotao Tang

Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, China

Introduction : Extracellular vesicles (EVs) hold significant potential for sepsis treatment; however, their therapeutic efficacy can be compromised by the formation of plasma protein corona (PC) upon entering the circulatory system. This study aims to characterize the composition and function of the plasma PC and to explore how chitosan oligosaccharide (COS)‐mediated electrochemical modification can inhibit PC formation around EVs, thereby enhancing their therapeutic potential for sepsis.

Methods : Small EVs derived from umbilical mesenchymal stem cells (MSCs) and HEK293F cells were harvested via serum‐free 3D culture. A dynamic system simulated blood circulation for plasma PC formation, characterized by NTA, Raman spectroscopy, proteomics, and super‐resolution imaging. Additionally, COS was utilized for electrochemical modification of EVs to assess their inhibitory effect on PC formation and influence on macrophage uptake. A septic mouse model was established to evaluate the in vivo distribution and therapeutic efficacy of COS‐modified EVs.

Results : Following plasma PC formation, MSC‐EVs and HEK293F‐EVs showed increased particle size and decreased zeta potential. Raman spectroscopy revealed significant changes in EV composition, characterized by increased protein peak intensities. Proteomic analysis identified 263 and 209 plasma proteins on MSC‐EVs and HEK293F‐EVs, respectively, with notable enrichment of immunoglobulins and complement proteins. Super‐resolution imaging further confirmed the presence of plasma proteins like IGHG1, C3 and APOA1 around EVs. Additionally, PC formation enhanced macrophage uptake of EVs and activated inflammatory responses. To address this issue, we modified EVs with COS, which significantly reduced PC formation. Consequently, COS modification effectively inhibited macrophage uptake and the inflammatory response triggered by the PC. IVIS imaging indicated that COS‐modified EVs had decreased distribution in the liver and spleen but enhanced uptake in the brain, heart, lungs, and kidneys of both normal and septic mice. Importantly, COS modification improved survival rates compared to unmodified MSC‐EVs and enhanced protective effects on the lungs, heart, and kidneys in sepsis.

Summary/Conclusion : Plasma proteins readily form a PC on EVs, while COS modification can reduce the adsorption of immunoglobulins, complements, and others, thereby inhibiting plasma PC formation. This modification decreases EV uptake by the mononuclear phagocyte system, enhancing their biodistribution in extrahepatic organs and subsequently ameliorating multi‐organ damage in sepsis.

Tracking the extracellular vesicle biomolecular corona in physiological environments

Angelo Musicò a,b , Andrea Zendrini a,b , Santiago Gimenez Reyes c,d , Valentina Mangolini a,b , Lucia Paolini b,e , Miriam Romano a,b , Andrea Papait f,g , Antonietta Rosa Silini h , Paolo Di Gianvincenzo c , Arabella Neva i , Marina Cretich J , Ornella Parolini f,g , Camillo Almici i , Sergio E. Moya c , Annalisa Radeghieri a,b , Paolo Bergese a,b

a Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy. b CSGI, Center for Colloid and Surface Science, 50019 Florence, Italy. c Soft Matter Nanotechnology, Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), Paseo de Miramon 194, Donostia‐San Sebastián, Spain. d Instituto de Fisica del Sur (IFISUR‐CONICET), Av. Alem, Bahia Blanca, Argentina. e Department of Medical and Surgical Specialties, Radiological Sciences and Public Health (DSMC), University of Brescia, Brescia, Italy. f Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, 00168 Rome, Italy. g Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, 00168 Rome, Italy. h Centro di Ricerca Eugenia Menni, Fondazione Poliambulanza Istituto Ospedaliero, 25124, Brescia, Italy. i Laboratory for Stem Cells Manipulation and Cryopreservation, Department of Transfusion Medicine, ASST Spedali Civili of Brescia, 25123, Brescia, Italy. j Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”—National Research Council of Italy (SCITEC‐CNR), 20131 Milan, Italy. k National Inter‐university Consortium of Materials Science and Technology (INSTM), Via Giuseppe Giusti 9, 50121 Firenze, Italy

Introduction : The biomolecular corona (BC) is now recognized as a key determinant of the identity and function of extracellular vesicles (EVs). The concept of the BC has evolved to encompass not only protein coronas but also a broader range of interactions, such as those between EVs and lipoproteins, all of which collectively shape EV functionality and identity. Studying such interactions without perturbing them remains a significant challenge. Indeed, the soft nature of EVs implies that the weak forces keeping them assembled are—in terms of nature and energy—analogous to those driving the BC formation. This makes the study of EV‐BC complexes directly in solution highly appealing, as attempting to separate them from their environment may lead to alterations in either the EVs or the BC.

Methods : We developed a Fluorescence Correlation Spectroscopy methodology that enables tracking the dynamics of the biomolecular corona in situ under close‐to‐physiological conditions. This technique was successfully applied to study the formation of BC on red blood cell (RBC)‐ and amniotic membrane Mesenchymal Stromal Cell‐ derived EVs. Both EV types were separated and characterized by applying state‐of‐the‐art methods and MISEV 2023 guidelines and then challenged with human plasma depleted of extracellular nanoparticles. Furthermore we probed the interactions between RBC‐EVs and various classes of lipoproteins.

Results : The technique enabled us to show for the first time that, under physiological conditions, (i) EVs from different sources exhibit distinct corona dynamics, (ii) RBC‐EVs interact differently with various lipoprotein classes (up to 100% of interacting EVs), and (iii) these interactions are modulated by the molecular components of human plasma.

Summary/Conclusion : This work introduces a powerful tool for studying the biomolecular corona of EVs, providing a deeper understanding of extracellular vesicles at the nanoscale and opening for enhanced characterization of EV biomolecular corona in a variety of experimental conditions.

Funding : MIUR—PRIN 2017E3A2NR_004 project; European H2020 BOW project (H2020‐EIC‐FETPROACT‐2019, ID: 952183); NFFA‐Europe‐Pilot Transnational Access Activity (ID 101007417), by CIC biomaGUNE, proposal ID 362. PID2020‐114356RB‐I00 project from the Ministry of Science and Innovation of the Government (Spain); Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali—INSTM.

TGF‐β‐activated cancer‐associated fibroblasts remodel the tumour microenvironment through extracellular vesicle surface‐associated proteins

Chao Li 1 , Agustin Enciso‐Martinez 1,2,3 , Lizhe Zhu 1 , Sarah A. Rotman 4 , Peter A. van Veelen 4 , Roman I. Koning 5 , Hailiang Mei 6 , Peter ten Dijke 1

1 Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands; 2 Amsterdam UMC, University of Amsterdam, Biomedical Engineering & Physics; Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands; 3 Amsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry; Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Meibergdreef 9, Amsterdam, The Netherlands; 4 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands; 5 Electron Microscopy Facility, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands; 6 Department of Biomedical Data Sciences, Sequencing Analysis Support Core, Leiden University Medical Center, Leiden, The Netherlands

Introduction : Cancer‐associated fibroblasts (CAFs) are a major component of tumour stroma in various cancer types. The cytokine transforming growth factor‐β (TGF‐β) is frequently present at high levels in the tumour microenvironment and is a potent inducer of CAF activation. Activated CAFs produce and remodel the extracellular matrix and release soluble factors and extracellular vesicles (EVs) to communicate with other cell types in the tumour microenvironment. The content of EVs, including proteins and nucleic acids, has mainly been studied for its association with EV function. However, the EV surface can function as a versatile platform for presenting surface proteins and extracellular matrix components, which directly mediate intercellular communication. The EV surface is in direct contact with extracellular space, enabling EVs to act as sponges that adsorb additional proteins and matrix components after release. In this study, we highlight the surface of CAF‐derived EVs and reveal the functions and mechanisms of EV surface‐associated proteins in promoting immune evasion and cancer development.

Methods : EVs were isolated from quiescent and TGF‐β‐activated CAFs using size exclusion chromatography for quantitative proteomic analysis and functional studies. The presence of EV surface‐associated protein was validated by super resolution microscopy and western blot. The biological functions of EVs were tested on spheroid models with cancer cells, CAFs and CD8+ T cells, and monocultures. The interactions between EV surface proteins and cell receptors were investigated using a proximity ligation assay.

Results : TGF‐β can induce CAF activation and upregulate the quantity of EV proteins. Quantitative proteomic analysis revealed that the top upregulated proteins are matrix proteins, including TSG6. The upregulated matrix proteins and extracellular matrix components localized on the EV surface and interacted with receptors on EVs; for example, hyaluronan, which colocalized with CD44+ EVs. The EV surface‐associated TSG6 and hyaluronan interacted with cell receptors CD44 and ALK5 and induced receptor clustering, thereby triggering signalling cascades before the internalization of EVs. This process can sustain CAF activation and induce immunosuppression in CD8+ T cells.

Summary/Conclusion : EV surface proteins and matrix components from TGF‐β‐activated CAFs interact with receptors and induce receptor clustering on target cells, thereby further inducing CAF activation and immunosuppression in CD8+ T cells.

25 April 8.30–10.00 Physiology and Pathology

Session Title OF11 Cancer and Metastasis

Cancer and metastasis intro talk

Invited Speaker: David C. Lyden

Weill Cornell Medicine, USA

Targeting Rab27B‐mediated extracellular vesicle release evokes a DNA damage response in ER‐positive breast cancer

Lien Lippens 1,2 , Robin Boiy 1,2 , Bruno De Geest 3 , Ilkka Miinalainen 4 , Pekka Rappu 5 , Olivier De Wever 1,2 , and An Hendrix 1,2

1 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium; 2 Cancer Research Institute Ghent, Ghent, Belgium; 3 Department of Pharmaceutics, Ghent University, Ghent, Belgium; 4 Biocenter Oulu, University of Oulu, Oulu, Finland; 5 Department of Life Technologies and InFLAMES Flagship, University of Turku, Turku, Finland Lien Lippens and Robin Boiy are equally contributing first authors

Introduction : Rab27 GTPases and their effector proteins play an important role in exocytosis and the late endocytic pathway, including endosomal transport and maturation, and regulate exosome secretion through trafficking of multivesicular endosomes (MVEs). We previously demonstrated that the small GTPase Rab27B enhances growth, invasion and metastasis in oestrogen receptor (ER)‐positive breast cancer cells in vitro, and elevated expression of Rab27B is associated with poor prognosis and metastasis in ER‐positive breast cancer patients. However, the underlying Rab27B‐dependent mechanisms remain to be investigated.

Methods : EVs were separated using Optiprep density gradient centrifugation and analysed using state‐of‐the‐art techniques, including TEM, AF4‐MALS, ONI, and LC‐MS/MS. The Rab27B‐dependent EV secretion pathway in ER‐positive breast cancer cells was targeted using overexpression of mutated isoforms of Rab27B. Furthermore, short hairpin constructs targeting the Rab27B‐dependent EV secretion pathway at multiple levels were used. Functional effects, survival and proliferation were assessed in vitro (in 2D and 3D) and in vivo.

Results : Small EV and large EV subsets differ in protein composition and biogenesis route. Proteomic analysis of EV subtypes revealed Rab27B‐depleted small EVs, originating from the plasma membrane and therefore a subclass of ectosomes, and Rab27B‐enriched large EVs, with a suggested endosomal origin. Importantly, although Rab27B was enriched in large EVs, it is essential to steer the formation of both small Rab27B‐depleted ectosomes and large Rab27B‐enriched exosomes. Furthermore, we show that perturbation of functional Rab27B results in a DNA damage response severely impacting cell proliferation and survival.

Summary/Conclusion : Overall, our study reveals a novel role for Rab27B in the secretion of small ectosomes in ER‐positive breast cancer cells. Furthermore, we demonstrate that targeting Rab27B elicits a DNA damage response and impairs cancer cell survival, identifying this protein as a target for novel anti‐cancer strategies.

Funding : This work was supported by the Fund for Scientific Research Flanders and Kom op tegen Kanker (Stand Up to Cancer), the Flemish cancer society.

Extracellular vesicle‐mediated release of beta‐2 adrenergic receptor by prostate cancer cells and its impact on tumour microenvironment

Krizia Sagini 1,2 , Mughilan Selvarajah 1,2 , Alicia Llorente 1,2,3

1 Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Norway, 2 Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, Norway, 3 Department for Mechanical Electronics and Chemical Engineering, Oslo Metropolitan University, Norway

Introduction : The beta‐2 adrenergic receptor (ADRB2) belongs to the G protein‐coupled receptor (GPCR) family. Studies indicate that GPCRs can be secreted within extracellular vesicles (EVs), enabling their horizontal transfer to recipient cells. In advanced prostate cancer (PCa), ADRB2 plays a key role in neuroendocrine differentiation, a process linked to poorer prognosis and tumour progression. This study aims to determine if PCa cells release ADRB2 in EVs and to assess how this impacts the tumour microenvironment.

Methods : EVs were isolated from the conditioned media of the highly metastatic PC‐3 cell line by differential centrifugation, obtaining large‐medium EVs and small EVs (sEVs) when 10,000 × g and 100,000 × g centrifugal force, respectively, were used. In certain cases, EVs were further purified by high‐resolution density gradient. EV characterization was performed with immunoblotting, radioligand binding assay, and nanoparticle tracking analysis (NTA). For some experiments, cells were treated with the ADRB2 agonist isoproterenol or the antagonist propranolol.

Results : Our findings reveal that PC‐3 cells constitutively release ADRB2 in annexin A2‐ and annexin A6‐positive sEVs. Exposure to the agonist isoproterenol reduced ADRB2 levels in EVs, while the antagonist prevented this reduction. NTA confirmed that this decrease was not due to an overall suppression of EV secretion by isoproterenol. Additionally, using a stable cell line expressing fluorescent ADRB2‐neonGreen, we showed that ADRB2 relocalized from the plasma membrane to cytosolic vesicular structures upon incubation with isoproterenol.

Summary/Conclusion : Our findings suggest that PC‐3 cells release ADRB2 through plasma membrane‐derived sEVs, as these vesicles contain annexins. Moreover, altering ADRB2's localization at the plasma membrane (agonist treatment) reduces its extracellular release. Further experiments with ADRB2‐neonGreen are ongoing to shed light on the role of EVs in ADRB2's horizontal transfer within the tumour microenvironment.

Delivery of melanoma cell‐derived miR‐92b‐3p by extracellular vesicles promotes the formation of carcinoma‐associated fibroblasts through the downregulation of PTEN

Stefanie Kewitz‐Hempel 1 , Nicola Windisch 1 , Gerd Hause 2 , Lutz Müller 3 , Cord Sunderkötter 1 , Dennis Gerloff 1

1 Department of Dermatology and Venereology, Martin‐Luther‐University Halle‐Wittenberg, Halle (Saale), Germany, 2 Biocenter, Martin‐Luther‐University Halle‐Wittenberg, Halle (Saale), Germany, 3 Department of Internal Medicine IV, Hematology and Oncology, Martin‐Luther‐University Halle‐Wittenberg, Halle (Saale), Germany

Introduction : In melanoma, carcinoma‐associated fibroblasts (CAFs) represent a significant cellular component of the tumour microenvironment, exhibiting the capacity to facilitate tumour growth and metastatic dissemination of malignant cells. Tumour cells are capable of influencing cells in the microenvironment by the release of extracellular vesicles (EVs). It has been demonstrated that EVs transfer miRNAs, which are small, non‐coding RNAs that regulate protein expression post‐transcriptionally. The objective of this study was to ascertain whether and by what molecular mechanisms EV‐delivered miRNAs from melanoma cells contribute to the formation of CAFs.

Methods : EVs from melanoma cell lines and normal melanocytes were purified by ultracentrifugation and size exclusion chromatography in accordance with the MISEV guidelines. Primary human dermal fibroblasts (NHDFs) were incubated with EVs, inducing the formation of CAFs. The characterization of CAFs was based on their biological functions and gene expression profiles. Next‐generation sequencing was employed to identify specific miRNAs present in the melanoma cell‐derived EVs.

Results : The uptake of melanoma cell‐derived EVs by NHDFs in vitro induces a CAF‐like phenotype. This phenotype is defined by an increased expression of CAF marker genes (IL‐8, αSMA, and FAP). This was accompanied by an increase in proliferation and cell migration. Next‐generation sequencing revealed a marked enrichment of miR‐92b‐3p in melanoma cell‐derived EVs relative to EVs derived from normal melanocytes (NHEMs). Consequently, an accumulation of miR‐92b‐3p was observed in NHDFs following incubation with melanoma cell‐derived EVs. The overexpression of miR‐92b‐3p in NHDFs resulted in the emergence of a CAF‐like phenotype that was comparable to that observed in NHDFs incubated with EVs derived from melanoma cells. Inhibition of miR‐92b‐3p by locked nucleic acids (LNAs) resulted in the prevention of the previously observed CAF‐like phenotype. As a potential target of miR‐92b‐3p, we identified the tumour suppressor PTEN. Consequently, treatment with melanoma cell‐derived EVs or overexpression of miR‐92b‐3p results in a reduction in PTEN protein expression in NHDFs. Furthermore, the knockdown of PTEN in NHDFs by siRNA resulted in an increased expression of CAF markers.

Summary/Conclusion : The delivery of miR‐92b‐3p from melanoma cells to fibroblasts via an EV‐mediated transfer has been demonstrated to contribute to the formation of a CAF‐like phenotype by targeting PTEN.

Exploring the role of exercise‐induced extracellular vesicles in breast cancer: mechanisms and implications

Agnese Brokāne 1 , Diana Skorinkina 1 , Kristaps Eglītis 2 , Pawel Zayakin 1 , Aleksandrs Dumešs 1 , Elza Bojāre 1 , Agata MLynska 3 , Jānis Eglītis 2 , Alicia Llorente 4 , Aija Linē 1

1 Latvian Biomedical Research and Study Centre, Latvia, 2 Latvian Oncology Center, Latvia, 3 National Cancer Institute, Lithuania, 4 Oslo University Hospital, Norway

Introduction : A growing number of studies show that physical exercise enhances physical and cognitive capabilities, thus improving the quality of life and disease outcomes in cancer patients. Several lines of evidence suggest that EVs mediate tissue crosstalk during exercise; however, the effects of exercise‐induced EVs on cancer are poorly understood. Here, we investigated the RNA cargo and impact of exercise‐induced EVs from breast cancer (BC) patients who performed regular high‐intensity interval training during neoadjuvant chemotherapy within the CancerBeat clinical trial (ClinicalTrials.gov: NCT06522971 ) and healthy endurance runners on the proliferation and gene expression profile of patient‐derived BC cells.

Methods : Blood samples from BC patients and runners were collected before and immediately after an exercise session. EVs were isolated from plasma samples by SEC, and their quality and quantity were assessed by NTA, TEM, and western blotting. EV RNA sequencing libraries were prepared with the QIAseq miRNA Library Kit. Patient‐derived BC cell cultures were established from the surgical specimens and characterized by immunofluorescence. The effects of EVs on BC cells were explored by CCK‐8 viability assay and whole transcriptome sequencing.

Results : EV RNA sequencing analysis revealed several mRNAs, tRFs and miRNAs with tumour suppressor properties that were upregulated in EVs collected immediately after an exercise session. Post‐exercise EVs from healthy runners tended to inhibit the proliferation of patient‐derived BC cells, while the effects of EVs from BC patients were diverse, with some samples inhibiting and some stimulating the proliferation of BC cells. In CB18T BC cells, EVs from both BC patients and healthy runners regulated the expression of genes involved in collagen fibril organization, tube and vasculature development, whereas the gene sets involved in oxidative phosphorylation, RNA and lipid metabolism were differentially regulated by pre‐ and post‐exercise EVs from BC patients and healthy controls.

Summary/Conclusion : Post‐exercise EVs from healthy runners may have a beneficial effect on inhibiting the growth of cancer cells; however, pre‐operative physical activity in some BC patients may stimulate the release of cancer‐derived EVs, causing distinct effects in cancer cells than exercise‐induced EVs from healthy runners.

Funding : EEA and Norway Grants project No. EEA‐RESEARCH‐164 (CancerBeat) and ERDF project No. 006.

25 April 8.30–10.00 Technologies and Methods

Session Title OF12 EV Hybrids and Mimetics

EV hybrids and mimetics for cancer therapy: challenges and future perspectives

Invited Speaker: Giorgia Pastorin,

Professor & Head of Department of Pharmacy & Pharmaceutical Sciences, Faculty of Science, National University of Singapore

Single‐particle tracking analysis of extracellular vesicles, synthetic vesicles and hybrid vesicles for a deconvoluted understanding of uptake and transfection efficiency

Mette Galsgaard Malle 1 , Bo Volf Brøchner 1 , Ping Song 1 , and Jørgen Kjems 1,2

1 Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark, 2 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Introduction : Presently, synthetic nanoparticles like liposomes and lipid nanoparticles (LNPs) are extensively utilized for drug delivery, yet they suffer from drawbacks such as toxicity and adverse immune reactions. Natural extracellular vesicles (EVs) offer promise as native, safe, and versatile carriers for drugs. However, incorporating large molecules into EVs remains challenging. In this study, we introduce a novel method for loading EVs by programmable fusion between EVs and liposomes loaded with mRNA. Furthermore, we track individual vesicles in live cells for both synthetic particles, EVs and fused hybrids for a direct understanding of the uptake.

Methods : EVs are isolated from C2C12 cell culture and from bovine milk. The EVs were characterized following the MISEV 2018 guidelines. RNA loading was evaluated by QRT‐PCR, and transfection efficiency was evaluated by delivering fused hybrid EV‐liposome encapsulated mRNA encoding mCherry to HEK cells. The single‐particle tracking is performed on fluorescently labelled particles and EVs using HILO microscopy and analysed by an in‐house developed Python script.

Results : EVs are anchored to a magnetic bead surface using programmable DNA handles. Using another set of complementary DNA handles, EVs and mRNA‐containing liposomes are fused, washed, and subsequently released from the beads. Hence, we can collect purified hybrid EVs loaded with mRNA. We track the individual particles in live single cells. Here we can directly observe EVs and hybrids fuse to the cell surface and track the delivered particles. We find distinct spatial‐temporal patterns for synthetic particles and EVs. Interestingly, hybrid particles have an intermediate spatial‐temporal pattern, reflecting both the synthetic and natural origin. Furthermore, we investigate a variety of fusion strategies to construct hybrid particles for an improved uptake. In vivo delivery of mRNA within hybrid particles is ongoing.

Summary/Conclusion : Single‐particle tracking offers a unique direct evaluation of the underlying delivery and cell‐interaction mechanism. Constructing hybrid particles is not only a more native‐like way of delivery, but using single‐particle tracking, we can determine the mechanistic interaction differences. We anticipate that this method represents a significant advancement for targeted EV‐mediated therapy and natural drug delivery.

Funding : Lundbeck Foundation grant R380‐2021‐1393 and Danish National Research Foundation grant 135.

Comparative proteomics of extracellular vesicles and liposomes show cancer‐related proteins and biomarkers

Ishman Kaur 1,4 Tad Wu 1,2 , Yunxi Chen 1,2 , Laura Kienzle 1 , Thupten Tsering 1,2 , Vahé Nerguizian 4 , and Julia V. Burnier 1,2,3

1 Research Institute of the McGill University Health Centre, McGill University, Montreal, QC 2 Department of Pathology, McGill University 3 Gerald Bronfman Department of Oncology, McGill University 4 Department of Electrical Engineering, École de technologie supérieure

Introduction : Extracellular vesicles (EVs) play an important role in metastasis by communicating with the tumour microenvironment through cellular uptake by recipient cells. EV parameters that influence this process include membrane and cargo proteins, lipid composition, and physicochemical properties. However, due to the heterogeneity of EVs and their technically challenging isolation procedure, studying individual EV characteristics and their properties is challenging. Liposomes are synthetic vesicles that share features with EVs, like a lipid bilayer and biomolecule encapsulation capabilities, offering an alternative approach for studying EVs. We aimed to generate liposomes that encapsulate EV‐derived proteins and compare their proteomic signature to natural EVs. This study has clear implications for modelling cancer metastasis.

Methods : SW620‐derived small EVs (sEVs) were isolated by filtering the conditioned media followed by ultracentrifugation. EV proteins were extracted using RIPA buffer followed by detergent removal. Liposomes with lipid formulation DMPC: CHOL (1:1) were synthesized using microfluidics, and proteins extracted from EVs were encapsulated. Samples were characterized in respect to their average mean diameter, size distribution, and zeta potential using ZetaView. SDS‐PAGE and BCA were performed to calculate the encapsulation efficiency of the proteins in the liposomes. Liposome internalization was visualized using Incucyte imaging. Finally, EV proteins as well as proteins extracted from the loaded liposomes were subjected to high‐resolution mass spectrometry.

Results : Liposomes were synthesized with an sEV‐like size distribution (137.9–250.1 nm) to mimic sEVs, and those with a higher encapsulation efficiency showed a closer zeta potential to sEVs (−32.57 mV). EV‐specific important markers such as CD63, CD9 and HSP70 were effectively encapsulated, with an efficiency of up to 35%. In a separate experiment, HSP70 encapsulation achieved up to 49% efficiency with optimized lipid formulations, demonstrating the impact of lipid composition on loading capacity. Furthermore, internalization of liposomes has been detected by Incucyte.

Summary/Conclusion : We successfully encapsulated EV proteins into liposomes. The encapsulation efficiency clearly shows the potential of liposomes to act as efficient carriers of significant cancer biomarkers. Moreover, such a comparative proteomic analysis of the specific protein profiles encapsulated in liposomes will not only help to explain the biology of EVs but also help further functional validation studies for understanding cancer metastasis.

Synthetic extracellular vesicles: differential effects of membrane and cytosolic proteins on physicochemical properties and functions

Yunxi Chen 1,2 , Noélie Douanne 1 , Kyle Dickinson 1 , Rubén López Salazar 1 , Thupten Tsering 1,2 , Tad Wu 1,2 , Ishman Kaur 1,3 , Chaymaa Zouggari Ben El Khyat 1,3 , Vahé Nerguizian 3 , and Julia V. Burnier 1,2

1 Research Institute of the McGill University Health Centre, Canada, 2 McGill University, Canada, 3 École de technologie supérieure, Canada

Introduction : The biology of extracellular vesicles (EVs) is intricate, involving surface interactions with recipient cells and cargo transfer that drives further downstream effects. Cancer cell‐derived EVs, for instance, have been implicated in organotropic metastasis, yet the underlying mechanisms are not fully understood. EV research remains challenging due to their high heterogeneity and lengthy isolation processes. Liposomes, widely used as delivery vehicles, share structural similarities with EVs, particularly in their lipid bilayer composition. We propose a novel synthetic extracellular vesicle (SV) system using liposomes to mimic EV size and zeta potential (surface charge) and then selectively load proteins to study their downstream effects.

Methods : MP41‐derived small EVs (sEVs) were isolated, and their physicochemical properties (size, zeta potential, and morphology) were characterized to guide the design of SVs. Total MP41 cell membrane and cytosolic proteins were extracted and loaded separately (Mem‐SV, Cyto‐SV) or in combination (Complete‐SV) into SVs using a microfluidic chip and characterized for EV‐mimicry. SV‐incorporated proteins were profiled using mass spectrometry. In vitro cell uptake by hepatocytes was analysed to determine the properties for optimized internalization. Biodistribution and tissue‐specific accumulation of sEV/SVs in a mouse model followed by tail vein injection was studied by in vivo, ex vivo, and histopathological imaging.

Results : All SVs were synthesized efficiently (E12 particles/mL/min) with an sEV‐like size distribution (100 to 200 nm). Mem/Complete‐SVs exhibited a zeta potential comparable to sEVs, with a more negative charge than that of Empty/Cyto‐SVs. Over 2500 proteins were identified across protein‐loaded SVs. Protein encapsulation efficiency was found to be influenced by the physical properties of proteins. Preferentially encapsulated proteins had a significantly smaller mean molecular weight and higher mean isoelectric point. In vitro, encapsulation of membrane proteins in SVs enhanced cell uptake, with Mem/Complete‐SVs showing higher cell internalization than Empty/Cyto‐SVs. In vivo, the liver and spleen showed the most sEV/SV enrichment. Analysis of cell type‐specific uptake in tissue sections is underway.

Summary/Conclusion : This study developed a customizable synthetic EV model to investigate individual EV‐associated factors in intercellular communication, enhancing our understanding of EV biology and informing targeted nanomedicine strategies.

Funding : Canadian Institutes of Health Research grant. Fonds de recherche du Québec—Santé.

Stable anti‐fouling nanospheres to standardize concentration measurements of extracellular vesicles

Presenter: Jelle van den Beukel

Amsterdam University Medical Center, The Netherlands

Introduction : Measuring concentrations of extracellular vesicles (EVs) is difficult due to their heterogeneity, which leads to incomparable and irreproducible data. To overcome this problem, nanospheres of known concentration could help track the dilution and volume of the EV‐containing sample during analysis. However, adding nanospheres to body fluids results in non‐specific adsorption of proteins, and with that, the formation of a protein corona. This interaction leads to a decrease in the concentration of nanospheres over time, resulting in unstable concentrations of nanospheres. Our aim is to develop nanospheres coated with anti‐fouling polymer brushes that can prevent non‐specific adsorption of proteins present in EV‐containing (bio) fluids.

Methods : Polystyrene nanosphere cores were functionalized using surface‐initiated photoinduced electron transfer‐reversible addition‐fragmentation chain‐transfer polymerization (SI‐PET‐RAFT), with three different monomers: N‐(2‐hydroxypropyl)methacrylamide (HPMA), methacrylate phosphocholine (MPC), and carboxybetaine methacrylate (CBMA). Functionalization was monitored using dynamic light scattering (DLS), X‐ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The functionalized nanospheres were incubated in 6 mg/mL fluorescently labelled bovine serum albumin solution and fluorescently labelled human blood plasma to monitor their anti‐fouling properties and stability over time.

Results : Functionalization of 220 nm polystyrene nanospheres increased their diameter to 260 nm (HPMA), 250 nm (MPC), and 350 nm (CBMA). XPS confirmed the chemical composition, and TEM showed core‐brush structures on the surface of all functionalized nanospheres. Anti‐fouling tests in two different fluorescent protein solutions demonstrated no binding of proteins to the HPMA‐coated nanospheres. In contrast, MPC nanospheres became slightly fluorescent in human blood plasma, and CBMA nanospheres became fluorescent in human blood plasma. Stability measurements in human blood plasma showed a stable concentration for the HPMA and MPC nanospheres up to 5 h, whereas the CBMA nanospheres decreased in concentration.

Summary/Conclusion : Stable anti‐fouling nanospheres can be produced in size ranges of 250–260 nm. Concentrations of HPMA and MPC nanospheres are stable in undiluted human blood plasma for at least 5 h. This stability shows that coated nanospheres can be used as internal calibrants or quality controls for instruments used to detect nanoparticle concentrations in biofluids, such as flow cytometers.

Funding : Dutch Research Council (NWO), VIDI 19724.

25 April 14.30–16.00 Disease and Therapy

Session Title OT13 EV Biomarkers 2

Harnessing circulating tumour extracellular vesicles for longitudinal monitoring of cancer evolution and therapeutic response in prostate cancer

Invited Speaker: Irene Casanova Salas

Vall d'Hebron Institute of Oncology (VHIO), Spain

In vitro characterization of vesicles released by prostate cancer cells dictates PSMA/STEAP1‐positive large‐EVs as targets to immunocapture tumour‐derived EVs ex vivo

Lorenzo Messa 1 , Alessia Marinelli 1 , Nicola Dalla Valle 1 , Yari Ciani 1 , Federico Vannuccini 1 , Orsetta Quaini 1 , Vera Mugoni 1 , Isabella Pesce 1 , Caterina Nardella 1 , Consuelo Buttigliero 2 , Marco Maruzzo 3 , Umberto Basso 3 , Orazio Caffo 4 , Dolores Di Vizio 5 , Francesca Demichelis 1

1 Department of Cellular, Computational, and Integrative Biology; University of Trento; Trento, Italy, 2 Department of Oncology, University of Turin, San Luigi Gonzaga Hospital, Torino, Italy, 3 Medical Oncology Unit, Department of Oncology, Istituto Oncologico Veneto IOV IRCCS, Padova, Italy, 4 Department of Medical Oncology, Santa Chiara Hospital, Trento, Italy, 5 Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars‐Sinai Medical Center, Los Angeles, CA, USA

Introduction : Prostate cancer (PCa) is a leading cause of cancer deaths worldwide due to drug‐resistance mechanisms. Recent studies pinpointed the potential of tumoural extracellular vesicles (t‐EVs) circulating in PCa patients’ bloodstream to gather information on disease status and therapy response. Yet, isolation of t‐EVs from the bulk of circulating particles remains challenging. To this purpose, small‐EV isolation through PSMA‐ or STEAP1‐based immunocapture approaches has been explored but requires further optimization.

Methods : Broad size range EVs (small < 200 nm; large > 200 nm) were isolated through sequential centrifugations (2800 × g , 10,000 × g , 100,000 × g ) of cell culture media and platelet‐poor plasma (PPP) samples and purified by iodixanol gradient ultracentrifugations. In vitro, EVs were isolated from a panel of PCa cell lines recapitulating different disease subtypes. Ex vivo samples were processed from healthy donors ( n = 5) and metastatic PCa patients’ PPPs ( n = 4) collected with standardized procedures at multiple clinical sites within the PRIME multi‐institutional programme. Small and large EVs were counted using dynamic light scattering and tunable resistive pulse sensing, respectively, and analysed through western blotting, flow cytometry, and imaging techniques.

Results : We confirm PSMA and STEAP1 as relevant prostate‐specific proteins secreted in EVs from multiple PCa cell lines, whose abundance reflects their androgen receptor status. We found PSMA and STEAP1 present not only in small EVs, as previously reported, but also in large EVs. Via high‐resolution imaging and flow cytometry, we show surface exposure of PSMA and STEAP1 with higher prevalence in the bulk of large EVs (> 50%) compared to small EVs (< 5%). Preliminary results show their presence also ex vivo, in large‐EVs purified from the plasma of a pilot cohort of metastatic PCa patients.

Summary/Conclusion : The higher relative abundance of PSMA‐/STEAP1‐positive large EVs compared to small EVs may suggest an overall lower heterogeneity of large EV subpopulations. This supports the implementation of PSMA/STEAP1‐based immunocaptures that, by including large‐EVs and co‐targeting two surface antigens, would allow the isolation of a higher amount of circulating t‐EVs and the acquisition of useful tumour‐related information.

Funding : Accelerator Award 2018 (ID 22792) and Investigator Grant (ID 29370) from AIRC; Cancer Research UK (CRUK, ID A26822); NIH R01CA287075‐01A1; Dipartimenti di Eccellenza 2023‐2027 MUR.

A novel platform for isolating brain cell‐specific extracellular vesicles to profile the cellular environment in multiple pathologies of the brain

Presenter: Richard Lobb

The University of Queensland, Australia

Introduction : Brain cells, including glial cells like microglia, astrocytes, and oligodendrocytes, are essential in maintaining neural health and responding to injury and disease. Extracellular vesicles (EVs) released by these cells can cross the blood‐brain barrier into the bloodstream, carrying molecular cargo reflective of the brain's cellular environment. Given the trace levels of these EVs in blood, highly sensitive detection methods are essential for their capture and analysis. This study presents a nanopillar array‐based platform designed to selectively capture and analyse EVs from specific brain cell types, enabling comprehensive profiling of the brain from blood across central nervous system (CNS) conditions, such as traumatic brain injury (TBI), neurodegenerative diseases, and brain cancer.

Methods : The platform uses a nanopillar array fabricated via electron beam lithography, enhancing EV capture by maximizing surface area and interaction time. Functionalized with antibodies targeting specific brain cell markers, the nanopillars enable selective capture of EVs from microglia, astrocytes, and other glial cells. Following capture, surface‐enhanced Raman scattering (SERS) nanoparticles with Raman reporters and detection antibodies allow for multiplexed single‐molecule analysis. Platform validation included nanoparticle tracking, western blotting for EV and cell‐specific markers, and electron microscopy to confirm EV integrity, purity, and cellular origin.

Results : The platform effectively isolated EVs from various brain cell types, detecting specific markers tied to CNS pathologies. In glioblastoma, captured EVs were enriched for glioma stem cell markers (CD24, CD44, CD133, and EGFR), highlighting their role in monitoring disease relapse. In TBI models and patients, microglia‐derived EVs and brain EVs with specific cytokine profiles provided insights into immune responses and neuroinflammation. In neurodegenerative disease models, EV profiles revealed molecular features of disease progression, underscoring the platform's sensitivity to detect changes in the brain environment over time.

Summary/Conclusion : This study introduces a high‐resolution, non‐invasive platform for isolating and characterizing brain cell‐specific EVs, offering a valuable tool for profiling pathological processes in CNS disorders. The findings support the platform's potential for monitoring cell‐specific contributions to CNS pathologies, aiding in diagnostics, disease tracking, and therapeutic evaluation across multiple brain conditions.

Funding : Mark Hughes Foundation.

Detection and monitoring of prostate cancer using activity‐based biomarkers from plasma extracellular vesicles

Cuno Laurin Lange 1 , Jan Van Deun 1 , Giuseppe Di Fonzo 1 , Sven Wach 2 , Helge Taubert 2 , Andreas Baur 1 , Bernd Wullich 2

1 Department of Dermatology, Uniklinikum Erlangen, Germany 2 Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Germany

Introduction : Prostate cancer (PCa) is the most common male cancer with an incidence of 25.1% in Germany. Serum PSA is the standard screening marker, having a high sensitivity (90%) but low specificity (20%), often leading to avoidable, invasive procedures. One of the treatments of metastatic PCa is the PSMA radionuclide, targeting and killing prostate tumour cells. Nevertheless, most tumours relapse. We have developed an activity‐based test assay based on pEV‐associated proteases (PAA) as well as proteomic analysis of non‐enzymatic factors, and evaluated its performance to detect patients with PCa and monitor PSMA radionuclide treatment.

Methods : pEV were isolated via dual‐mode chromatography (DMC). Plasma EV‐associated protease activity (PAA) (patent in progress) was determined using a FRET‐peptide‐based fluorescence assay. Proteomic content was analysed using a commercial proximity extension assay (Olink Proteomics) for 3000 factors. To correct for confounders, we included a matched control group with similar medical profiles. Machine learning algorithms were employed.

Results : When comparing PCa patients ( n = 245) with control groups ( n = 210) (obtained informed consent) applying the PAA and proteomics, an AUC of 0.88–0.9 was achieved, as compared to an AUC of 0.67 assessing the PSA. Notably, we observed increased pEV protease activity in non‐cancer controls, emphasizing the importance of including additional markers, like the PSA, to classify test subject. When monitoring PSMA radionucleotide‐treated patients, the PAA correctly documented the decrease of tumour cells, similar to the PSA. However, unlike the PSA, the pEV‐based PAA seemed to recognize tumour escape earlier.

Summary/Conclusion : The PAA could be used for early detection of PCa as well as therapy‐ and relapse monitoring, potentially recognizing the minimal residual disease (MRD). Due to its high accuracy, reproducibility and simplicity, the PAA might be integrated into diagnostic cancer management. However, test confounders potentially related to inflammation need to be corrected for.

Funding : Bundesministerium für Bildung und Forschung (BMBF), Bayerisches Staatsministerium für Wirtschaft (Bayern Innovativ) and the Hahn Schickard Institute.

Circulating extracellular vesicle isomiR networks to predict therapy response in patients with multiple myeloma

Cristina Gómez‐Martín 1,2 , Esther E. E. Drees 1,2 , Monique A. J. van Eijndhoven 1,2 , Nils J. Groenewegen 1,2,3 , Steven Wang 1,2 , Sandra A. W. M. Verkuijlen 1,2 , Jan R. T. van Weering 4 , Ernesto Aparicio‐Puerta 5‐8 , Leontien Bosch 1,2 , Kris A. Frerichs 2,9 , Christie P. M. Verkleij 2,9 , Marie J. Kersten 2,9 , Josée M. Zijlstra 2,9 , Daphne de Jong 1,2 , Catharina G. M. Groothuis‐Oudshoorn 10 , Michael Hackenberg 3,5‐8 , Johan R. de Rooij 3 , Niels W. C. J. van de Donk 2,9 , D. Michiel Pegtel 1–3

1 Amsterdam UMC location: Vrije Universiteit Amsterdam, Department of Pathology, Amsterdam, The Netherlands, 2 Cancer Center Amsterdam, Program Imaging and Biomarkers, Amsterdam, The Netherlands, 3 ExBiome B. V., Amsterdam, The Netherlands, 4 Amsterdam UMC location: Vrije Universiteit Amsterdam, Department of Human Genetics, Amsterdam, The Netherlands, 5 Computational Epigenomics and Bioinformatics, Department of Genetics, University of Granada, Spain, 6 Bioinformatics Laboratory, Biotechnology Institute, Granada, Spain, 7 Instituto de Investigación Biosanitaria (ibs). GRANADA, University of Granada, Granada, Spain, 8 Excellence Research Unit “Modelling Nature” (MNat), University of Granada, Granada, Spain, 9 Amsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Hematology, Amsterdam, The Netherlands, 10 Department of Health Technology and Services Research, University of Twente, Enschede, The Netherlands

Introduction : Multiple myeloma (MM) is a largely incurable malignancy characterized by uncontrolled proliferation of clonal plasma cells in the bone marrow. While introducing immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies (e.g., daratumumab) has improved survival rates, most patients eventually relapse and become refractory to further treatments. To shift MM from an incurable to a chronic disease, there is an urgent need for biomarkers that accurately monitor therapeutic response and predict individual outcomes, especially in relapsed/refractory (RR) MM patients. The current standard for response monitoring involves measuring M‐protein and free light chains in blood and urine, alongside invasive bone marrow biopsies to confirm minimal residual disease (MRD) status. Therefore, there is a need for minimally invasive biomarkers that can better predict treatment responses in RRMM patients.

Methods : We used IsoSeek, an advanced small RNA‐sequencing method capable of single‐nucleotide resolution, to profile microRNA variants (isomiRs) from extracellular vesicles (EVs) isolated from the plasma of RRMM patients. A total of 96 samples from 49 MM patients (both with active MM and responding to treatment) and 48 samples from healthy donors were analysed. Machine learning was applied to develop classifier signatures for evaluating treatment responses in patients receiving daratumumab‐containing regimens. We further conducted targetome analysis to identify gene networks regulated by the isomiR classifiers.

Results : Our analysis identified a robust classifier signature for on‐treatment response evaluation with a promising area‐under‐the‐curve (AUC) of 0.98 (95% CI: 0.94–1.00). Another classifier, which includes miR‐148‐3p—a microRNA highly selective to plasma cells—was identified as a strong predictor of durable responses (greater than 6 months). This signature was also prognostic for progression‐free survival (PFS) and overall survival (OS), with a hazard ratio of 23.64 for PFS. Targetome analysis identified MYC and BCL2 as key targets of the isomiR networks.

Summary/Conclusion : This study proposes EV‐isomiR sequencing as a minimally‐invasive alternative to bone marrow biopsies, offering a liquid biopsy approach to predict treatment response and outcomes in RRMM patients. Our approach only requires 1 mL of plasma and is scalable to diagnostic platforms, showing promise for clinical implementation and personalized treatment strategies.

Funding : Stichting Cancer Center Amsterdam [CCA2021‐9‐77, CCA2023‐9‐93], Spanish Government [AGL2017‐88702‐C2‐2‐R], NWO Perspectief Cancer‐ID and TKI‐health Holland AQrate.

25 April 14.30–16.00 EV Sources and Basic Biology

Session title OF14 EVs of different sources

They are not alone: EVs and viruses sharing and shaping the extracellular space

Invited Speaker: Janis A. Müller

Philipps University Marburg, Germany

Human milk‐derived extracellular vesicles reduce NF‐kB pathway activation and NLRP3 inflammasome formation in vitro and in vivo

Jueqin Lu, Jasmyne A. Storm, Mon Fran Obtial, Sanoji Wijenayake

Department of Biology, The University of Winnipeg, Canada

Introduction : Human milk contains extracellular vesicles (MEVs), which withstand gastrointestinal digestion and can cross the blood–brain barrier. However, whether MEVs influence neuroinflammation requires exploration. We characterized the molecular underpinnings of how MEVs attenuate the Nuclear Factor Kappa‐B (NF‐κB) pathway activation and NLR family pyrin domain containing 3 (NLRP3) inflammasome formation in vitro and in vivo in response to acute and chronic stress, respectively.

Methods : MEVs were isolated from pooled unpasteurized human donor milk using differential ultracentrifugation combined with serial filtration. MEVs were characterized as per MISEV 2023 via transmission electron microscopy, nanoparticle tracking analysis, and western immunoblotting against CD9, CD63, syntenin‐1, and calnexin. All experimental protocols were approved by the local Human Ethics and Animal Care Committees. Aim 1: Immortalized human microglia (ATCC: HMC3) were polarized using 10 ng/mL of IFN‐γ. The cells were supplemented with 200 µg of MEVs 24 h post IFN‐γ treatment and were harvested at 6, 12, and 24 h. Aim 2: Female Long Evans breeders (Charles Rivers, n = 6/diet) were placed on a control diet (CHD, Research Diets, D12450J) and a high‐fat diet (HFD, Research Diets, D12492) for 4 weeks pre‐gestation, gestation, and lactation. At postnatal day 4, neonates born to these dams were assigned to two treatment groups ( n = 2/sex/litter/treatment). Group 1: pups that received a dose of 1 × 10 12 MEVs/g/BW. Group 2: control pups. Neonates underwent treatment 2×/day for 7 days. The hypothalamus and pre‐frontal cortex were collected for analysis. Transcript and protein abundance of TLR4, IKK, IκBα, NF‐κB, NLRP3, caspase‐1, IL‐1β, and IL‐18 were analysed via RT‐qPCR and westerns.

Results : IFN‐γ polarization of human microglia and maternal HFD exposure in neonates lead to NF‐κB pathway activation and NLRP3 inflammasome formation. MEV administration attenuates these pro‐inflammatory cascades and promotes homeostasis. Groups that received MEVs consistently had lower levels of TLR4, p‐Iκκ α/β (S176/180), p‐IκBα (S32), p‐NF‐κB (S536), cleaved‐caspase‐I, and NLRP3 in vitro and in vivo.

Summary/Conclusion : Human MEVs elicit pro‐survival benefits that mitigate acute and chronic pro‐inflammation. Our study highlights the therapeutic potential of MEVs in reducing neuroinflammation and identifies critical checkpoints in the NF‐κB pathway that is targeted by MEVs at the cell and systems level.

Mesenchymal stromal cells overexpressing hypoxia‐inducible factor 1 alpha and notch 1 intracellular domain release small extracellular vesicles with potential for cardiac repair

Presenter: Marc Buigues

Institute of Research Hospital La Fe, Spain

Abstract unavailable

Dual miRNA enriched MSC‐sEV formulation as a novel regenerative modality to enhance skin and hair regeneration

Sujata Mohanty 1* , Yashvi Sharma 1 , Seema Kashyap 2 , Aastha Singh 3 , Ritu Kulshreshtha 3

1 Stem Cell Facility‐ DBT‐Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi 110029, India; 2 Department of Ocular Pathology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India; 3 Department of Bioscience and Engineering, Kusuma School of Biological Sciences, IIT Delhi, India

Introduction : Non‐healing chronic wounds present a significant socio‐economic burden, with treatment options often limited to supportive care. Regenerative medicine offers promising alternatives, particularly with Mesenchymal Stem Cell (MSC)‐derived small Extracellular Vesicles (sEV), known for their regenerative and immunomodulatory properties. Among the bioactive components in sEV are microRNAs (miRNAs), which can influence cellular functions independently or synergistically. Notably, miR125b‐5p and miR145a‐5p regulate key pathways involved in wound healing and skin restoration. The study aimed to evaluate the regenerative potential of these miRNAs, individually and in synergy, to determine their efficacy in skin regeneration, while also exploring the underlying molecular mechanisms targeted by sEV.

Methods : WJ‐MSCs, isolated with donor consent (IC‐SCR/120/21(R)), were cultured in serum‐free media. miR125b‐5p and miR145a‐5p were introduced into sEV via lipofection, and a miR‐enriched sEV formulation (under patenting) was developed to assess synergistic effects. sEV were isolated using ultracentrifugation and characterized per MISEV, 2023 guidelines. In vitro assays, including proliferation, migration, ROS reduction, and immunomodulation, were conducted on human cell lines. In vivo, a full‐thickness excisional wound model in Wistar rats (226/IAEC‐1/2019) was established. Wound healing was evaluated through closure rates and histopathological analysis. Molecular pathways were examined using dual‐luciferase, qPCR, and immunofluorescence, followed by stability testing across different durations and temperatures, and incorporation into silk and amniotic membrane‐based scaffolds.

Results : The results demonstrated that miR‐enriched sEV were highly effective in vitro, with significantly enhanced functionality when used in synergy. miR125b‐5p was especially efficient in macrophage polarization and ROS reduction, while miR145a‐5p played a key role in regeneration. In vivo, the multi‐miRNA formulation significantly accelerated wound closure, achieving ∼95% closure by Day 14. miR125b‐5p mediated immunomodulation via the IL‐6R/NFkB pathway, while miR145a‐5p supported dermal and hair regeneration through the TGFb2/SMAD4 pathway. These sEV were also found to be stable for up to 1 month at 4°C, and comparable to fresh sEV in efficacy for up to 6 months at −20°C and −80°C. Moreover, their incorporation in a scaffold promoted their sustained release in animal.

Summary/Conclusion : This study highlights the therapeutic potential of miR125b‐5p and miR145a‐5p‐enriched sEV for immune‐related and degenerative diseases, as well as their promising applications in bioengineering and cosmetic therapies.

The exRNA Atlas v3 map of extracellular RNA in human biofluids sheds light on the exRNA ‘dark matter’ beyond previously annotated genomic regions

R. Sreyya Sudharsan 1 , Robert Fullem 1 , Federico Vannuccini 2 , David CY Chen 1 , Andrew Jackson 1 , Jessie Arce 1 , Ryan K. Perez 1 , Abbas Hakim 3 , Peter De Hoff 3 , Srimeenakshi Srinivasan 3 , Martin Ng 4,5 , Louise C. Laurent 3 , Saumya Das 6 , Robert L. Raffai 4,5 , Julie A. Saugstad 9 , Varduhi Petrosyan 1 , Alain Charest 10 , Kendall Van Keuren‐Jensen 11 , Jeffrey L. Franklin 8 , Robert J. Coffey 8 , Ionita Ghiran 7 , Francesca Demichelis 2 , Matt Roth 1 , Aleksandar Milosavljevic 1

1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA; 2 Department of Cellular, Computational, and Integrative Biology (CIBIO), University of Trento, Trento, Italy; 3 Department of Obstetrics, Gynecology, and Reproductive Sciences and Sanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, California, USA; 4 Division of Vascular and Endovascular Surgery, Department of Surgery, University of California San Francisco, San Francisco, California, USA; 5 Department of Veterans Affairs, Surgical Service (112G), San Francisco VA Medical Center, San Francisco, California, USA; 6 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; 7 Department of Anesthesia, Pain and Critical Care Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; 8 Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA; 9 Oregon Health & Science University, Portland, Oregon, USA; 10 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; 11 Bioinnovation and Genome Sciences Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA

Introduction : exRNA Atlas version 3 contains uniformLy processed publicly accessible extracellular small RNA‐seq profiles from > 10,000 samples. These samples cover a number of biofluids and disease conditions, as well as sample preparations for total exRNA, EV‐enriched, or NVEP‐enriched exRNA. Meta‐analyses of previous Atlas versions provided a global map of exRNA expression in human biofluids based on GENCODE and extracellular RNA Binding Protein (exRBP) annotations. Using exRNA sequencing coverage, we identify all genomic loci that show exRNA expression in human biofluids, thereby illuminating the previously inaccessible exRNA ‘dark matter’ and empowering more comprehensive analyses of exRNA.

Methods : We developed an extracellular Genomic Location (exGL) Registry to provide stable identifiers for exRNA loci and their mappings across reference sequences. Loci that met exRNA evidence thresholds were then registered through the exGL registry. These exGLs include those outside annotated regions and short fragments within longer annotated transcripts that are suggestive of exRBP or other carrier‐mediated protection in the extracellular space. Average normalized exRNA abundance was computed for all exGLs, including metadata specific abundances by donor condition, exRNA source, library preparation kits, and RNA isolation kits.

Results : We processed and annotated 3400 plasma exRNA‐seq samples that resulted in the registration of > 10 million distinct exGLs that cover > 5% of the genome. We demonstrate that library preparation and RNA isolation methods influence the ‘visibility’ of many exGLs. Despite these factors, concordance of loci across studies is observed, with a core set of 0.5 million exGLs detectable in at least 10 independent datasets, suggesting opportunities to broaden exRNA analyses to orders of magnitude more loci than currently considered. To empower such analyses, we provide a suite of resources, including exGLs and their annotations.

Summary/Conclusion : The Atlas v3 exGL analyses and annotations, provide the first comprehensive map of the > 5% of the genome that encodes exRNA present in human biofluids and carried by EVs and NVEPs and associated with exRBPs. We extend the Atlas as a resource to facilitate the studies of exRNA biology and case‐control analyses through the prism of exGLs, overcoming the current biases due to non‐exRNA‐specific annotations.

Funding : U54 DA036134 /DA/NIDA NIH HHS/USA.

25 April 14.30–16.00 Technologies and methods

Session title OF15 Comparability and Reproducibility

Building an infrastructure for sustainable reproducibility of EV research

Invited Speaker: Rienk Nieuwland

Amsterdam University Medical Center, The Netherlands

Automated gating for reproducible measurements of extracellular vesicles by flow cytometry

Ishwor Thapa 1 , Yohan Kim 2 , Fabrice Lucien 2 , Hesham Ali 1

1 College of Information Science and Technology, University of Nebraska at Omaha, USA; 2 Department of Urology, Mayo Clinic, USA

Introduction : Nanoscale flow cytometry (nFCM) is an emerging technology used to quantify circulating extracellular vesicles (EVs) directly from various biofluids. Gating refers to the process of selecting fluorescently labelled EVs of interest acquired from the nFCM, and it is often performed manually with expertise. Therefore, manual gating is user‐dependent, time‐consuming and error‐prone. We propose a novel pipeline for processing nFCM files that includes quality control checks and automated gating of EV sub‐populations. This approach significantly enhances the reproducibility of the gating process, making it possible to use gated EVs as a reliable signal for further analysis.

Methods : Cell‐free urine ( n = 20, each with three replicates) was analysed using the standardized and calibrated Apogee A60 Micro‐Plus using the MIFlowCyt‐EV guideline and fluorescent antibodies against Prostate Specific Membrane Antigen (PSMA) and Six‐Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) proteins. Using the R programming language, we developed automated gating strategies to enumerate marker‐positive EVs by analysing the tapering of fluorescence of the background noise. The starting position of the gate is then extended based on the size exclusion criteria of EVs. Additionally, one side of the gate is further split to enable linear scaling of fluorescence with the size. We have developed a web application that applies the automated gating and performs quality checking of event acquisition.

Results : We evaluated the results of automated gating performed on sixty nFCM files by comparing the counts of positive EVs obtained from automated and manual gating performed by two reviewers with different levels of gating expertise. The correlation between manual gating counts was 0.78 ( p < 0.001), while it was higher for the second reviewer and the automated gating (Rho = 0.84 and p < 0.001). The intraclass correlation value between automated and manual gating counts was significant ( p < 0.001).

Summary/Conclusion : The results obtained from the automated gating pipeline demonstrate that it accurately identifies disease specific EVs in an unbiased manner and produces results comparable to those of manual gating by experienced users. Additionally, the method can be extended to reveal unique sub‐populations of EVs that can serve as significant features in biomarker discovery.

Mapping bacterial extracellular vesicle research: insights, best practices and knowledge gaps

Niké Guilbert 1,2 , Amélie Vander Cruyssen 1,2 , Nele De Langhe 1,2,3,4 *, Sofie Van Dorpe 1,2,5 *, Sarah Deville 1,2 , Sándor Dedeyne 1,2 , Quentin Roux 1,2 , Hannelore Denys 2,6 , Linos Vandekerckhove 3,4 , Olivier De Wever 1,2 , An Hendrix 1,2

1 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium; 2 Cancer Research Institute Ghent, Ghent, Belgium; 3 HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, Ghent, Belgium; 4 Department of General Internal Medicine and Infectious Diseases, Ghent University Hospital, Ghent, Belgium; 5 Department of Gynecology, Ghent University Hospital, Ghent, Belgium; 6 Medical Oncology, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent, Belgium. * These authors contributed equally

Introduction : Bacterial extracellular vesicles (BEV) play a pivotal role in interkingdom communication, shaping unique ecosystems that influence both physiological and pathological processes. Given their emerging significance, the study of BEV has gained increasing attention, revealing tremendous potential for clinical applications. However, the diverse nature of BEV—including variations in their biophysical and biochemical characteristics, as well as their complex cargo—poses significant challenges. We performed a systematic analysis to shed light on the global BEV research landscape and map current practices in BEV research, highlight critical knowledge gaps and offer actionable recommendations.

Methods : We analysed all 845 original BEV research articles published from 2015 to 2021, encompassing a total of 3338 experiments. For each experiment, we employed a detailed checklist comprising 233 parameters related to the source, preparation protocols, and characterization methods. The extracted data is publicly accessible through the EV‐TRACK platform (https://evtrack.org/).

Results : Our analysis revealed a concerning inconsistency in nomenclature, with fewer than 20% of studies utilizing the term ‘bacterial extracellular vesicle.’ Meanwhile, the variety of BEV preparation methods used is extensive, with 934 unique protocols reported. Strikingly, only 24.5% of experiments included both particle and protein characterization, and a significant one‐quarter failed to report any form of characterization. Consequently, biophysical and biochemical characteristics of BEV preparations remain largely unknown. As an initiating endeavour, we catalogue commonly explored BEV‐enriched/depleted proteins. Furthermore, the average EV‐METRIC score—a measure for quality and transparency—of BEV studies plateaued from 2018 onwards, contrasting with the exponential increased in EV‐METRIC observed in eukaryotic EV studies since the release of the MISEV2018 guidelines.

Summary/Conclusion : Analysis of over 800 studies reveals that MISEV2018 best practices are underutilized in the emerging field of BEV, reflected by a low average EV‐METRIC score of 14.5%. We integrate specific experimental parameters into the EV‐TRACK knowledgebase, adapting the tool to meet specific BEV research needs. As the field of BEV research continues to expand, prioritizing transparency and reproducibility will be essential to unlock biological insights and to advance BEV‐based applications.

Funding : This work was supported by UGent, FWO, Horizon Europe MSCA grant agreement No. [722148] and ERC grant agreement No. [101045156].

Stripping the adsorbed protein corona may improve immune detection of EVs

András I Försönits 1 , Eszter Á Tóth 1,2,3 , Tünde Bárkai 1 , Tamás Visnovitz 1 , Xabier Osteikoetxea 1,4 , Edit I Buzás 1,4,5

1 Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, Hungary, 2 The Buda Hospital of the Hospitaller Order of Saint John of God, Hungary, 3 Department of Rheumatology and Clinical Immunology, Semmelweis University, Hungary, 4 HCEMM SU Extracellular Vesicles Research Group, Hungary, 5 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Hungary

Introduction : Our group was among the pioneers in demonstrating that a biomolecular corona forms around extracellular vesicles (EVs) in biofluids. However, its impact on the accessibility of EV surface molecules remains unclear.

Methods : This study aimed to evaluate whether the EV corona interferes with the immunodetection of EV surface molecules. Various approaches were applied for removing the EV corona to enhance immunodetection of EVs. We constructed an artificial EV corona using EVs derived from HEK293T‐PalmGFP cells and Cy5‐labelled human plasma proteins. The EVs were exposed to EDTA, Tween‐20, and beta‐mercaptoethanol and subjected to size exclusion chromatography and high ionic strength washes (NaCl, LiCl, and KCl). The effectiveness of corona removal was assessed by measuring the co‐localization rate of plasma proteins with EVs. We evaluated fluorescent annexin‐V binding and annexin‐V‐based affinity capture of EVs, both with and without high ionic strength washing. Additionally, EVs derived from serum‐containing and serum‐free conditioned medium of THP‐1 cells were measured with a MACSPlex kit to assess this effect.

Results : We found clear evidence of EV corona stripping following high ionic strength washes. These washes significantly reduced the co‐localization of plasma proteins with EV membranes ( p < 0.0001, Mann–Whitney U test), indicating substantial corona removal. Notably, annexin‐V binding ( p < 0.01, paired t ‐test) and annexin‐V affinity capture ( p < 0.05, paired t ‐test) both increased for THP‐1 and blood plasma‐derived EVs after high ionic strength washing. The improved detection was specific to EVs derived from serum‐containing media, with no effect observed in serum‐free conditions. Enhanced immunodetection was noted for 10 out of 37 measured EV markers in plasma‐derived EVs following high ionic strength washing.

Summary/Conclusion : Our findings suggest that stripping the protein corona of EVs with high ionic strength solutions can enhance immunodetection of certain EV markers, providing a potential method to improve the accuracy of EV identification. Additionally, corona stripping could enable the re‐decoration of EV surfaces with bioactive molecules, potentially enhancing their therapeutic efficacy.

Funding : This research was funded by NVKP_16‐1‐2016‐0004, Hungarian National Research, Development and Innovation Office (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, Therapeutic Thematic Programme TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003 (National Cardiovascular Laboratory Program) 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 Research and Innovation Programme, grant agreement No. 739593.

EV Barcode: A Web Resource to Predict EV Source from EV cargo

Sriram Gummadi 1 , Suresh Mathivanan 1 , Pamali Fonseka 1

1 Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia

Introduction : Extracellular vesicles (EVs) are membrane‐bound vesicles released by all cell types. They play crucial roles in intercellular and intracellular communication, as well as in regulating physiological and pathological processes. Composed of a lipid bilayer, these vesicles are categorized into small and large EVs based on size and contain diverse cargo, such as proteins, nucleic acids, and metabolites. Their cargo reflects the physiological state of the parent cell and can profoundly influence recipient cells by modulating gene expression and signalling pathways. Research into EVs has expanded significantly, highlighting their potential as diagnostic biomarkers, drug delivery vehicles, and therapeutic tools. However, identifying the source of EVs in complex biological samples like blood, urine, and plasma remains challenging and costly due to their heterogeneous origins.

Methods : To address this challenge, we introduce EV Barcode, an open‐source web resource that employs machine learning techniques to predict the cellular origin of EVs based on their cargo composition. The predictive model was trained on a comprehensive dataset comprising over 5 million single‐cell data from more than 1000 samples representing diverse tissues, sourced from public repositories like the gene expression omnibus and the single cell portal. Model refinement included hyperparameter optimization, feature selection, and ensemble techniques, supported by rigorous cross‐validation to ensure robust performance. The resulting model has been integrated into the EV Barcode web platform, allowing users to input EV cargo data from omics studies and receive predictions regarding the cellular source.

Results : EV Barcode predicts the cellular origin of EVs from omics experiments, estimating the percentage contribution from each cell type and organ. Each prediction includes a p value indicating the statistical significance of the association. Additionally, EV Barcode features real‐time visualization capabilities, enabling users to explore cell‐type clusters and monitor dynamic changes in cluster size and composition across different experimental conditions.

Summary/Conclusion : EV Barcode addresses critical knowledge gaps in the field by providing a powerful tool for predicting the cellular origin of EVs in omics studies. This resource enhances our understanding of EV biology and accelerates the translation of EV research into clinical applications, advancing the potential of EVs in diagnostics and therapeutics.

25 April 14.30–16.00 Physiology and pathology/EV sources and basic biology

Session title OF16 Immunity and Inflammation

EVs and lymphocytes: mapping their interactions in vivo

Invited Speaker: Thomas Brocker

LMU Munich, Germany

Filaggrin insufficiency leads to accumulation of anti‐adhesive proteins associated with keratinocyte‐derived sEVs, which impacts their propensity for uptake by dendritic cells

Presenter: Adrian Kobiela

University of Gdańsk, Poland

Introduction : Atopic dermatitis (AD) is a highly prevalent inflammatory skin disease, where insufficiency in the late epidermal protein filaggrin is frequently observed in the lesional skin of patients, with direct and indirect impact on the skin barrier quality and function. Using filaggrin knockdown keratinocytes generated by shRNA interference (shFLG), we have previously observed that filaggrin insufficiency greatly impacts cellular compartments related to exosomes/small extracellular vesicles (sEVs), but the significance of this is unknown.

Methods : ShC and shFLG keratinocytes were grown in DMEM, and sEVs were isolated from keratinocyte‐conditioned media by ultracentrifugation protocol. We used mass spectrometry to detect proteins in the isolated fractions, the Alpha Fold2 tool for protein binding modelling, and functional uptake assays (with prior PKH67 labelling). Dendritic cell (DC) models were generated by monocyte differentiation in IL‐4/GM‐CSF, and LPS (a maturing factor) was added when needed; IL‐10 was used to generate tolerogenic DCs.

Results : We established that filaggrin insufficiency in keratinocytes results in changes in the content of sEV‐associated proteins, and specifically, increased association of anti‐adhesive proteins (tenascin C; TNC and matrillin‐2; MATL2) with sEVs. At the functional level, such an increased abundance of TNC and MATN2 resulted in a reduction in shFLG‐sEV uptake by all DC subsets studied. We identified that these proteins belong to the fibronectin 1 network and can interact with fibronectin 1 (FN1), likely competing with sEVs adhesion to the cell surface. Accordingly, the rescue experiments with excess recombinant human rhFN1 demonstrated an increase in sEV uptake for shFLG, but not for shC‐derived sEVs.

Summary/Conclusion : An increase in the anti‐adhesive proteins within the sEV corona resulting from filaggrin insufficiency results in changes in the propensity of keratinocyte‐released sEVs to undergo cellular uptake by dendritic cells. This change may affect the ability of keratinocytes to transfer antigens and innate signals and, as such, contribute to less effective pathogen clearance in AD.

Funding : POIR.04.04.00‐00‐21FA/16‐00 project, carried out within the First TEAM program of the Foundation for Polish Science, co‐financed by the European Union under the European Regional Development Fund (awarded to DGO). UK Medical Research Council, NIHR Oxford Biomedical Research Centre and NIHR Clinical Research Network (GSO).

Plasma extracellular vesicles control neutrophil inflammation via CD39/CD73‐mediated ATP cleavage

Presenter: Martina Fabiano

Johns Hopkins University, USA

Introduction : Neutrophils are key players in acute inflammation, exerting defensive functions by producing reactive oxygen species and cytokines, phagocytosing microbes and releasing NETs. However, to avoid tissue damage, neutrophil activity has to be controlled, mainly by their removal from the inflamed area by macrophage‐mediated phagocytosis of apoptotic cells. Herein, we studied whether plasma extracellular vesicles (pEVs) modulate neutrophil activation and their interaction with macrophages.

Methods : pEVs were purified from healthy donor plasma by size‐exclusion chromatography followed by centrifugation. Analysis of EV and co‐isolate markers revealed that our pEV preparations were highly pure and contained canonical EV markers, as revealed by immunoblot and single particle characterization by SP‐IRIS and super‐resolution microscopy. Neutrophils were isolated from healthy donors' blood by a standard density gradient separation method. Neutrophil degranulation (CD11b/CD66b surface expression) and oxidative burst (dihydrorhodamine oxidation) were analysed by flow cytometry (FC) following N‐Formyl‐Met‐Leu‐Phe (fMLP) stimulation. The presence of the enzymes CD39 and CD73 in pEVs isolated from plasma was analysed by immunoblot and super‐resolution microscopy. The rate of ATP hydrolysis was determined by measuring the decrease in extracellular ATP concentration using luminometry. The AMPase activity of pEVs was assessed by exposing pEVs to AMP and measuring AMP concentrations at 30 and 60 min by AMP‐Glo. Macrophage and neutrophil interaction was analysed by confocal microscopy, and cytokines were measured by ELISA.

Results : Results show that pEV treatment inhibited both neutrophil oxidative burst and degranulation. We demonstrate that the oxidative burst regulation is mediated by the biochemical activity of the extracellular nucleotidases CD39 and CD73, which are present on the surface of the pEVs. These enzymes degrade inflammatory extracellular ATP into the anti‐inflammatory molecule adenosine. Finally, we observed that pEV‐coated neutrophils interact more with macrophages, as compared to untreated neutrophils, and imprinted an anti‐inflammatory signature on these, decreasing IL‐6 and promoting IL‐10 secretion.

Summary/Conclusion : In conclusion, pEVs inhibit neutrophil activation by CD39‐CD73 ATP cleavage and enhance neutrophil‐macrophage interaction, thus acting as homeostatic mediators favouring inflammation resolution.

Funding : Argentinean National Agency for Science and Technology Promotion (ANPCYT) under Grants No. PICT 2019 2526.

ESCRT‐0‐mediated fine‐tuning of PD‐L1 secretion via small extracellular vesicles

Xiao‐Le Wang 1,2 , Hou‐Fu Xia 1,2 , Wei Zhang 1,2 , Gang Chen 1,2

1 State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China 2 Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan, 430079, China

Introduction : To elucidate the intricate mechanisms governing the secretion of programmed death‐ligand 1 (PD‐L1) in small extracellular vesicles (sEVs) and to further explore its role in establishing a tumour‐promoting microenvironment and inducing systemic immunosuppression.

Methods : Flow cytometry was employed to analyse the expression levels of sEV PD‐L1 in tumour tissues and peripheral blood samples from patients with oral squamous cell carcinoma (OSCC). A tissue microarray was utilized to detect the expression levels of proteins associated with sEV secretion in OSCC tissues. The secretion of sEV PD‐L1 by tumour cells under conditions of ESCRT‐0 activation and silencing was assessed using flow cytometry and nanoparticle tracking analysis (NTA). Co‐immunoprecipitation (Co‐IP) was conducted to identify molecules implicated in the sorting of sEV PD‐L1. Western blot and immunofluorescence assays were applied to investigate the mechanisms regulating the secretion of sEV PD‐L1 by these molecules. Ultimately, animal models were established to evaluate the therapeutic efficacy of targeting ESCRT‐0 members or tumour‐derived sEV PD‐L1 in a tumour‐bearing mouse model.

Results : STAM2, a component of the ESCRT‐0 complex, plays a pivotal role in the trafficking of PD‐L1 to sEVs. Through GST‐pulldown assays, we demonstrate that STAM2, rather than STAM1 or HRS, directly interacts with PD‐L1, forming a STAM2‐PD‐L1 complex on the plasma membrane. This complex further recruits another ESCRT‐0 protein, HRS, on endosomes, thereby facilitating the routing of PD‐L1 to sEVs. In vitro studies reveal that knockout of STAM2 significantly suppresses the expression of PD‐L1 in OSCC cell‐derived sEVs, and these sEVs markedly inhibit the proliferation of CD8+ T cells, underscoring the central role of STAM2 in regulating sEV PD‐L1 secretion. Moreover, silencing STAM2 enhances the therapeutic efficacy of PD‐L1 blockade in tumour models, as inhibiting STAM2 simultaneously suppresses membrane PD‐L1 recognition and endosomal HRS recruitment.

Summary/Conclusion : ESCRT‐0 members sequentially participate in the secretion of sEV PD‐L1, highlighting their crucial role in modulating this process. This finding offers valuable insights into diseases characterized by dysregulated PD‐L1 secretion, particularly in the context of cancer.

When cellular couriers spark universal reactions: unveiling the consistent immunogenicity of diverse human EVs in primates

Presenter: Olesia Gololobova

Johns Hopkins Medicine, USA

Introduction : Extracellular vesicles (EVs) have the inherent ability to transfer bioactive molecules, which has positioned them as promising candidates for therapeutic applications, including targeted drug delivery, regenerative medicine, and vaccine development. However, the clinical translation of EV‐based therapies necessitates a thorough understanding of their biocompatibility and immunogenicity, especially when EVs are derived from different cell types and administered across species barriers.

Methods : In this study, we evaluated the immunogenicity of EVs derived from three distinct human cell types: Expi293F cells (a derivative of the 293 human embryonic kidney cell line), umbilical cord mesenchymal stem cells (MSCs), and red blood cells (RBCs). EVs were enriched and purified using a combination of differential centrifugation, gel‐exclusion chromatography, and ultrafiltration techniques. EVs were administered intravenously to Macaca nemestrina (pig‐tailed macaque). Particles were characterized using nanoflow cytometry (NFCM), nanoparticle tracking analysis, microfluidic resistive pulse sensing (MRPS), SDS‐PAGE and immunoblotting, SP‐IRIS, transmission electron microscopy (TEM), and single‐molecule localization microscopy. Immune responses were measured by assessing the levels of IgG responses against intact EVs in macaque plasma. Cytokine production in plasma was checked using an MSD assay.

Results : Characterization of EVs confirmed that Expi293F‐ and MSC‐derived EVs exhibited typical morphology, size distribution, and expression of characteristic EV proteins, as assessed by TEM, NFCM, MRPS, and immunoblotting. However, RBC‐derived EVs showed co‐isolation of likely platelet‐derived EVs and displayed a bimodal size distribution, indicating a heterogeneous population. All three EV types elicited measurable immune responses in the macaque models, as evidenced by the production of anti‐IgG antibodies. This uniform immunogenicity across different EV sources indicates that EVs from diverse cellular origins can trigger adaptive immune responses when administered across species barriers. Additionally, ex vivo PBMC biodistribution studies revealed that EVs interacted preferentially with specific blood cell populations, particularly B‐cells (for Expi293F and MSC EVs), but no interaction was found for RBC EVs.

Summary/Conclusion : We demonstrated that EVs derived from Expi293F cells, MSCs, and RBCs can induce immune responses in a non‐human primate model. This underscores the importance of thoroughly evaluating the immunogenicity of EV‐based therapies, especially when considering cross‐species applications and careful consideration in the clinical translation of EV‐based therapeutics.

Saturday, 26th April

26 April Plenary 3 and featured abstract

Exosomes as critical mediators of cell migration

Plenary Presenter: Alissa M. Weaver, MD, PhD

Vanderbilt University School of Medicine, USA

Non‐lytic and active emission of cellular fragments carrying whole genome DNA during cancer cell migration

Presenter: Thupten Tsering

University Health Centre, Montreal, Canada

Introduction : Migrating cancer cells emit a variety of extracellular vesicles (EVs), including small EVs (sEVs), large EVs known as migrasomes and cellular fragments. These EVs may carry bioactive molecules that play roles in intercellular communication and tumour progression. We previously developed EV‐ADD, a database on EV‐associated DNA (Tsering et al. 2019, JEV), and we also demonstrated the presence of EV‐associated DNA at the single EV level using transmission electron microscopy (TEM) (Tsering et al. 2024, Current Protocols). However, the molecular mechanisms underlying the packaging and release of DNA within these EVs remain unclear.

Methods : In this study, we investigated the molecular composition of cellular fragments released during migration in cancer and non‐cancer cell lines. These non‐lytic cellular fragments were isolated using ultracentrifugation and characterized using NanoSight, atomic force microscopy, TEM and western blots. To determine their contents, we performed whole‐genome sequencing (WGS) and proteomics analysis. WGS findings were validated using droplet digital PCR and Sanger sequencing. To further understand the migration‐dependent release of these fragments, we treated them with migration‐inhibitory drugs (cytochalasin B, CK‐636, dynasore).

Results : Scanning electron microscopy and live‐cell time‐lapse imaging revealed distinct cellular fragments released by breast cancer cells, but not by normal epithelial cells. WGS analysis showed that these cellular fragments carry DNA representing the complete genomic sequence of the parent cells and reflecting their mutational status. Proteomics analysis identified several membrane, cytoplasmic, and mitochondrial proteins, including integrin alpha‐2 (ITGA2), integrin alpha‐3 (ITGA3), integrin beta‐1 (ITGB1), basigin (BSG), filamin‐C (FLNC) and malate dehydrogenase 2 (MDH2). Analysis of the top 100 proteins revealed distinct protein profiles in these cellular fragments compared to other EV subtypes (small and large EVs). Notably, migration‐inhibitory drugs significantly reduced the biogenesis of cellular fragments, while the exosome inhibitor GW4869 had no effect. We have termed these cellular fragments ‘Motile Vesicles’ (MoVes).

Summary/Conclusion : This study is the first to demonstrate that non‐apoptotic and active emission of cellular fragments carrying DNA mirror the complete genome of the host cancer cells. Our findings provide novel insights into the molecular cargo of cellular fragments released during cell migration and highlight their potential role in cell‐to‐cell communication and metastasis.

Syntenin controls extracellular vesicle‐induced tumour migration by regulating the expression of adhesion proteins on small extracellular vesicles

Barnabas Irmer 1,2 , Allegra Angenendt 1,2 , Luc Camoin 3 , Stéphane Audebert 3 , Christiane Geyer 1 , Mirjam Gerwing 1 , Hanna Spiessbach 1 , Mira Hebel 1 , Émilie Baudelet 3 , Darius Wlochowitz 4 , Uwe Hansen 1 , Annalen Bleckmann 1,2 , Pascale Zimmermann 3,5 , Kerstin Menck 1,2,3,4

1 University of Muenster, Germany, 2 University Hospital Muenster, Germany, 3 Centre de Recherche en Cancérologie de Marseille, France, 4 University Medical Center Goettingen, Germany, 5 K. U. Leuven, Belgium

Introduction : Despite knowing that cancer‐derived small extracellular vesicles (sEVs) support tumour growth, the specific roles of different sEV subpopulations remain unclear. Syntenin has been identified as one of the main regulators of endosomal‐derived sEVs. Our study investigates how syntenin affects the protein cargo and tumour‐supporting function of sEVs in breast cancer.

Methods : Small EVs were isolated by differential ultracentrifugation from syntenin‐deficient murine 4T1 and human MCF‐7 breast cancer cells and comprehensively characterized based on the MISEV guidelines using NTA, electron microscopy and immunoblots. Syntenin knockout (KO) and corresponding wild‐type (WT) sEVs from 4T1 cells were further characterized by mass spectrometry for their proteomic cargo, and differentially expressed proteins were validated by immunoblotting. The influence of Syntenin silencing on the adhesive capacity of breast cancer sEVs was tested in vivo by biodistribution analysis in syngeneic mice and in vitro by cell and sEV adhesion assays.

Results : We detected 178 down‐regulated proteins on sEVs upon Syntenin KO. Pathway enrichment analysis suggested a strong enrichment of adhesion‐related processes. Accordingly, sEVs from Syntenin‐deficient 4T1 and MCF‐7 cells showed a reduced expression of several focal adhesion and cell‐cell junction proteins. In line, Syntenin silencing reduced the fibronectin‐binding capacity of sEVs from both cell lines, which was mediated by sEV‐associated Integrin alpha‐V/beta‐3 (αVβ3). Compared to sEVs from WT cells, Syntenin KO sEVs showed decreased tropism towards the fibronectin‐rich liver microenvironment in vivo, provided less adhesive support for 4T1 cells and thereby failed to induce cancer cell migration, which appeared to be independent of EV uptake.

Summary/Conclusion : Syntenin regulates the proteomic cargo and concomitantly the adhesive, organotropic and pro‐migratory properties of sEVs in breast cancer.

Funding : Else Kroener‐Fresenius‐Foundation (project 2019_A162), Deutsche Forschungsgemeinschaft (DFG, project ME 4573/1‐1) and the Heidenreich‐von‐Siebold programme of the University Medical Center Goettingen.

26 April 8.30–10.00 Technologies and methods/EV sources and basic biology

Session title OS17 Engineering of EVs

EVs for lung repair and drug delivery

Invited Speaker: Ke Cheng

UNC‐Chapel Hill & NC State University, USA

Protein corona role in the targeting properties of extracellular vesicles for engineering therapeutic biomimetic nanoparticles

María Sancho‐Albero 1,2,3,4 , Alba González 2 , Irene Orera 5 , Víctor Sebastián 1,2,3,4 , and Jesús Santamaría 1,2,3,4

1 Instituto de Investigación Sanitaria de Aragón (IIS Aragón), Spain; 2 Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC‐U. Zaragoza, Spain; 3 Networking Research Center in Biomaterials, Bioengineering and Nanomedicine (CIBERBBN), Spain; 4 Department of Chemical and Environmental Engineering, U. Zaragoza, Spain; 5 SCT Proteómica, Instituto Aragonés de Ciencias de la Salud (IACS), Spain

Introduction : Extracellular vesicles (EVs) have the ability to selectively transfer information between cells, playing a crucial role in intercellular communication processes. This makes them an innovative option for the transport of drugs, molecules, and nanoparticles to target cells, with promising applications. The use of EVs as vehicles for the transport of nanotechnological products has been exploited in the last years for the development of selective therapeutic strategies against cancer.

Methods : This work presents the key factors that influence the final fate of EVs loaded with therapeutic products (catalytic palladium nanosheets, therapeutic platinum nanoparticles and photothermal gold nanoparticles). Among the parameters evaluated, we have assessed how the presence of the therapeutic payload determines EVs stability, aggregation and protein corona formation when they are incubated in biological fluids (using transmission electronic microscopy (TEM), nanoparticle tracking analysis (NTA) and zeta potential). The composition of the protein corona associated with the EVs has been studied by spectroscopic techniques and bioinformatic approaches. Finally, we have determined how these parameters and the biological identity of the protein corona govern their uptake by the cancer cells, and we have compared it with macrophages (using confocal microscopy and spectroscopic techniques).

Results : Our results indicate that noble metal nanostructures can be generated inside EVs using a mild methodology that is based on CO‐mediated reduction of the corresponding ionic precursor directly inside the EVs. This procedure preserves the integrity, morphology, structure and functionality of the exosomal membrane. However, this therapeutic cargo significantly determines the formation of the protein corona associated with the EVs. This protein corona is also different based on the nanomaterial encapsulated (Pd nanosheets, Pt nanoparticles, and Au nanoparticles) and plays a key role in the internalization by target cancer cells and macrophages.

Summary/Conclusion : This study provides data evidencing the key factors that govern the final fate of EVs loaded with therapeutic nanomaterials and how this cargo affects their biological and physicochemical behaviour in biological fluids. These findings will guide future research to create specific EV‐based drug delivery platforms able to be accumulated specifically in cancer target cells, reducing their recognition by the immune system and decreasing the undesired side effects.

Targeting the tumour microenvironment: a click chemistry‐based surface‐functionalization method and a therapeutic‐loading strategy for artificially produced erythrocyte‐derived extracellular vesicles

Presenter: Maria Chiara Ciferri

University of Genova, Italy

Introduction : Because of their ability to transport functional cargoes and the possibility to modify their surface to incorporate ligands, EVs have been recently considered valuable delivery vehicles, especially for targeted therapy. Particularly, erythrocyte‐derived nanoparticles, which are beneficial in yield and bioavailability, have shown promising potential as efficient targeted drug delivery systems.

Methods : in this study, we propose a two‐step strategy for Nanoerythrosome (NanoEs, artificial erythrocyte‐derived vesicles) engineering: (i) surface functionalization by a copper‐free click chemistry approach with a fluorescent peptide able to target an oncofoetal variant of the fibronectin (Extra Domani B (EDB)‐fibronectin), normally absent in healthy adult tissues but upregulated in various pathological conditions, including cancer; (ii) loading with Paclitaxel (PTX) by sonication. Imaging flow cytometry was employed to evaluate the achievement of membrane functionalization. Then the internalization of anti‐EDB‐NanoEs vs naïve NanoEs by EDB‐positive and negative cells was evaluated through an organ‐on‐chip technology in a millifluidic environment.

Results : Click chemistry turned out to be a successful approach for NanoE surface engineering, producing about 60% of Click‐NanoEs, and we observed a higher internalization of functionalized NanoEs compared to the naïve ones by EDB‐expressing cells. The fluorescent anti‐EDB peptide was shown to significantly recognize EDB‐fibronectin both in its free and NanoE‐conjugated form, thus demonstrating that the conjugation by click chemistry doesn't affect the targeting ability. Moreover, the loading efficiency of PTX was analysed by HPLC‐MS, revealing a 1.5% drug encapsulation, and at the same time, the cytotoxic effect of PTX‐NanoEs, in comparison to naïve (empty) NanoEs, was evaluated by MTT assay. PTX‐loaded NanoEs showed a significant reduction of viability, indicating the achievement of the therapeutic effect.

Summary/Conclusion : These data suggest that click chemistry for NanoE surface functionalization, and sonication for PTX encapsulation represent two valid and powerful strategies to produce engineered NanoEs directed toward the tumour microenvironment.

Funding : This work was supported by Worldwide Cancer Research under Grant (Reference 24‐0042).

Modulating the binding affinity of aptamer‐based loading constructs enhances EV‐mediated CRISPR/Cas9 delivery

Charlotte V. Hegeman 1 , Omnia M. Elsharkasy 2 , Tom A.P. Driedonks 2 , Kate R.J. Friesen 3 , Pieter Vader 2 , Olivier G. de Jong 1, 2 .

1 Utrecht University, The Netherlands. 2 University Medical Center Utrecht, The Netherlands. 3 University of Oxford, United Kingdom

Introduction : The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, targeted delivery of the large Cas9‐sgRNA ribonucleoprotein (RNP) complexes remains challenging due to immunogenicity, negative charge, and rapid degradation. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intercellular delivery vehicles. EVs exhibit the natural ability to carry RNA and proteins across biological barriers and can be engineered to load and deliver biotherapeutic molecules and target specific tissues.

Methods : To load Cas9‐sgRNA RNPs into EVs, sgRNAs containing MS2 aptamers and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs) were expressed alongside Cas9 and VSV‐G in HEK293T cells. To study the effect of binding affinity on Cas9‐sgRNA delivery, various mutations affecting binding affinity were made in both the interacting sgRNA MS2‐hairpin and the RNA‐binding domain of the MCPs. To separately study the effects of affinity on cargo loading and release, a UV‐sensitive photocleavable protein (PhoCl) was included in the MS2‐CD63 construct to maximize cargo release. We used a previously published fluorescent Cas9 stoplight reporter system to measure Cas9 RNP delivery (De Jong et al. Nature Communications 2020).

Results : We confirmed that adaptation of the sgRNAs did not adversely affect their functionality by direct transfection. Comparing Cas9‐sgRNA delivery of the modulated sgRNAs revealed that adapting binding affinity highly affects functional delivery (0.5% to 22.2%). A similar effect on functional delivery was seen after modifying the affinity of the RNA‐binding domain of the MCP. After UV‐treatment, photocleavable MS2‐PhoCl‐CD63 fusion proteins revealed similar Cas9 delivery for most affinities, with the exception of the lowest affinity. This indicates that affinity can be substantially lowered before negatively affecting cargo loading and that cargo release was a limiting factor in aptamer‐mediated Cas9 delivery for most of these sgRNAs.

Summary/Conclusion : Here, we describe a novel way to optimize EV‐mediated loading and delivery of Cas9‐sgRNA RNPs. Our results demonstrate that EVs are capable of functional Cas9‐sgRNA complex delivery and that modulation of binding affinity can be used to increase delivery efficiency.

Engineering extracellular matrix‐bound nanovesicles secreted by human brain organoids with different regional identity

Chang Liu 1 , Xingchi Chen 1 , Changchun Zeng 2 , Li Sun 3 , Yan Li 1

1 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering; 2 Department of Industrial and Manufacturing Engineering, FAMU‐FSU College of Engineering; 3 Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, 32306, FL, USA

Introduction : Extracellular vesicles (EVs), including the small‐sized subpopulation exosomes (30–150 nm) are membrane‐bound vesicles that show significance in intercellular communications and high therapeutic potential (e.g., immunomodulation and neuroprotection) in treating neurological disorders. Most studies have been focusing on generating EVs from tissue or monolayer culture. In this study, EVs were isolated from the spent media of 3D brain organoids differentiated from human induced pluripotent stem cells (hiPSCs). In particular, a new type of EV subpopulation, matrix‐bound nanovesicles (MBVs), was isolated from the decellularized extracellular matrix of brain organoids to compare with supernatant EVs (SuEVs).

Methods : The hiPSC‐derived forebrain cortical organoids (iFCo) and the hiPSC‐derived hindbrain cerebellar organoids (iHCo) were differentiated for 15‐35 days. SuEVs and MBVs were harvested on Days 15 and 35. The EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, and western blot. The EV cargo was analysed by proteomics and lipidomics. In vitro functional assay was performed using an oxygen and glucose deprivation (OGD) model based on human brain organoids.

Results : Nanoparticle tracking analysis showed that MBVs were larger than SuEVs in the range of 100–150 nm. The organoids generated 10‐fold more MBVs than SuEVs. SuEVs contained more enriched miRNA cargo than MBVs, and miRNA relative abundance in the organoid EVs changed during maturation. The forebrain and hindbrain organoid SuEVs were highly overlapped in their protein cargo based on proteomics analysis. The comparison between SuEVs and MBVs revealed more membrane proteins, including integrins, in MBVs. Lipidomics data showed that MBVs were enriched in glycerophospholipids and sphingolipids, leading to lipid membrane rigidity and recruitment of integral membrane proteins, which may contribute to MBV deposition in the matrix. In vitro OGD model revealed stronger recovery effects of MBVs than SuEVs at the same dose. The effects were exerted by regulating autophagy, reactive oxygen species scavenging, and anti‐inflammation.

Summary/Conclusion : This study has significance in advancing our understanding of the communication of EVs with the human brain tissue and in the design of cell‐free based therapy for treating neurological disorders such as ischaemic stroke and Alzheimer's disease.

Funding : This project is supported by NSF CAREER 1652992 and NIH R01NS125016 of USA.

26 April 8.30–10.00 Physiology and pathology/EV sources and basic biology

Session title OS18 EV Neurobiology 1

EV‐mediated glia‐neuronee signalling: enhancing axonal energy metabolism and synaptogenesis

Invited Speaker: Zu‐Hang Sheng

National Institute of Neurological Disorders and Stroke (NINDS), NIH, USA

Alcohol‐exposed hepatocyte‐derived exosomes sensitize microglia to a pro‐inflammatory phenotype via miRNA‐122 transfer

Veronika Brezani, Radhika Joshi, Marti Ortega‐Ribera, Mrigya Babuta, Viliam Brezani, Adam Zivny, Gyongyi Szabo

Beth Israel Deaconess Medical Center & Harvard Medical School, USA

Introduction : Alcohol misuse induces inflammation both in the liver and brain. We showed that alcohol increases circulating exosomes containing miR‐122, a hepatocyte‐enriched miRNA, in humans and mice. Hepatic inflammation has been shown to induce encephalopathy and neuroinflammation. Therefore, we hypothesized that exosomes may mediate liver‐brain communication through miR‐122 transfer, particularly in the context of alcohol‐induced neuroinflammation in Alzheimer's disease (AD).

Methods : Exosomes were isolated from mouse serum or supernatants of ethanol‐exposed hepatocytes, size and numbers were characterized by nanoparticle tracking analysis, and exosome markers were determined by western blot. BV‐2 mouse microglia were treated with hepatocyte exosomes before lipopolysaccharide (LPS) stimulation, and cytokine levels were assessed at 24 h. WT and APP/PS1 mice received daily alcohol binges (3.5 g/kg) for 30 days.

Results : Alcohol increased exosome biogenesis and release from mouse hepatocytes. Furthermore, we found that the mature form of miRNA‐122 was enriched in the exosomes derived from ethanol‐treated hepatocytes compared to control exosomes. Co‐culture of PKH26‐stained hepatocyte exosomes with BV‐2 microglia confirmed microglial uptake of hepatocyte exosomes. Furthermore, exosomes from alcohol‐treated hepatocytes sensitized microglia to LPS stimulation to produce significantly increased MCP‐1, CXCL2, CCL3, and TNF‐α compared to control exosomes. Transfection of microglia with an miR‐122 mimic resulted in pro‐inflammatory sensitization and significantly higher protein levels of MCP‐1 and CCL3 at baseline, which were further elevated by LPS treatment. This supports our hypothesis that the transfer of miR‐122 from ethanol‐treated hepatocytes to microglia increases the pro‐inflammatory responses in microglia. In APP/PS1 mice, alcohol administration increased the number of circulating exosomes and miR‐122 levels in the brain compared to WT mice. Alcohol‐treated APP/PS1 mice showed increased levels of ALT, indicating liver damage and increased inflammatory markers. Moreover, we showed worsened amyloid beta pathology in AD brains after alcohol exposure.

Summary/Conclusion : Alcohol increases circulating EVs and miRNA‐122 expression in the brain of APP/PS1 mice. Alcohol‐induced hepatocyte‐derived exosomes sensitize microglia to pro‐inflammatory responses via miR‐122 transfer. Our results suggest an exosome‐mediated crosstalk between hepatocytes and microglia in alcohol use and AD. The impact of the hepatocyte‐microglia crosstalk in AD and alcohol use deserves future investigations.

Funding : This project is supported by NIH grants 5R01AG072899 and 5R01AA020744.

APOE4/4‐induced human microglia secrete extracellular vesicles enriched for proteins associated with immune response and proteasomal pathways

Yang You 1 , Son N. Nguyen 2 , Sean D. Mann Jr 1 , Prakruthi Vadakattu 1 , Takahisa Kanekiyo 1 , Maria Paz. Gonzalez‐Perez 2 , Seiko Ikezu 1 , Scott A. Shaffer 2 , Wayne W. Poon 3 , Tsuneya Ikezu 1*

1 Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA. 2 Mass Spectrometry Facility, University of Massachusetts Chan Medical School, Shrewsbury, MA, USA. 3 Department of Neurobiology and Behaviour, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, California, USA. *Co‐responding author.

Introduction : The APOE4 allele is the strongest genetic risk factor for late‐onset Alzheimer's disease (AD). Recent studies demonstrated APOE as a key driver to induce disease‐associated microglia and may in turn affect extracellular vesicle (EV) biogenesis, leading to neuronal vulnerability and neurodegenerative risk. However, the contribution of microglial APOE4 to AD pathogenesis and EV‐mediated disease progression remains poorly understood. Here we conduct a comprehensive proteomics analysis using APOE isogenic human induced pluripotent stem cells (iPSCs)‐derived microglia (iMG) and microglial EVs (iMDEV) to explore the impact of APOE4 on regulating the microglial response and the protein composition of EVs in AD pathology.

Methods : Three pairs of APOE3/3 and APOE4/4 isogenic iPSC lines were generated by CRISPR/Cas9 and differentiated into iMG. Fibrillar Aβ was employed to induce the disease associated microglia phenotype between the APOE3/3 and APOE4/4 backgrounds. iMDEVs were isolated from homeostatic and Aβ‐induced iMG by size exclusion chromatography and characterized by nano‐flow cytometry. Data‐independent‐acquisition mass spectrometry (DIA‐MS) was performed to analyse the proteomic profiles of APOE isogenic iMG and iMDEVs under homeostatic and Aβ‐treated conditions. The iMG/iPSC‐neuronee and iPSC‐organoid co‐culture model was utilized to investigate the contributions of APOE isogenic microglia and iMDEVs to AD pathogenesis.

Results : APOE isogenic iPSC lines were successfully differentiated into mature microglia. APOE4 altered the microglial phagocytic function compared to APOE3. Additionally, the production of EVs was significantly higher in APOE4 iMG than APOE3 iMG. Using DIA‐MS, we totally identified over 6000 iMG proteins and 600 iMDEV proteins in homeostatic and fibrillar Aβ‐treated iMGs. Proteomics analysis revealed increased levels of MHC class I proteins in APOE4 iMG compared to APOE3 iMG under homeostatic conditions. Upon fibrillar Aβ treatment, APOE3 iMG exhibited elevated expression of interferon‐γ signalling and MHC class II proteins, while APOE4 iMG showed induction of cGAS‐STING and apoptosis‐related pathways. Notably, fibrillar Aβ remarkably increased the expression of proteasomal proteins in APOE4 iMDEVs compared to APOE3 iMDEVs. The microglial co‐culture model further demonstrated that Aβ‐treated APOE4 iMG and iMDEVs resulted in neurotoxicity and impaired neuronal activity.

Summary/Conclusion : APOE4 impairs the microglial response, triggers cGAS‐STING signalling and modifies the protein cargos of iMDEVs in an AD iMG and organoid model.

Brain pericytes: from origins to implications in cell‐cell communication within the neurovascular unit through extracellular vesicles

Julien Saint‐Pol

Univ. Artois, UR 2465, Blood‐Brain Barrier Laboratory (LBHE), F‐62300 Lens, France

Introduction : The BBB is a natural barrier located in the endothelial cells of the cerebral microvessels (BMECs) that restricts exchanges between the bloodstream and the cerebral parenchyma. The brain pericyte, which shares a common basement membrane with BMECs, has been shown to be the major cell type involved in the induction and maintenance of BBB main features. However, nothing is known about the role of BP‐derived EVs in the regulation of the BBB's main features.

Methods : EVs from hBP supernatants have been isolated through ultracentrifugation with or without size exclusion chromatography, and have been characterized by transmission electron microscopy, atomic force microscopy, tunnelling resistive pulse sensing (size and zeta potential), dynamic light scattering, proteomics and lipidomics approaches. The BBB main features after exposure to hBP‐derived EVs have been followed by mass spectrometry approaches, as well as permeability to reference compounds, transendothelial electric resistance (TEER), immunofluorescence, Western blot and functional assays.

Results : The protein pattern of hBP is modified once cocultured with BMECs and highlights a metabolic switch induced by BMECs on hBP. A stimulation of EV biogenesis is observed, indicating a potential need for hBP‐derived cell‐cell communication through EVs. The mechanisms of interaction between BMECs and hBP‐derived EVs are in favour of clathrin‐coated pit‐mediated endocytosis. Under inflammation, hBP‐derived EVs exhibit a modified protein pattern compared to untreated EVs and induce a dysregulation of trans‐endothelial electric resistance (TEER) on BMECs. Moreover, hBP‐derived small EVs regulate the BBB's main features through (i) a reduction of paracellular permeability to sodium, (ii) the promotion of the TEER and (iii) the spatial organization of tight junction proteins in endothelial cells in a dose‐dependent manner.

Summary/Conclusion : Despite a former reputation of contaminant for BBB modelling in vitro, hBP remains of importance for maintaining the BBB phenotype, and BMECs also modulate the hBP functions and developmental fates. This work also highlights the intriguing role of hBP‐derived EVs in regulating BBB features.

Funding : This project is funded by the French Research National Agency (ANR‐21‐CE14‐0002).

APOE4 drives neuroinflammation and lipid dysbiosis in Alzheimer's disease by modulating lipid compositions and cell adhesion molecules in brain‐derived extracellular vesicles

Presenter: Zhengrong Zhang

Mayo Clinic, USA

Introduction : Extracellular vesicles (EVs) are key mediators in transferring pathological proteins associated with Alzheimer's disease (AD). The apolipoprotein E (APOE) gene, particularly the ε4 allele, is a major genetic risk factor for late‐onset AD. However, the influence of APOE genotype on the biological characteristics and cargo composition of brain‐derived extracellular vesicles (BDEVs) in AD remains poorly understood.

Methods : In this study, BDEVs were isolated from human AD temporal cortex with APOE3/3 and APOE4/4 genotypes ( N = 3–20 per group). Nanoflow cytometry and super‐resolution microscopy were used to evaluate the tau load within these BDEVs. EV‐mediated tau transmission was assessed in aged human Tau KI and APPNL‐G‐F: Tau KI mice in vivo. BDEV uptake and tau seeding were evaluated in iPSC‐derived neuronees through live imaging. Additionally, shotgun lipidomics and data independent acquisition proteomics were analysed for the lipidome and proteome of BDEVs, respectively. Key molecules linked to APOE genotypes were identified through integrated weighted gene co‐expression network and trait correlation analysis.

Results : APOE4/4 BDEVs exhibited a higher proportion of phosphorylated tau (pS396) at single EV levels. Upon injection of 300 pg tau of BDEVs into aged Tau KI and APPNL‐G‐F: Tau KI mice, APOE4/4 BDEVs significantly increased AT8+ phosphorylated tau levels and induced neuroinflammation in vivo. Moreover, APOE4/4 BDEVs demonstrated stronger neuronal uptake and tau transfer compared to APOE3/3 BDEVs, impairing the neuronal activity of iPSC‐derived neuronees. Lipidomics revealed that APOE4/4 BDEVs were significantly enriched in pro‐inflammatory long‐chain polyunsaturated fatty acids (PUFA) compared to APOE3/3 with Braak stage association. Furthermore, gene ontology pathway analysis using differentially expressed proteins in APOE4/4 BDEVs showed increased activity in oxidative phosphorylation and sterol‐binding. Multi‐omics integrated data revealed that upregulation of PUFA was significantly associated with elevated cell adhesion molecules and ATPase transporter activity, with neural cell adhesion molecule as one of the key molecules. Notably, neutralizing antibodies against the molecule effectively reduced APOE4/4 BDEV internalization and mitigated EV‐mediated tau pathology in recipient cells.

Summary/Conclusion : Our comprehensive analysis suggested that AD APOE4/4 BDEVs may enhance transmissibility of tau pathology and neuroinflammation via enriched pro‐inflammatory PUFA and cell adhesion molecules. Targeting these pathways presents a promising therapeutic strategy to mitigate disease progression, particularly in APOE4 individuals.

26 April 8.30–10.00 Physiology and pathology/EV sources and basic biology

Session title OS19 Microbial EVs

Breaking barriers: microbial EVs at the crossroad of inflammation

Invited Speaker: Claudia Günther

UKER, Medizinische Klinik 1, Germany

Role of extracellular vesicles in placenta‐brain dialogue during congenital human cytomegalovirus infection

Charlène Martin 1 , Mathilde Bergamelli 1 , Hélène Martin 1 , Alexandre Favereaux 2 , Cécile Malnou 1

1 Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity) INSERM UMR1291 – CNRS UMR5051 ‐ Université Toulouse III, CHU Purpan—BP 3028, 31024 Toulouse CEDEX 3; 2 Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

Introduction : Human cytomegalovirus (HCMV) is the most common virus transmitted in utero and the leading cause of brain malformations and deafness of infectious origin, with no reliable prognostic methods available to date. The placenta, one of the targets of HCMV replication, is a key organ during congenital infection, playing a major role in maternal‐foetal exchanges, particularly through the secretion of extracellular vesicles (EVs) into both maternal and foetal compartments. Placental EVs have multiple functions during pregnancy, and their secretion and composition are disrupted in many pregnancy‐related pathologies. However, until now very few studies have examined the role of placental EVs during viral congenital infections. Our team previously demonstrated that EVs from HCMV‐infected cytotrophoblasts increase foetal neural progenitor cell susceptibility to infection. We now aim to investigate the direct impact of placental EVs on brain development, by studying the contribution of placental EVs to human neural stem cell phenotype.

Methods : Placental EVs were isolated from the culture supernatant of primary cytotrophoblasts, previously infected or not by HCMV, using differential ultracentrifugations followed by an iodixanol/sucrose gradient. miRNA sequencing analysis was performed to characterize EV content. Functional assays were performed by incubating a model of human neural stem cells with various amounts of EVs for different durations. Growth and viability were evaluated by trypan blue exclusion, proliferation by Ki67 immunofluorescence, migration via wound healing assay and differentiation with different approaches such as immunofluorescence, qRT‐PCR and proteomic analysis.

Results : While NSC growth, viability, and proliferation remained unaffected upon EV incubation, significant differences were observed in the migration and neuronal differentiation capacities of NSCs treated with EVs from HCMV‐infected cytotrophoblasts compared to uninfected ones. Moreover, the miRNA composition of placental EVs revealed an enrichment of miRNAs linked to central nervous system development upon infection.

Summary/Conclusion : Our results support the hypothesis that placental EVs directly influence foetal brain development, a process altered upon HCMV infection. The virus may perturb foetal brain development by modulating placental EV miRNA composition, which, in turn, would affect foetal neural progenitor cell phenotype through paracrine signalling. These insights provide a new perspective on how HCMV can impact foetal brain development during congenital infection.

Probiotic extracellular vesicles: exploring their utility as a preventative and potential treatment approach for colorectal cancer‐associated cachexia

Presenter: Kyle P. Bramich

La Trobe University, Australia

Introduction : Probiotic species of bacteria are typically introduced into the body through various food sources and can confer a beneficial effect on the host. Over the past decade, extensive research has revealed the therapeutic potential of probiotic extracellular vesicles (EVs) in several disease contexts. Previously, EVs derived from various common probiotic bacterial strains have been shown to promote apoptosis of colorectal cancer cells. However, to the best of our knowledge, no one has investigated the functionality of probiotic EVs in the context of cancer cachexia. In this study, we attempted to investigate the ability of probiotic EVs to attenuate cancer‐associated cachexia.

Methods : Isolation of L. delbrueckii EVs (LEVs) and S. thermophilus EVs (SEVs) was achieved using tangential flow filtration and differential centrifugation coupled with ultracentrifugation. Characterisation of isolated EVs was further confirmed by microscopy, and proteomic analysis of EV samples was performed using mass spectrometry (LC‐MS/MS). Major in vitro experiments were performed with C26 and HCT116 cell lines. In vivo studies employed the use of the genetically engineered mice model ApcMin/+.

Results : The uptake of LEVs and SEVs by C26 and HCT116 was confirmed using microscopy and fluorescence assays. The metabolic activity and clonogenicity were also reduced in cells following incubation with probiotic EVs. Interestingly, oral gavage of LEVs and SEVs to ApcMin/+ mice prevented body weight loss and decreased the formation of intestinal polyps. Moreover, skeletal muscle fibre cross‐sectional area was greater in the treatment groups, and there was a reduction in the abundance of muscle atrophy markers. Proteomic analysis also revealed cargo from probiotic EVs may play roles in a range of metabolic processes including peptide and cellular amide metabolism, tRNA metabolic processes, and carboxylic acid metabolism.

Summary/Conclusion : These findings indicate that probiotic EVs can be taken up by cancer cells and elicit a functional response. Proteomic analysis revealed the multiplicity of L. delbrueckii EV protein cargo and the associated diverse roles in various important biological processes. We have also demonstrated that ingestion of LEVs and SEVs minimised the development of intestinal adenomas and alleviated the cachexic phenotype in mice.

Bacterial extracellular vesicles as protective decoys: a novel mechanism of resistance

Marie Burt 1 , Georgia Angelidou 2 , Christopher Nils Mais 3 , Christian Preußer 4 , Timo Glatter 2 , Thomas Heimerl 3 , Gowtham Boosarpu 1 , Elke Pogge 4 , Janis A Müller 5 , Gert Bange 3 , Mareike Lehmann 1 , Nicole Paczia 2 , Bernd Schmeck 1,3,6,7 , Anna Lena Jung 1,7

1 Institute for Lung Research, Philipps‐University Marburg; 2 Max Planck Institute for terrestrial Microbiology; 3 Center for Synthetic Microbiology (SYNMIKRO), Philipps‐University Marburg; 4 Institute for Tumor Immunology, Philipps‐University Marburg; 5 Institute of Virology, Philipps‐University Marburg; 6 Department of Medicine, Pulmonary and Critical Care Medicine, Marburg; 7 Core Facility Flow Cytometry—Bacterial Vesicles, Philipps‐University Marburg

Introduction : The continuous emergence of multidrug‐resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae (Kp) being a prominent threat. We conducted a comprehensive study on K. pneumoniae ’s antibiotic resistance mechanisms, focusing on bacterial extracellular vesicles (bEVs) and polymyxin B (PB), a last‐resort antibiotic. Additionally, we examined polymyxin B nonapeptide (PB‐NP), a derivative of PB that retains binding ability but lacks antimicrobial activity.

Methods : Our research demonstrates that bEVs protect bacteria from polymyxins. SEC‐purified bEVs derived from PB‐stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation compared to bEVs from unstressed or PB‐NP‐treated Klebsiella. Notably, these bEVs also shielded bacteria from other bacterial families. This protective effect was validated ex vivo and in vivo using precision‐cut lung slices and Galleria mellonella models. In all models, bEVs protected K. pneumoniae from PB and reduced the associated stress response at the protein level, as determined by proteomics.

Results : Significant changes in the lipid composition of bEVs upon PB treatment were observed through an untargeted lipidomics approach, affecting their binding capacity to PB as determined by triple quadrupole mass spectrometry. The altered binding capacity of bEVs from PB‐stressed K. pneumoniae was linked to a reduction in lipid A content within the released vesicles, but not PB‐NP‐induced vesicles. Despite the reduction in lipid A per vesicle, the overall increase in the number of vesicles resulted in enhanced protection, as the total number of PB binding sites increased. This discovery unravels the mechanism behind the altered PB protective efficacy of bEVs from PB‐stressed K. pneumoniae compared to control bEVs. The lipid A‐dependent protective effect against PB was further confirmed using artificial vesicles, which also bound PB and successfully protected Klebsiella from PB ex vivo and in vivo.

Summary/Conclusion : Our findings indicate that stress‐induced bEVs act as protective shields for bacteria by binding to polymyxins via their lipid A, effectively serving as decoys and preventing antibiotic interaction with the bacterial cell surface. These insights into the mechanisms underlying antibiotic cross‐protection offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug‐resistant bacterial infections.

From bacteria with love: extracellular vesicles as intranasal postbiotics in allergy prevention and treatment

Agnieszka Razim 1,2 *, Astrid Laimer‐Digruber 1,3 , Anna M. Schmid 1 , Tanja V. Edelbacher 3 , Magdalena E. Paschal 1 , Tamara Weinmayer 1 , Michael Thaler 1 , Magdalena E. Skalska 4 , Paweł Migdał 2 , Mattia Morandi 5 , Catherine Daniel 6 , Dagmar Srutkova 7 , Martin Schwarzer 7 , Jiri Hrdy 8 , Monika Ehling‐Schulz 3 , Sabina Górska 2 , Aleksandra Inic‐Kanada 1 , Ursula Wiedermann 1 , Irma Schabussova 1

1 Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria; 2 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Poland; 3 Institute of Microbiology, Centre of Pathobiology, Department of Biological Sciences and Pathobiology, University of Veterinary Medicine, Vienna, Austria; 4 Marian Smoluchowski Institute of Physics, Medical Physics Department, Jagiellonian University, Poland; 5 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Czech Republic; 6 University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, Center for Infection and Immunity of Lille, France; 7 Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Czech Republic; 8 Institute of Clinical Immunology and Allergology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic

Introduction : Escherichia coli O83 (EcO83) is a probiotic strain used clinically to prevent nosocomial infections and diarrhoea in neonates and to reduce allergic sensitization when administered orally. However, concerns about systemic infection and gene transfer limit the use of live probiotics. This study investigates extracellular vesicles (EVs) derived from EcO83 (EcO83‐EVs) as a safer alternative and focuses on their characterisation and therapeutic potential in reducing allergic airway inflammation.

Methods : EcO83‐EVs were isolated by ultracentrifugation, and their number, morphology, zeta potential, and cargo (content of proteins, DNA, RNA, lipids, and LPS) were analysed. The immunomodulatory potential was analysed in human nasal epithelial cells, mouse splenocytes and bone marrow‐derived dendritic cells (BMDCs). The anti‐allergic effect of intranasally administered EcO83‐EVs was analysed in a mouse model with ovalbumin‐induced allergic airway inflammation in vivo.

Results : EcO83‐EVs are spherical nanoparticles (∼110 nm) containing LPS and 1120 proteins, with 136 enriched in the EVs compared to the parent bacteria. These vesicles increased the expression of proteins related to oxidative stress and inflammation in human nasal epithelial cells and activated the TLR2/4/5 and NOD1/2 receptors. In vivo, EcO83‐EVs interacted with nasal‐associated lymphoid tissue, were internalised by airway macrophages and stimulated neutrophil recruitment. They activated the NF‐κB signalling pathway, resulting in the production of nitric oxide. Intranasal administration of EcO83‐EVs reduced airway hyperreactivity, eosinophilia, Th2 cytokine production and mucus secretion in mice.

Summary/Conclusion : EcO83‐EVs show significant potential as a postbiotic alternative to live bacteria and represent a safer option for the prevention of allergic airway inflammation. These results suggest that EcO83‐EVs could be used in mucosal immunisation and targeted immunotherapy strategies. Further research is needed to explore their clinical applications in allergy prophylaxis and treatment.

Funding : The Government of Lower Austria project Danube‐Allergy Research Cluster (P17); the FWF project P 34867; the MSCA‐PF project LactoVES (No. 101066450); OEAD: CZ 04/2024, CZ 07/2023, CZ 15/2023, RS 08/2022.

26 April 8.30–10.00 Disease and Therapy

Session title OS20 EVs in Preclinical Models

Clinical application of EV‐based therapeutics

Invited Speaker: Mario Gimona

Paracelsus Medical University Salzburg and Ludwig‐Boltzmann Institute for Nanovesicular Precision Medicine, Austria

Therapeutic poxvirus triggers the secretion of extracellular vesicles with immunomodulatory, anti‐tumoural activity

Lucas Walther 1,2 , Kuang‐Jing Huang 1 , Vincent Mittelheisser 1 , Marie‐Christine Claudepierre 2 , Caroline Tosch 2 , Magali Rompais 3 , Jules Deforges 2 , Alexandre Durand 4 , Corinne Crucifix 4 , Christine Carapito 3 , Eric Quémeneur 2 , Jacky G. Goetz 1 , Karola Rittner 2# and Vincent Hyenne 1#

1 INSERM UMR_S1109, Tumor Biomechanics Lab, Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Equipe Labellisée Ligue contre le Cancer, Strasbourg, France. 2 Transgene SA, 400 Boulevard Gonthier d'Andernach, Parc d'innovation, 67400, Illkirch‐Graffenstaden, France. 3 Labouratoire de Spectrométrie de Masse Bio‐Organique, Institut Pluridisciplinaire Hubert Curien, UMR 7178, CNRS‐Université de Strasbourg, Strasbourg, France. 4 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Integrative Structural Biology, 1 Rue Laurent Fries, 67404, Illkirch Cedex, France. # Co‐authors

Introduction : Virus‐based therapies show promise in cancer as immunotherapeutic vectors. We hypothesize that immune cells infected with therapeutic poxvirus release modified extracellular vesicles (EVs) with anti‐tumour potential. To test this, we studied how Modified Vaccinia Ankara (MVA) affects EV secretion from human PBMC and whether EVs secreted by the armed MVA‐infected mouse dendritic cell line (DC2.4) elicit a T‐cell mediated anti‐tumour response in vivo.

Methods : Comparing different methods to isolate EVs secreted by MVA‐infected cells, we found that only 0.1 µm filtration, combined with ultracentrifugation or size exclusion chromatography, reliably separates small EVs from viruses despite their structural similarities. The characteristics of EVs were analysed by NTA, cryo‐EM, mass spectrometry, immunoassays and flow cytometry, and their therapeutic potential was assessed in vitro and in vivo in a mouse tumour model.

Results : After infection of PBMC with MVA, we first observed an increased EV secretion and detected changes in endogenous proteins as well as the presence of viral proteins within these EVs, suggesting that they could contribute to therapeutic effects. To test this hypothesis, we isolated EVs from DC2.4 infected with an MVA vaccine vector (MVA‐OVA) encoding the ovalbumin epitope SIINFEKL as a model antigen, together with mIL‐12 and mCD40L immunostimulatory payloads. We showed these EVs express tetraspanins and MVA‐encoded payloads, including SIINFEKL presented by major histocompatibility complex class I on their surface. Importantly, those EVs, unlike EVs coming from DC2.4 infected with an empty MVA, reduced tumour growth similarly to the vaccine vector alone in the E.G7‐OVA mouse lymphoma model. Finally, we detected OVA‐specific CD8+ T cells in the blood of EV‐treated mice and demonstrated in vitro the ability of such armed EVs to directly activate and stimulate these OVA‐specific CD8+ T cells.

Summary/Conclusion : Altogether, we showed that murine dendritic cells infected with the MVA‐OVA vector secrete immunostimulatory EVs able to trigger a CD8+ T cell response and efficiently reduce tumour growth in an E.G7‐OVA model. Our results suggest that EVs contribute significantly to the anti‐tumoural response induced by therapeutic viral vectors and pave the way for promising therapeutics.

Funding : This PhD project is co‐funded by Transgene S.A.

Regulation of parasite virulence by extracellular vesicles during malaria infections

Presenter: Pierre‐Yves Mantel

CK‐CARE AG—Center for Allergy Research and Education, Davos, Switzerland

Introduction : People infected with plasmodium falciparum, the parasite responsible for malaria, are more susceptible to bacterial infections. In fact, bacterial infections are a common and often fatal complication of malaria infection. In particular, the neutrophil bactericidal activity is impaired, leading to deficient resistance mechanisms. The factors causing neutrophil dysfunction remain unknown. Extracellular vesicles (EVs) are small membrane vesicles released by Plasmodium falciparum infected red blood cells (iRBCs). These vesicles contain a subset of proteins, and nucleic acids that are derived from the RBC as well as the parasite. EVs are involved in numerous physiological processes. We have shown that iRBCs release vesicles, which not only regulate parasite development but also mediate the host‐pathogen interactions. EVs contain RBC‐derived miRNAs that regulate gene expression in the recipient cells. The most abundant miRNA is miR451a. Here, we investigated the contribution of EVs in regulating neutrophil function during malaria in vivo.

Methods : We have generated a mouse model in which miR‐451a is specifically knocked out in RBCs. The mice are then infected using P. yoelii to mimic anaemia and later on, with Salmonella typhimurium to mimic co‐infections. The immune response in the blood, spleen, and bone marrow of the infected mice was characterized by using high‐dimensional flow cytometry. We next performed histology to characterize the immune pathology. Finally, we characterized the circulating EVs by proteomics and transcriptomics.

Results : We showed that the parasites develop normally and grow in all the different stages in the miR451a − / − RBCs. However, the parasites were rapidly cleared in the anaemia model, whereas in the WT the parasites kept growing and the mice showed signs of anaemia. In addition, a group of immature neutrophils characterize by the expression of Gr1 + CD11b + Sca1 + expends during malaria. Furthermore, we found that EVs were promoting ferroptosis in neutrophils and their dysfunction.

Summary/Conclusion : Here, we have demonstrated that miR451a is a key regulator of malaria disease severity and contributes to neutrophil dysfunction in vivo.

Funding : Swiss National Science Foundation, Novartis Foundation for medical‐biological research. Gottfried & Julia Bangerter‐Rhyner‐Stiftung.

Modulation of cancer EV secretion for improved immunotherapy outcomes in pancreatic cancer

Presenter: Carolina Dias

Universidade do Porto, Portugal

Introduction : Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5‐year survival rate of 10% due to inefficient treatment of patients diagnosed with inoperable tumours. It is urgent to develop new therapeutic strategies to improve patient outcomes. Here, we investigate how we can leverage intercellular communication by cancer extracellular vesicles (cEVs) to modulate the immune response in pancreatic tumours.

Methods : We used genetically engineered mouse models that spontaneously develop PDAC, enabling Rab27a knockout (KO) specifically in cancer cells. We validated our findings in human PDAC samples.

Results : We found that Rab27a KO leads to an earlier tumour onset and reduced survival, a phenotype that depends on a proficient immune system, as demonstrated by the recovery of the phenotype in an immune‐deficient background (Rag2 KO). Importantly, major alterations in Rab27a KO tumours are associated with immune response and inflammation. Specifically, we found that loss of thrombospondin 1‐positive cEVs (TSP1+ cEVs) disrupts the immune microenvironment, promoting angiogenesis and recruiting PSGL1‐rich immune cells. This cascade triggers the secretion of the pro‐inflammatory molecule MRP8 by pro‐inflammatory myeloid cells, which subsequently activate cancer‐associated fibroblasts to secrete IL‐6, inducing CD4+ T cell differentiation into a Th17 pro‐tumourigenic phenotype. By targeting inflammation with dexamethasone, depleting CD4+T cells, or IL‐17A, we significantly impaired tumour growth and improved survival, specifically in Rab27a KO tumours. Remarkably, injection of TSP1+ cEVs restored the phenotype of Rab27a KO tumours. Additionally, we showed that 25% of PDAC patients lack Rab27a expression, exhibiting high infiltration of MRP8+ cells and leading to a worse prognosis, compared to those with high Rab27a expression.

Summary/Conclusion : Our study highlights the critical role of Rab27a in modulating the immune microenvironment in PDAC. Rab27a loss disrupts immune balance, enhancing pro‐inflammatory and pro‐tumourigenic pathways. Importantly, targeting inflammation — whether through anti‐inflammatory drugs, immune cell modulation, or reintroduction of TSP1+ cEVs—shows potential to mitigate tumour progression and improve survival in PDAC. These insights offer a promising foundation for developing immunomodulatory therapies that may improve outcomes for PDAC patients, particularly those lacking Rab27a expression.

Funding : Portuguese Foundation for Science and Technology (2022.02284.PTDC), Programa Operacional Regional do Norte (NORTE‐01‐0145‐FEDER‐072678 ‐ Consórcio P.CCC) and Sociedade Portuguesa de Gastrenterologia (ID: 01/2021).

Development of in vivo genome editing technology using plant‐derived extracellular vesicles as drug delivery carriers

Tomohiro Umezu, Yui Nagamatsu, Taehun Hong, Takahide Niijima, Shin‐ichiro Ohno, Masahiko Kuroda

Tokyo Medical University, Japan

Introduction : We developed a drug delivery system (DDS) utilizing acerola‐derived extracellular vesicle‐like nanoparticles (AELNs) as carriers. Previously, we demonstrated that complexing AELNs with nucleic acid drugs, such as siRNA, enables effective oral delivery. In this study, we investigated whether complexing AELNs with CRISPR‐Cas9 ribonucleoprotein (RNP) complexes, administered orally or intranasally, could achieve targeted genome editing, particularly within the central nervous system (CNS).

Methods : AELNs were isolated from acerola juice by filtration and ultracentrifugation. For AELN/RNP complex formation, AELNs were incubated with Cas9 and guide RNA (gRNA) on ice for 30 min. This complex was then added to cultured cells, and genome editing efficiency was assessed 24 h post‐treatment. To specifically target the pathological GGGGCC repeat expansion in the C9orf72 gene, associated with amyotrophic lateral sclerosis (ALS), two gRNAs were designed to excise the repeat. Furthermore, to enhance targeting specificity, we incorporated the GLP2 peptide into the AELN/RNP complex, evaluating editing efficiency in HEK293 cells overexpressing the GLP2 receptor.

Results : The GLP2‐modified AELN/RNP complex significantly increased genome editing efficiency in GLP2 receptor‐expressing HEK293 cells compared to wild‐type HEK293 cells. Since GLP2 receptors are known to be expressed in the brain, these findings suggest that GLP2 could facilitate targeted delivery of AELN/RNP complexes to neural tissues. Using a mouse model, we further evaluated in vivo genome editing in the CNS by intranasally administering AELN/RNP complexes targeting the C9orf72 repeat. We successfully excised the pathological repeats in brain tissue, with markedly improved editing efficiency observed in mice treated with GLP2‐modified complexes. We are currently working toward achieving CNS‐targeted genome editing through oral administration.

Summary/Conclusion : This study demonstrates that AELNs hold significant potential as carriers for intracellular delivery of CRISPR/Cas9, enabling targeted genome editing of CNS disease‐related genes via non‐invasive routes. The success of CNS‐specific editing opens new therapeutic avenues for genetic diseases, particularly neurodegenerative disorders, and highlights the applicability of this technology for non‐invasive therapeutic interventions.

26 April 14.30–16.00 Disease and therapy

Session title OS21 EVs for Cancer Immunotherapy

EVs in immune suppression and tumour progression

Invited Speaker: Wei Guo

University of Pennsylvania, USA

A CD21 and phosphatidylserine‐targeting fusion protein binds extracellular vesicles, targets them to B cells and potentiates their T‐cell immunogenicity

Annemarijn Offens 1,2,3 , Loes Teeuwen 1,2,3 , Gozde Gucluler Akpinar 1,2,3 , Loïc Steiner 1,2,3 , Sander Kooijmans 4,5 , Doste Mamand 1,2 , Hannah Weissinger 1,2 , Alexander Käll 1,2 , Maria Eldh 1,2,3 , Oscar P. B. Wiklander 1,2 , Samir El‐Andaloussi 1,2 , Mikael C. I. Karlsson 1 , Pieter Vader 4 , Susanne Gabrielsson 1,2,3

1 Karolinska Institutet, Sweden, 2 Karolinska University Hospital, Sweden, 3 Center for Molecular Medicine (CMM), Sweden, 4 University Medical Center Utrecht, The Netherlands, 5 Regenerative Medicine Center, The Netherlands

Introduction : Extracellular vesicles (EVs) are known to modulate immune responses via their cargo molecules, such as proteins, lipids and nucleic acids. EVs from Epstein–Barr virus infected cells were shown to carry GP350 on their surface and target human CD21 (huCD21) present on B cells. Moreover, antigen (Ag)‐loaded EVs from bone‐marrow derived dendritic cells (BMDC) induce an antigen‐specific T cell response that depends on B cells. Here, we investigated whether BMDC EVs could be targeted to B cells by decoration with a soluble protein containing phosphatidylserine‐ and huCD21 binding domains, and if this could amplify the Ag‐specific CD8 T cell response.

Methods : Native EVs were produced from mouse BMDCs by ultracentrifugation, and mNeonGreen‐ or Nanoluc‐expressing EVs from Freestyle HEK293 cells by tangential flow filtration. EVs were decorated by incubation with the CD21‐binding protein or a mutated control. To investigate B cell targeting, fluorescently labelled BMDC EVs were incubated with splenocytes from mice expressing huCD21 and human PBMC or injected iv into huCD21 mice. Cells positive for EVs were detected by flow cytometry. Freestyle HEK293 EVs were detected in vitro by mNeonGreen expression and in vivo by luciferase activity. To measure immune responses, huCD21 mice were injected i.v. with ovalbumin‐loaded BMDC EVs twice, followed by analysis of ovalbumin‐specific T cells in the spleen. All experiments were approved by the appropriate committees.

Results : EVs decorated with CD21‐binding protein increased in vitro B cell targeting over 5‐fold more compared to undecorated or mutated protein decorated EVs. When decorated EVs were injected in vivo, fusion protein did not alter their biodistribution across organs while targeting them specifically to B cells in the spleen, blood and LNs. When mice were immunized with CD21‐targeted Ag‐loaded EVs, Ag‐specific CD8 T cells increased compared to the mice immunized with untargeted EVs.

Summary/Conclusion : EVs decorated with a CD21‐binding fusion protein target B cells and improve antigen‐specific T cell responses and support the application of phosphatidylserine‐binding fusion proteins for cell‐specific targeting of native EVs.

Funding : The study was funded by the Swedish Medical Research Council, the Swedish Cancer Society, the Cancer Research Foundations of Radiumhemmet, and the Karolinska Institutet's KID grant.

Targeted exosomal delivery of PD‐L1 siRNA in lung tumour immunotherapy

Farrukh Aqil 1 , Raghuram Kandimalla 1 , Omar Sadi Sarkar 1 , Disha Moholkar 1 , Margaret Wallen 2 , Chuanlin Ding 1 , Kavitha Yaddanapudi 1 , Ramesh Gupta 1,2

1 University of Louisville, Louisville, KY, USA. 2 3P Biotechnologies Inc., Louisville, KY, USA

Introduction : The host's innate and adaptive immune systems interact closely in cancer immunosurveillance, with PD‐L1 in the tumour microenvironment being a key immunotherapy target. Here, we developed an exosome‐polyethyleneimine matrix (EPM) nanoplatform for the delivery of PD‐L1 siRNA (siPD‐L1) to efficiently silence PD‐L1 in the lung tumour microenvironment leading to lung tumour inhibition.

Methods : Exosomes were isolated from bovine colostrum powder by rehydration and ultracentrifugation and characterized for size, polydispersity index (PDI), charge (Zetasizer), and surface protein markers (western blot). Four siPD‐L1 sequences, specific to mouse and human, were screened in vitro using LLC and A549 cells, respectively. The lead siPD‐L1 candidate was evaluated in vivo in syngeneic orthotopic lung tumour xenografts established with Lewis lung carcinoma LL/2‐Luc2 (50 × 103) cells in C57BL/6 mice. Ten days post‐inoculation, mice received intravenous treatments three times weekly with vehicle, scrambled siRNA, or FA‐EPM‐siPDL1 (15 µg/dose). Bioluminescence imaging was performed twice weekly to monitor tumour growth. At euthanasia, tissues were collected, and single‐cell suspensions from tumourous lungs were prepared for flow cytometry analysis of immunological parameters, including myeloid and T cell markers.

Results : The average particle size of exosomes was 66 ± 2.5 nm, and the entrapment efficiency of the EPM for siPD‐L1 (1–20 µg) was over 90%. Analysis of protein lysates showed a significant downregulation of PD‐L1 expression, ranging up to 80% in LLC‐1 cells and over 80% in A549 cells. The in vivo mouse study with syngeneic orthotopic LL/2‐Luc2 tumours demonstrates significant tumour growth inhibition (> 70%; p < 0.01) compared to vehicle or scrambled siRNA control. The average weight of tumour bearing lungs in the PD‐L1‐treated group was also reduced (approximately 60%; p < 0.01) compared to the control. Flow cytometry analysis revealed a marked decrease in PD‐L1 expression in tumour associate macrophages (TAMs) with FA‐EPM‐siPD‐L1 treatment. Additionally, siPD‐L1‐treated animals showed depletion of immunosuppressive tumour‐associated macrophages while anti‐tumoural gamma delta T cells were increased.

Summary/Conclusion : EPM‐based, tumour‐targeted siPD‐L1 delivery enhances immune surveillance and effectively slows lung tumour progression, establishing a promising nanoplatform for advancing siRNA therapeutics in cancer treatment.

Funding : Supported by the Immunotherapy CCII CoBRE pilot project (P20GM135004) and the Agnes Brown Duggan Endowment.

Mechanisms and functions of CSF1 association to extracellular vesicles in anti‐tumour immune responses

Shima Ghoroghi 1 , Louise Merle 1 , Luisa Loconte 1 , Nathalie Nevo 1 , Lea Guyonnet 2 , Jeremy Mesple 1 , Coralie Guérin 2,3 , Lorena Martin‐Jaular 1,3 , Alain Joliot 1 , Clotilde Théry 1,3

A. Joliot and C. Théry are co‐senior authors. 1 Institut Curie Research center, INSERM U932, PSL Research University, Paris, France; 2 Institut Curie Research Center, CurieCoreTech Cytometry, Paris, France; 3 Institut Curie Research center, CurieCoreTech Extracellular Vesicles, Paris, France

Introduction : Extracellular vesicles (EVs) are secreted lipid bilayer‐enclosed particles, which can transfer bioactive molecules to target cells and thus modulate their physiology. Tumour EVs can either activate or inhibit anti‐tumour immune responses. Recently, we showed that triple negative breast cancer (TNBC) cells release EVs bearing the macrophage‐colony‐stimulating factor CSF1, which promotes monocyte differentiation towards a distinct macrophage type with pro‐inflammatory features, correlating with better survival and a potentially anti‐tumoural immune infiltrate in TNBC patients (Tkach et al. 2022, PNAS 119: e2107394119). Here, we further studied the molecular bases of CSF1 association to EVs to explore the anti‐tumour therapeutic potential of CSF1‐EVs.

Methods : EVs were isolated by size exclusion chromatography from the conditioned medium of MDA‐MB‐231 TNBC cells or HEK293 cells engineered to express different forms of CSF1. EVs were quantified by nanoparticle tracking analysis, tagged CSF1 was analysed by western blotting and non‐tagged CSF1 by commercial LegendPlex. The E0771 C57Bl6 mouse mammary carcinoma was injected orthotopically in the mammary fat pad. Tumour size was measured by calliper, and immune infiltrate by spectral flow cytometry or immunohistochemistry.

Results : Injection of CSF1‐EVs from MDA‐MB‐231 reduced progression of E0771 in immunocompetent mice. The CSF1 gene is transcribed as two major isoforms, short S‐CSF1 and long L‐CSF1, both containing a transmembrane domain, but different extracellular glycosylation and cleavage sites. By expressing them separately in HEK293 cells, we demonstrated that S‐ and L‐CSF1 are associated with EVs through different mechanisms: S‐CSF1 is mainly covalently associated as a full‐length transmembrane protein, while L‐CSF1 is associated as a cleaved form through reversible binding to the EV surface without involvement of the transmembrane domain. Functionally, HEK293‐EVs bearing either L‐ or S‐CSF1 induced different differentiation patterns of human monocytes into macrophages in vitro. When injected in vivo, they induced different modifications in the immune cell infiltrates of the tumours, resulting in different effects on tumour growth in immunocompetent hosts.

Summary/Conclusion : Our results could lead to future therapeutic uses of CSF1‐bearing EVs as novel strategies of anti‐tumour immunotherapies.

Funding : INSERM, CNRS, Institut Curie, Fondation de France, PSL Research University, ANR, INCa, ARC.

Distinct role of large oncosomes in modulating bone marrow microenvironment and promoting metastasis through immune reprogramming in cancer progression

Taylon F. Silva 1 , Diana Kitka 1 , Blandine Victor 1 , Chen Qian 1 , Minhyung Kim 1 , Tatyana Vagner 1 , Giorgia Guerra 1 , Fatima Bedier 1 , Virginia Concato‐Lopes 1 , Catherine Grasso 1 , Sungyong You 1 , Fayyazz Sutterwala 1 , Caroline Jefferies 1 , Paola de Candia 2 , Michael Freeman 1 , Helen Goodridge 1 , Karen Cavassani 1 , Jlenia Guarnerio 1 , Dolores Di Vizio 1

1 Cedars‐Sinai Medical Center, Los Angeles, USA; 2 University of Naples Federico II, Naples, Italy

Introduction : Large oncosomes (LO) represent a distinct class of extracellular vesicles (EV) with unique features. They have been recently recognized as key players in cancer progression and metastasis due to their ability to reprogram recipient cell behaviour and induce vascular morphogenesis.

Methods : EV‐treated cells were analysed by RNA‐seq, qPCR, and ELISA. Cancer cell migration and recruitment were assessed using transwell migration, a 3D organoid bone model, and in vivo intracardiac injection. Orthotopic breast cancer and intratibial injections of 4T1.2 cells were conducted in Balb/cJ mice. Neutrophils were characterized by spectral flow cytometry, and IVIS imaging detected in vivo metastasis.

Results : LO from prostate and breast cancer exhibit enhanced potency in reprogramming bone marrow‐derived mesenchymal stem cells (BM‐MSCs) compared to exosomes (Exo), as observed by RNAseq. Mechanistically, LO induces a robust inflammatory response, including metastasis‐relevant chemokines and transcription factors involved in type I interferon responses, such as RSAD2 and IRF3/7. Importantly, this activation in response to LO was selectively induced in BM‐MSCs but not in fibroblast and epithelial cells. Using in vitro and in vivo orthogonal methods, we observed that LO‐treated BM‐MSC promotes the recruitment of cancer cells. Intravenous priming of orthotopic 4T1.2 tumour‐bearing mice with LO promoted three times more metastasis than Exo‐ or vehicle‐treated mice. Additionally, neutrophil chemotaxis was significantly promoted by cancer‐derived LO‐treated BM‐MSC. Neutrophils exposed to a conditioned medium from LO‐treated BM‐MSC significantly increased the expression of LOX‐1, Arginase‐1 and IL‐10, suggesting their differentiation into polymorphonuclear‐myeloid‐derived suppressor cells (PMN‐MDSC). We also observed an increase in the presence of PMN‐MDSC in the bone marrow of tumour‐bearing mice along with a complete lack of lymphocyte populations, indicating their immunosuppressive activity that could lead to pre‐metastatic niche formation.

Summary/Conclusion : The mechanisms by which cancer cells exploit IFN‐I responses to drive immune cells into dysfunctional immunosuppressive states remain unknown. This comprehensive study unveils the molecular pathways underlying the immune response to LO in the bone marrow, shedding light on mechanisms that could influence metastatic progression in prostate cancer. The findings underscore the unique role of LO in modulating the BM microenvironment, providing potential targets for therapeutic intervention.

Funding : National Institutes of Health R01CA218526 (DDV); DoD PC150836 (DDV).

26 April 14.30–16.00 Technologies and Methods

Session title OS22 Tracking of EVs

Through the looking glass: tracking EVs in vivo

Invited Speaker: Frederik J. Verweij

Utrecht University, The Netherlands

Manipulation of tumour extracellular vesicles biodistribution and uptake in vivo by a cell‐surface ‘EV‐Trap’

Vincenzo Verdi 1 , Maribel Lara Corona 1 , Pierre‐Michaël Coly 1 , Wendy van Straaten 2 , Daniele D'Arrigo 3 , Amandine Robac 1 , Erwan Boëdec 1 , Alessia Di Maggio 2 , Frederik J. Verweij 1,2 and Guillaume van Niel 1,4 Presented by Ela Babursah 1,4

1 Institut de Psychiatrie et Neurosciences de Paris, France. 2 Universiteit Utrecht, The Netherlands. 3 Abbelight Biotech Research Company, France. 4 Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers (CRCI 2 NA) France

Introduction : Tumour‐derived EVs are acknowledged as important cellular mediators that are involved in several hallmarks of cancer progression, such as tumour proliferation, immune system modulation, angiogenesis induction and the development of pre‐metastatic niches. However, there are still a lot of unanswered questions in the field about the function of EVs in vivo. A number of methods, most notably in the zebrafish embryo, have been developed to live‐track the dynamics and fate of tumour‐derived EVs at a single particle scale in vivo. Nonetheless, there are currently insufficient methods available to precisely direct and control the dispersal and targeting of tumour EVs in vivo, to either better understand intercellular communication during pathological development or improve EV‐based therapeutic strategies. To fill this gap, we present here a novel tool, the EV‐trap, for selectively capturing and locally accumulating circulating tumour EVs towards engrafted trapping cells or endogenous tissues.

Methods : We have generated, on one hand, stable lines of prostate cancer cells (PCa) expressing the pHluorin tag associated to tetraspanins C63 or CD9 and, on the other hand, cells and a zebrafish strain expressing an “EV‐trap”, consisting of a transmembrane domain exposing specific nanobodies at the cell surface. After full characterization of the PCa‐derived EVs, we have used imaging approaches to evaluate the trapping of this tool in vitro and in vivo using zebrafish embryos.

Results : We show here that modified PCa produce large amounts of EVs carrying pHluorin moieties. The EV‐trap expressing cells can selectively and specifically capture EVs within min, up to 20,000 EVs per cell without saturation. This capture is efficient in vivo for exogenous and endogenous EVs and redirects EVs from their intended destinations.

Summary/Conclusion : We have developed an ‘EV‐trap’ tool able to capture and redirect EVs thanks to the expression of a chimeric receptor. This tool will provide new insights into the function of EVs and possible new therapy avenues in a number of physiopathological processes. We are now adapting this tool to natural tumour antigens, such as PSMA, to gain a better understanding of the biological activities of tumour EVs, such as in the creation of pre‐metastatic niches.

Funding : Marie Curie Actions, proEVLifeCycle, Fondation ARC.

Multiplexed isotopically enriched tellurium‐based labelling of the EV proteome (MULTI‐TeLEV) allows high‐dimensional single‐cell profiling of EV uptake from six different EV sources in parallel

Patrick Hoelker 1 , Lisa Wessendorf 1 , Rahil Gholamipoorfard 2 , Yong Jia Bu 3 , Yoojeong Chun 3 , Selen Seyhan 4 , Mark Nitz 3 , Nima Abedpour 2 , Michael Hallek 1 and Daniel Bachurski 1

Patrick Hoelker, Lisa Wessendorf and Rahil Gholamipoorfard are co‐first authors. Nima Abedpour, Michael Hallek and Daniel Bachurski are co‐senior authors. Daniel Bachurski is the corresponding author.

1 University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Center for Molecular Medicine Cologne, CECAD Center of Excellence on Cellular Stress Responses in Aging‐Associated Diseases, Cologne, Germany, 2 Cancer Research Center Cologne Essen, Department I of Internal Medicine, Department of Translational Genomics, Faculty of Medicine, University of Cologne, University Hospital Cologne, Cologne, Germany, 3 University of Toronto, Toronto, Canada, 4 Dokuz Eylül University, Izmir, Turkey

Introduction : EV uptake heterogeneity across EV recipient and source cell types remains unexplored at the single‐cell level. Most uptake studies examine a single EV source, which misses potential interactions between EVs from different origins and their combined effects on recipient cells. To address this, we developed MULTI‐TeLEV, employing six isotopologues of L‐2‐tellurienylalanine (TePhe) for parallel, high‐dimensional single‐cell analysis of EV uptake from multiple sources. This approach, leveraging high‐parametric mass cytometry, reveals how EVs from distinct sources interact and influence uptake across 50 PBMC immune cell types and subpopulations.

Methods : Six TePhe isotopologues (122TePhe, 124TePhe, 125TePhe, 126TePhe, 128TePhe, and 130TePhe) labelled EVs from U937, THP‐1, Ramos, Jurkat, OSU‐CLL, and HEK293T cell lines, respectively. Cell‐conditioned media were generated under serum‐free conditions, and EVs were purified using the combination of 10 kDa ultrafiltration and size‐exclusion chromatography. One million healthy donor PBMCs were incubated with 5 × 10 11 TeLEVs for 16 h alongside single‐source EV conditions for comparison.

Results : As anticipated, total EV uptake decreased in co‐incubation with EVs from five different sources; however, uptake reduction was significantly source‐ and cell‐type‐specific. Myeloid dendritic cells and monocyte subsets (classical, non‐classical, and transitional) maintained high THP1‐TeLEV uptake, while Ramos B cell line TeLEV uptake was reduced by 50%, indicating differential immune cell tropism. Unexpectedly, low‐density neutrophils showed a 50%–100% increase in uptake from four EV sources, suggesting a priming mechanism for EV uptake.

Summary/Conclusion : In summary, MULTI‐TeLEV unveils the complex interactions and competitive dynamics of EVs from multiple sources, demonstrating patterns of selective uptake by diverse immune cell subtypes. This single‐cell approach highlights the influence of EV origin on uptake efficiency, revealing that specific cell types exhibit unique tropisms impacting cellular response and EV‐mediated signalling. These findings underscore the need for future EV assays to incorporate and evaluate combinations of EV sources to accurately reflect the nuanced biological interactions occurring in vivo, advancing our understanding of EV biology in multi‐source environments.

EVAnalyzer2: moving from in vitro to in vivo high‐content single‐vesicle imaging for quantitative serum and tissue pharmacokinetics of extracellular vesicles using C++ and artificial intelligence‐based image analysis

Melanie Schürz 1,2 , Joachim Danmayr 1 , Maria Jaritsch 1 , Tanja Plank 1,2 , Patricia Hrasnova 1,3 , Vesna Stanoljovic 1,2 , Heloisa Melo‐Benirschke 1 , Cristian Matea Tudor 1 , Nicole Meisner‐Kober 1,2

1 Paris Lodron University of Salzburg, Austria; 2 Ludwig Boltzmann Institute for Nanovesicular Precision Medicine at the PLUS, Salzburg, Austria; 3 Paracelsus Medical University Salzburg, Austria

Introduction : With the rapid growth of the EV field, rigorous characterization at the single vesicle level has become increasingly important to assure comparability and reliability of published data. To facilitate standardization, we recently developed ‘EVAnalyzer,’ an ImageJ plug‐in optimized for automated, quantitative image analysis from single vesicle imaging and cellular EV uptake data, which we published together with a robust protocol for routine EV applications. With over 3400 downloads, the program has become a tool of widespread use within the EV community.

Methods : Given this evident need in the field, we further progressed EVAnalyzer into EVAnalyzer2, which additionally enables tracking of EVs in huge, complex in vitro and in vivo models. Usage of near infrared dyes together with flexible image processing pipelines and rigorous object quantification enables the detection of EV signals over diverse autofluorescent structures and thereby enables sensitive tissue and serum pharmacokinetics. In addition, advanced object recognition and segmentation are enabled by the support of AI models that are entirely based on open‐source technology and can be easily trained and used without any deep AI knowledge. Moving into in vivo high‐content vesicle imaging, the increase in image sizes and the inherent growth in data volume make automated image and fast data processing inevitable. As these requirements hit the limits of the Java‐based image analysis software ImageJ, we changed to the more efficient programming language C++.

Results : New developments for EVAnalyzer2 will be presented together with application examples for quantitative serum and tissue pharmacokinetics. Additionally, inspired by the rigour and standardization work done by ISEV, especially in the field of flow cytometry, we also evaluate calibration beads in single vesicle imaging for the comparisons of EV numbers and brightnesses between different instruments and imaging platforms.

Summary/Conclusion : EVAnalyzer2, an open‐source high‐throughput image processing tool with a comprehensible user interface, allows the creation of highly flexible pipelines for standard image processing and thresholding and the inclusion of AI‐driven object detection and automated data processing options. This makes the analysis of EVs in complex in vitro and in vivo images accessible to everyone.

Funding : EV‐Quant (Salzburg, WISS2025, 20102‐F2100572‐FPR); EVTT (EU, EFRE/IWB 20102‐F1900731‐KZP); CONSONANT (Salzburg, WISS2025; F2200397‐KZP).

The making of bi‐specific extracellular vesicles for targeted tissue‐specific delivery

Madhusudhan Reddy Bobbili 1,2 , Philipp Sabler 2 , Núria Gimeno Magán 2 , Gordana Wozniak‐Knopp 1 , Johannes Grillari 1,2

1 Institute of Molecular Biotechnology, Department of Biotechnology and Food Science, BOKU University, Vienna, Austria. 2 Ludwig Boltzmann Institute for Traumatology, the Research Center in cooperation with AUVA, Vienna, Austria

Introduction : Despite recent advances with lipid nanoparticles (LNPs) for drug delivery, LNPs can display toxicity and can be limiting in targeted delivery. Thus, the ineffective drug delivery problem needs to be addressed urgently, which can be overcome by extracellular vesicles (EVs). To enhance EV‐targeted drug delivery, we employed intrinsically overexpressed proteins, CD81 and CD9, to serve for recognition of the desired target antigens.

Methods : Yeast libraries displaying mutant variants of the large extracellular loop of CD81 and CD9 have been selected for binders to EGFR and HER2 as an example target. Their specific interaction with EGFR and HER2 was confirmed in a mammalian display system. Derived sequences for EGFR were reformatted to full‐length CD81 and for HER2 into CD9 and expressed in EVs produced by HEK293 cells.

Results : To assess the novel functionality of antigen‐binding CD81 & CD9 LEL variants, internalization of such EVs into EGFR and HER2 over expressing cell lines such as SKBR‐3, N‐87, A431 and MDA‐MB‐468 was assessed. EVs derived from the wild‐type CD81 and CD9 production cell lines were included as controls. Indeed, we observed bispecific EVs targeted to EGFR & HER2 were significantly internalized in EGFR and HER2 overexpressing cells compared to single antigen‐expressing cells. Furthermore, compared to WT‐EVs, targeted EVs loaded with small molecules induce apoptosis when loaded with cytotoxic drugs. Currently, we are assessing the in vivo targeted drug delivery of bi‐specific EVs.

Summary/Conclusion : Developing bi‐specific targeting extracellular vesicles (EVs) could significantly enhance the efficiency of EV‐mediated delivery across diverse cell types and tissues. Leveraging the extensive versatility of this platform, it enables modular adaptation to a wide array of target antigens, which can be seamLessly replaced with full‐length CD81 and CD9 proteins to facilitate targeted EV delivery. Furthermore, we anticipate that this approach will reduce off‐target effects, thereby improving therapeutic specificity.

26 April 14.30–16.00 Physiology and pathology

Session title OS23 Neurobiology 2

Neurobiology II introduction talk

Invited Speaker: Roosmarijn E. Vandenbroucke

Ghent University, Belgium

Sex‐ and APOE‐4 dependent changes in cerebrospinal fluid extracellular vesicle miRNAs identify synaptic protein alterations in females with Alzheimer's disease

Presenter: Ursula S. Sandau

Oregon Health & Science University, USA

Introduction : Sex and APOE genotype are two AD risk factors, with females accounting for 60% of AD patients. Further, female APOE‐4 carriers have more brain atrophy and memory loss compared to age‐ and genotype‐matched males. Yet the mechanisms by which sex and APOE impact AD pathophysiology are unknown. Thus, we used human cerebrospinal fluid (CSF) extracellular vesicle (EV) miRNAs to identify genes altered in the human female AD brain.

Methods : Human CSF EV miRNAs from AD ( n = 28) and control ( n = 28) were assayed by qPCR. CSF EVs isolated by size exclusion chromatography were characterized by immunoblot for EV (e.g., CD81, flotillin) and non‐EV‐associated proteins (e.g., albumin), resistive pulse sensing, and transmission electron microscopy. TargetScan 7.2, miRDB, and Ingenuity Pathway Analysis were used for miRNA target prediction. MiR‐16‐5p and Reelin, SNAP‐25, and STXBP1 3’‐UTR luciferase reporter constructs were co‐transfected into HEK293T cells. Human hippocampus protein lysates from AD ( n = 34) and controls ( n = 22) were used to measure Reelin, SNAP‐25, and Syntaxin‐binding protein 1 (STXBP1) by immunoblot. Human frontal lobe brain sections from AD ( n = 4) and control ( n = 3) were co‐labelled for SNAP‐25 and miR‐16‐5p by dual immunohistochemistry‐miRScope.

Results : CSF EV preparations were positive for EV‐associated markers, and particles were of the expected size (∼50–100 nm). In CSF EVs, miR‐16‐5p was increased in AD females with the APOE‐3,4 genotype, but not in APOE‐3,3 AD females nor APOE‐3,4 and ‐3,3 AD males. The predicted targets of miR‐16‐5p include Reelin, SNAP‐25, and STXBP1, which are integral to synaptic transmission. We show that Reelin, SNAP‐25, and STXBP1 can be directly regulated by miR‐16‐5p in luciferase reporter assays and that protein levels are decreased in the hippocampus of AD females but not AD males. AI analysis shows that miR‐16‐5p and SNAP‐25 are also colocalized in cortical grey matter, and miR‐16‐5p trends towards increasing in AD.

Summary/Conclusion : Alterations in CSF EV miRNAs are indicative of AD pathophysiology, including synaptic dysfunction, and may thus identify novel protein targets to treat AD. Also, considering that AD pathophysiology is sex‐ and APOE genotype‐dependent, these factors need to be considered in drug development and treatment strategies.

Funding : NIA R01AG079356, NIA RF1AG059392; OHSU Circle of Giving; Collins Medical Trust.

Neuronal extracellular vesicles carrying APOE downregulate filament actin polymerization signalling to inhibit synapse formation in Alzheimer's disease

Presenter: Zhixin Ma

Sun Yat‐sen University, China

Introduction : Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accounts for the majority of dementia cases. Synaptic loss is a key factor driving cognitive dysfunction in AD. The APOE gene is strongly associated with increased AD risk. Synapse formation relies on filamentous actin (F‐actin) polymerization signalling, but it is unclear whether APOE modulates this process in AD. Extracellular vesicles (EVs) mediate inter‐neuronal communication and may play a crucial role in AD progression. However, the impact of neuronal EVs on synapses in the AD brain remains poorly understood.

Methods : WT and APP/PS1 (AD model) primary neuronee‐derived EVs were isolated by multi‐step ultracentrifugation, termed WTNEVs and APPNEVs. Nanoparticle flow cytometry, electron microscopy, and Western blot confirmed EV purity and characteristics. Proteomics and bioinformatics identified proteins differentially expressed in APPNEVs. Synapse formation and the APOE‐Rac1‐N‐WASP‐Arp2/3 signalling pathway were analysed using Western blotting and super‐resolution microscopy. Stereotaxic injections of EVs into mouse brains, followed by fluorescence staining, enabled in vivo analysis of synapse formation.

Results : Compared to WTNEVs, APPNEVs were released at higher concentrations. APPNEVs reduced PSD95 and synaptophysin expression, significantly decreasing the number of synapses, particularly mature synapses, in neuronees. Proteomic analysis revealed elevated levels of APOE protein in APPNEVs, suggesting its involvement in the synaptogenesis signalling pathway. Further investigation demonstrated that APPNEVs, through APOE cargo, downregulated neuronal GTP‐Rac1 levels, attenuated N‐WASP‐mediated phosphorylation of Arp2, and inhibited Arp2/3 complex‐driven F‐actin branching, thereby impairing synapse formation. In vivo experiments confirmed that APPNEVs suppressed synapse formation and downregulated PSD95 and synaptophysin expression. Additionally, inhibiting APOE function prevented APPNEVs from downregulating Rac1‐N‐WASP‐Arp2/3 signalling or impairing synapse formation, both in vitro and in vivo.

Summary/Conclusion : EVs released by APP/PS1 neuronees carry APOE to recipient healthy neuronees, where they inhibit synapse formation by downregulating Rac1‐N‐WASP‐Arp2/3‐mediated F‐actin polymerization. This disruption exacerbates synaptic deficits and accelerates AD progression.

Funding : National Natural Science Foundation of China (32200638); Basic and Applied Basic Research Fund of Guangdong Province (2021A1515110512 and 2023A1515010090); Shenzhen Science and Technology Innovation Program (JCYJ20230807110316034 and JCYJ20210324134612035); Research Start‐up Fund of the Seventh Affiliated Hospital, Sun Yat‐sen University (ZSQYBRJH0021).

Microglial extracellular vesicles promote synaptic pruning via C1q deposition at the synapse

Martina Gabrielli 1,2 , Giulia D'Arrigo 1 , Giulia Cutugno 1 , Maria Teresa Golia 1 , Francesca Sironi 3 , Marta Lombardi 1 , Camilla Halimi 1 , Roberto Frigerio 4 , Marina Cretich 4 , Elisabetta Battocchio 1 , Sara Francesca Colombo 1,2 , Cristiana Barone 5 , Emanuele Azzoni 5 , Roberta Ghidoni 6 , Caterina Bendotti 3 , Rosa Chiara Paolicelli 7 , Claudia Verderio 1,2

Martina Gabrielli and Claudia Verderio are co‐senior authors.

1 Institute of Neuroscience, National Research Council of Italy, Via Raoul Follereau 3, 20854, Vedano al Lambro, Italy. 2 NeuroMI Milan Center for Neuroscience, University of Milano‐Bicocca, Milan, Italy. 3 Research Center for ALS, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri, 2, 20156 Milano, Italy. 4 National Research Council of Italy, Institute of Chemical Science and Technologies (SCITEC‐CNR), Via Mario Bianco 9, 20131 Milan, Italy. 5 School of Medicine and Surgery, University of Milano‐Bicocca, Monza, Italy 6 Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, 25125 Brescia, Italy. 7 Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

Introduction : The complement factor C1q is released by microglia, localizes on weak synapses and acts as a tag for microglia‐mediated synaptic pruning, a fundamental process for proper circuit refinement across early postnatal life, and is reactivated in neurodegeneration. However, how C1q tags synapses at specific times remains elusive. We explored the possible involvement of extracellular vesicles (EVs) released by microglia in C1q delivery to synapses designated for removal.

Methods : We optimized a protocol to extract large and small EVs from the interstitial fluid of mouse brain tissue. By western blot, TRPS, single molecule arrays (SimoA) and confocal imaging, we measured the amount, cell source and complement cargo of EVs extracted from WT postnatal pups and adult mice brains, and from C9orf72KO adult brains displaying enhanced microglia‐mediated pruning. Then, we performed in vitro and ex vivo studies to enquire about microglial EVs involvement in C1q deposition to the synapse and synaptic engulfment, using approaches to increase or reduce microglial EVs production by EVs supplementation, C9orf72 silencing or pharmacological inhibition (GW4869).

Results : We show that microglia release more EVs carrying C1q in C9orf72KO adult mice, providing a link between microglial EVs production and synaptic removal. Moreover, we report that production of C1q carrying microglial EVs peaks during the pruning period in the early postnatal hippocampus (postnatal day P17), confirming a positive correlation between EVs production and synaptic removal in a physiological setting. In neuronee‐microglia co‐cultures, microglial EVs, labelled by the fluorescent dye mCLING, make preferential contacts with synapses, deliver C1q to pre‐synapses that externalize phosphatidylserine and promote synaptic removal. Interestingly, C9orf72KO microglia engulf more synaptic terminals and decrease synaptic density to a greater extent compared to WT microglia, whereas inhibition of EVs release by GW4869 restores normal pre‐synaptic density, providing mechanistic evidence linking EVs release to synaptic remodelling.

Summary/Conclusion : This study identifies microglial EVs as delivery vehicles for C1q to synapses targeted for removal and implicates abnormal EVs production from microglia in neurodevelopmental and age‐related disorders characterized by dysregulated synaptic pruning. Analysis of specific subpopulations of brain EVs opens new perspectives to the understanding of EVs roles in physiology and pathology.

Funding : RF‐2016‐02361492 Italian Ministry of Health.

Extracellular vesicles modulate the amyloid β aggregation in Alzheimer's disease via surface‐expressed prion protein

Mohsin Shafiq 1# , Andreu Matamoros‐Angles 1 , Ladan Amin 2 , Stefano Da Vela 3 , Susanne C. Meister 1 , Behnam Mohammadi 1 , Emina Karadjuzovic 1 , Alexander Hartmann 1 , Milou Rooijakkers 1 , Ilka Egger 1 , Ayesha Zafar 1 , Angela Correia 4 , Matthias Schmitz 4 , Bente Siebels 5 , Hartmut Schlüter 5 , Isidre Ferrer 6 , Susanne Krasemann 1 , Hermann C. Altmeppen 1 , Inga Zerr 4 , Carolin Seuring 7 , David A. Harris 2 , Dmitri Svergun 3 , Markus Glatzel 1#

Mohsin Shafiq and Markus Glatzel are co‐senior authors.

1 Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf, Germany, 2 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA, 3 European Molecular Biology Laboratory Hamburg, Germany, 4 Department of Neurology, University Medical Center Goettingen, Germany, 5 Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany, 6 Bellvitge University Hospital, IDIBELL, L'Hospitalet de Llobregat, Spain, 7 Multi‐User CryoEM facility, Centre for Structural Systems Biology, Hamburg, Germany

Introduction : Extracellular vesicles (EVs) expressing cellular prion protein (PrPC—a neuronal ly expressed Aβ receptor) have previously been shown to accelerate Aβ aggregation. However, mechanistic and structural details of this modulation are still not well understood. It also remains unclear whether the surface PrPC molecules on EVs mediate the EV−Aβ interaction in AD brain. Here, we aim to understand structural aspects of EV−Aβ interaction, the role of PrPC in EV‐mediated Aβ fibrillation, Alzheimer's disease (AD)‐related changes in PrPC expression in brain‐derived EVs (BDEVs), and whether the association of EVs with Aβ plaques is driven by surface PrPC molecules of EVs.

Methods : PrPC‐expressing (WT) and ‐deficient (KO) EVs were derived from WT and PrPC‐KO Neuro‐2a cells. Mouse brain‐derived EVs (BDEVs) of WT and KO phenotypes were also isolated from WT and PrPC‐KO mice. EVs were incubated with recombinant Aβ in various settings to study the EV‐mediated Aβ fibrillation using small‐angle x‐ray scattering (SAXS), super‐resolution microscopy (SRM), and cryogenic electron tomography (CryoET). PrPC expression of BDEVs from frontal cortex autopsies of AD and age‐matched controls was studied by immunoblotting and proteomics. We also isolated plaque‐associated proteins from a human cohort and from an amyloid pathology mouse model with a wild‐type background (5xFADPrnp+/+) or a PrPC knock‐out background (5xFADPrnp−/−) to study the correlation between expression levels of EV markers and PrPC in Aβ plaques. Expansion microscopy was employed to visualize the EV markers and PrPC in Aβ plaques in both systems.

Results : SAXS, EM, and SRM highlighted potent Aβo‐sequestering activity of the WT‐EVs compared to the KO‐EVs. 3D reconstructions of EV‐Aβ complexes from CryoET highlight empirical involvement of PrPC in the EV‐Aβ interaction. We observed a positive correlation between BDEVs‐associated Aβ amounts and BDEVs‐PrPC levels. In the plaque isolates prepared from the human cohort and the amyloid mouse models, the expressions of EV markers were positively correlated with PrPC expression.

Summary/Conclusion : Our data suggests a clear role of EV‐expressed PrPC in the association of EVs to the amyloid plaques in AD. Taken together with the mouse model and in vitro data, we conclude that PrPC expression on EVs is crucial for their Aβ sequestration leading to plaque formation in AD.

26 April 14.30–16.00 EV sources and basic biology

Session title OS24 Cross‐Kingdom Communication

EVs in cross‐species interactions: biology and applications

Invited Speaker: Amy Buck

University of Edinburgh, UK

Nicotiana benthamiana EVs: new frontiers in drug delivery systems

Presenter: Mahmoud Eldahshoury

Leeds Beckett University, UK

Introduction : Extracellular vesicles (EVs) are promising drug delivery nanocarriers, though research is mostly focused on costly, hard‐to‐scale mammalian models. This study investigates small EVs from nicotiana benthamiana (Nb), a plant model used in biopharma, as a scalable, cost‐effective, and biocompatible alternative. Plant EVs allow for easy genetic manipulation, potentially enabling transgenic plants to produce disease‐targeted EVs, offering an affordable, ethical drug delivery approach.

Methods : Nb EVs were isolated via ultracentrifugation and iodixanol gradient following MISEV guidelines. EV size and morphology were assessed by nanoparticle tracking analysis (NTA) and electron microscopy, while proteomics and transcriptomics characterized EV content and functions. Cytotoxicity assays (MTT, LIVE/DEAD) tested interactions with SH‐SY5Y neuroblastoma cells, and tetraspanin8‐GFP‐labelled EV uptake was analysed by flow cytometry and FlowSight. Loading efficiency was confirmed using Exofect kits and Tx‐red‐tagged siRNA, and BACE1‐targeting siRNA‐loaded EVs’ delivery efficiency was evaluated by fluorescence microscopy, qPCR, and flow cytometry.

Results : NTA and electron microscopy revealed an enrichment of vesicles under 200 nm, with negative staining confirming the presence of characteristic cup‐shaped vesicles. Western blot and proteomic analyses confirmed the presence of a CD63 protein homologue (tetraspanin‐8) and the absence of the SYP121 protein (associated with a distinct EV subpopulation in plants). Proteomic analysis further revealed a notable abundance of homologues to mammalian EV markers, indicating significant cross‐kingdom similarities in EV composition. In vitro, Nb EVs were non‐cytotoxic and were efficiently internalized by SH‐SY5Y cells. When loaded with BACE1‐targeting siRNA, Nb EVs significantly reduced BACE1 mRNA and protein levels compared to controls (free siRNA and unloaded EVs). These results highlight Nb‐derived EVs as efficient, low‐immunogenic, and cost‐effective carriers for RNA‐based therapeutics.

Summary/Conclusion : This study provides compelling evidence for the potential of nicotiana benthamiana EVs as a drug delivery platform. The findings demonstrate that Nb EVs are not only readily taken up by mammalian cells but also effectively deliver functional cargo in vitro. These attributes underscore their promise as a scalable and affordable alternative to conventional EV‐based delivery systems, with particular relevance to RNA‐based therapeutics.

Funding : This study was funded by Leeds Beckett University.

Pomegranate‐derived nanovesicles as a promising therapy for inflammatory diseases: evidence from mouse models of acute pancreatitis and atopic dermatitis

Christian M. Sánchez‐López 1,2 , Miquel Martínez‐Navarrete 3 , M. Isabel Torres‐Cuevas 4 , Antonio J. Guillot 3 , Ana Melero 3 , Salvador Pérez‐Garrido 4 , Antonio Marcilla 1,2

1 Àrea de Parasitologia, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot (Valencia), Spain. 2 Joint Research Unit on Endocrinology, Nutrition and Clinical Dietetics, Health Research IIS La Fe‐Universitat de València, Valencia, Spain. 3 Àrea de Farmàcia i Tecnologia Farmacèutica, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot (Valencia), Spain. 4 Departament de Fisiologia, Universitat de València, Burjassot (Valencia), Spain

Introduction : Pomegranate is an edible fruit that has garnered increasing scientific interest due to its potential health benefits. Our research group first described that pomegranate juice (PgJ) contains nanovesicles (NVs) with antioxidant, anti‐inflammatory, and wound‐healing properties in vitro, highlighting their therapeutic properties (1). We now further evaluate the therapeutic potential of PgNVs in two mouse models of inflammatory diseases like acute pancreatitis and atopic dermatitis.

Methods : PgNVs were isolated using size exclusion chromatography (SEC) and characterized through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Acute dermatitis (AD) and chronic dermatitis (ChD) were induced in CD1‐Swiss female mice via oxazolone (OXA) topic administrations, with PgNVs administered subcutaneously on the same days as OXA. Acute pancreatitis (AP) was induced in C57BL/6J mice by administering three intraperitoneal L‐arginine injections (3 g/kg body weight) at 1 h intervals; a single subcutaneous dose of PgNVs was given prior to the first L‐arginine injection.

Results : In AD mice, PgNVs administration significantly lowered IL‐4 and IL‐1β levels, although it did not lead to marked changes in skin condition. In contrast, ChD mice treated with PgNVs exhibited notable reductions in skin histological damage, including reductions in epidermal thickening, collagen deposition, and inflammatory cell infiltration, along with increased IL‐10 and decreased IL‐1β levels. In the AP model, PgNVs markedly reduced oedema and inflammatory infiltrates, while limiting the rise in plasma amylase activity observed after AP induction. The improvements in AP were strongly associated with the regulation of IL‐6 expression and PgNV's antioxidant activity through NQO1 upregulation.

Summary/Conclusion : Our findings suggest that PgNVs have significant therapeutic potential for inflammatory diseases, effectively modulating cytokine profiles and reducing markers of inflammation in ChD and AP models. These results support PgNVs as a promising natural therapeutic with anti‐inflammatory and antioxidant properties, meriting further investigation for clinical applications. (1) C.M. Sánchez‐López et al. Food Funct 13 (2022) 12870–12882.

Funding : This work was supported by Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Spain (Grants PID2020‐114530GA‐I00 and PID2023‐146116NB‐I00 and ERDF/EU), and Conselleria d'Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Valencia, Spain (Grant CIPROM/2023/54). C.M.S.‐L. is the recipient of a postdoctoral fellowship funded by AEI /10.13039/501100011033.

Microalgae‐derived extracellular vesicles as multifunctional cosmetic ingredients against oxidative‐ and UVB‐related damages

Paola Gargano 1,2,* , Giorgia Adamo 1 , Sabrina Picciotto 1 , Daniele P. Romancino 1 , Monica Salamone 1 , Angela Paterna 3 , Estella Rao 3 , Samuele Raccosta 3 , Mauro Manno 2,3 , and Antonella Bongiovanni 1,2

1 Cell‐Tech HUB at Institute for Research and Biomedical Innovation, National Research Council of Italy (CNR), Palermo, Italy; 2 EVE Biofactory at Cell‐Tech HUB, National Research Council of Italy (CNR), Palermo, Italy; 3 Cell‐Tech HUB at Institute of Biophysics, National Research Council of Italy (CNR), Palermo, Italy

Introduction : The increasing demand for green, sustainable, and solvent‐free cosmetic ingredients is prompting companies to explore natural biotechnological approaches. Microalgae have recently been the focus of intensive research in the cosmetic sectors due to their bioactive compounds (e.g., pigments and vitamins). These compounds, as well as the whole microalgae extract, are already used as cosmetic ingredients, but this approach presents big limitations in terms of economic and environmental sustainability: the microalgal biomass used during the extraction process is discarded as waste. A patented and eco‐compatible solution is extracellular vesicles (EVs) isolated from microalgae culture medium, named (nano)algosomes (Alg). During the isolation process microalgae are separated from the medium and used to start a new cultivation. In our previous studies, Alg showed high biocompatibility, anti‐inflammatory bioactivity, and a peculiar tissue tropism. Here we explore the role of Alg in protecting skin cells against oxidative‐ and UVB‐related damages.

Methods : Alg are isolated from Tetraselmis chui cultures through tangential flow filtration (TFF) and widely characterized following MISEV 2023 guidelines. We set up our experiment using two skin cell lines, fibroblast (NHDF) and human melanoma cells (A375).

Results : Exposure of cells to oxidative stress (i.e., TBH) or UVB rays induces an increase of ROS levels, leading to cellular death. Pre‐treatment of cells with Alg demonstrated their ability in restoring cell viability (photoprotective role) and in rebalancing ROS levels (antioxidant activity). Moreover, UVB rays’ or α‐melanocyte‐stimulating hormone's exposure promotes melanin synthesis in A375, but the pre‐treatment with Alg showed a reduction in melanin content, demonstrating an anti‐melanogenic effect. Western blot analysis confirmed that Alg can reduce the expression levels of tyrosinase, a key enzyme in melanogenesis.

Summary/Conclusion : These findings open a promising new direction for cosmetic and cosmeceutical formulations using natural biotechnological ingredients. Alg protects skin cells from oxidative stress, inflammation, and UVB‐related damage while reducing melanin content. This positions Alg as an effective depigmenting agent for whitening formulations, a multifunctional cosmetic ingredient with intrinsic protective benefits, and a potential delivery system for active ingredients, enhancing their stability, penetration, and efficacy.

The Sdp‐SH3b2 domain contained in extracellular vesicles from the probiotic Lactobacillus johnsonii N6.2 inhibits murine norovirus replication

Danilo R. da Silva, Asra B. Sharjeel, Melissa Jones, Claudio F. Gonzalez, Graciela L. Lorca

University of Florida

Introduction : The internalization of Lactobacillus johnsonii N6.2 extracellular vesicles (EVs) by cells results in a significant induction of the 2’,5’‐oligoadenylate synthetase (OAS) pathway. It also induces expression of IFI44L, MX1, MX2 and DDX60. In this work, we evaluated whether the antiviral response induced by L. johnsonii N6.2 EVs has an inhibitory effect on an RNA viral insult using murine norovirus (MNV‐1) as the viral infection model.

Methods : L. johnsonii N6.2 was grown in MRS broth to the late exponential growth phase, and EVs were isolated from the supernatant. EVs were purified through differential ultracentrifugation and quantified using Nanosight NS300. Selected proteins enriched on the proteomic analyses of EVs were cloned, overexpressed and purified as His‐tagged fusion proteins using nickel affinity chromatography. To evaluate the role of EVs on MNV‐1 infection, murine RAW 246.7 macrophage cell lines were utilized. For the in vitro treatments, L. johnsonii N6.2 EVs were added at increasing concentrations (1 × 108 to 1 × 1010 particles/mL). MNV‐1 was tested at an MOI = 5. For in vivo experiments, C57BL/6 mice were housed in the University of Florida animal facilities. The animal protocols were approved by the Institutional Animal Care and Use Committees at the University of Florida.

Results : We found that RAW 264.7 Macrophages treated with EVs significantly decreased the levels of the MNV‐1 genome. These results were consistent with an increase in expression of Oas1b, Oas2, Oasl, Mx1, Mx2 and Ifi44l (6 h post infection). Out of six proteins enriched in EVs, we found that the SH3b2 domain of Sdp was the only protein effector molecule able to recapitulate the activation of the OAS pathway. In C57BL6 mice, the administration of live L. johnsonii N6.2, EVs, and Sdp‐SH3b2/liposomes significantly decreased MNV‐1 titres in the distal ileum, in contrast to the controls with PBS and liposomes alone that did not affect MNV‐1.

Summary/Conclusion : These results establish that the SH3b2 domain of Sdp, which is enriched in L. johnsonii derived EVs, is an effector molecule in EVs that can orchestrate the control of viral infections in vivo.

Funding : This study was partially funded by the National Institute of Diabetes and Digestive and Kidney Diseases at NIH award R01DK121130.

Sunday, 27th April

27 April Plenary 4 and featured abstract

RNA in exosomes to extracellular vesicle therapeutics—a 20‐year journey

Plenary Presenter: Jan Lötvall

University of Gothenburg, Sweden

Fetoplacental‐derived extracellular vesicles deliver conceptus‐derived antigens to maternal secondary lymphoid tissues for T cell recognition

Juliana S Powell, William J Shufesky, Adriana T Larregina, Yoel Sadovsky, Adrian E Morelli

University of Pittsburgh, USA

Introduction : Pregnancy constitutes an ‘immunological paradox,’ where despite the competent maternal immune system, the semi‐allogeneic foetus evades rejection. Mechanisms at the maternal‐foetal interface and the mother's secondary lymphoid tissues (SLTs) that dodge the immunologic attack on the foetus, remain unknown. Passage of small extracellular vesicles (EVs) is a mechanism of cell‐to‐cell communication that transfers antigens (Ags), regulatory mediators, and RNAs. In transplantation, graft‐derived EVs carried through blood or lymph promote anti‐donor T‐ and B‐cell responses in the maternal SLTs. Thus, akin to transplantation, fetoplacental‐derived EVs transported via blood could represent a mechanism by which fetoplacental Ags and immune‐regulatory mediators could be delivered to maternal SLT immune cells for T and B cell recognition.

Methods : We tested whether fetoplacental EVs could transport paternal Ags to maternal SLTs.

Results : In female BALB/c mice impregnated with mOVA C57Bl/6 (B6) males, the surrogate paternal Ag OVA accumulated in maternal SLTs. In a mouse model in which EVs released by the fetoplacental unit are labelled with the green fluorochrome mNeonGreen linked to the small EV‐associated marker CD81, mNeonGreen‐CD81 was detected by fluorescence microscopy in APCs in the maternal spleen. Immunoprecipitation followed by western blot of EVs from primary cultures of trophoblasts from BALB/c females impregnated with mOVA B6 males revealed OVA on EVs. Administration of these trophoblast‐derived EVs to virgin females induced defective activation and proliferation of OVA‐specific CD4 (OT‐II) and CD8 (OT‐I) TCR transgenic T cells in the spleen. In Rab27a‐/‐ mice in which vesicle release is deficient, OVA‐specific OT‐I proliferation is reduced. In B6 females impregnated with Act‐mOVA males, paternal Ag, OVA, is retained in maternal tissues until at least post‐partum (PP) day 30. In humanized mice, i.v. injection of CM‐DiI (red)‐labelled EVs from primary human trophoblasts were captured by human antigen presenting cells (APCs) in the spleen.

Summary/Conclusion : Therefore, systemically released fetoplacental EVs carry paternal/trophoblast Ags that reach the maternal APCs in the mother's SLTs, where the Ags are presented in a manner that promotes deficient activation of T cells as occurs during a normal pregnancy. Funding: National Institutes of Health, USA. 5R01AI168142‐03, 5R01AI148690‐05, 5R01AI174273‐03.

How EV isolation impacts protein cargo and function

Presenter: Christian Preußer

Philipps University Marburg, Germany

Introduction : A key question persists in EV research since its beginnings: Are we isolating vesicles or actively shaping them through our methods? While the MISEV guidelines provide a classification framework, they assume that purification mainly affects contamination rather than the properties of EVs as the analyte. Recent studies suggest, however, that ‘EV‐specific’ features may depend on isolation methodology and thus represent artefacts. Here, we compare EV isolation methods in terms of proteomic composition and functional consequences, focusing on the canonical enzymatic activity of EV‐associated membrane‐bound metalloproteinases.

Methods : EVs were isolated in individual batches ( n = 5) from peritoneal fluid of ovarian cancer patients (ascites) or ES‐2 cell culture supernatants using four workflows: ultracentrifugation (UC) with differential UC (UC‐UC) or density gradient UC (UC‐DG), and tangential flow filtration (TFF) followed by ultrafiltration (TFF‐UF) or size exclusion chromatography (TFF‐SEC). EVs were characterized using orthogonal approaches, including electron microscopy and deep label‐free mass spectrometry, to assess the impact of isolation on protein composition. Functional assays using established FRET‐based fluorescent peptides were performed to evaluate EV‐associated metalloproteinase activity.

Results : The isolation methods significantly influenced particle concentration, shape and the EV‐associated proteome. TFF‐based techniques yielded the highest particle concentrations, whereas UC‐UC showed the highest batch variability. TFF‐UF exhibited substantial contamination with putative non‐EV components, while UC‐DG and TFF‐SEC achieved more selective enrichment of canonical EV markers. Functionally active proteases (ADAMs and MMPs) were detected in all samples and verified by appropriate inhibitors, although with isolation‐dependent variations. These findings suggest that rigid adherence to MISEV classifications may be insufficient for evaluating or applying a given isolation method.

Summary/Conclusion : The comprehensive analysis of EVs from biofluids and cell culture supernatants—examining shape, surface markers, proteome content, and metalloproteinase activity—highlights the need to reassess EV‐associated protein classifications and potential contaminants. Our findings stress the crucial link between purification methods and EV functions. The MISEV guidelines should serve as a flexible framework rather than rigid rules, emphasizing the importance of critically evaluating methods for accurate EV interpretation.

Funding : This research was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; 416910386 GRK 2573/2; UKGM‐3/2020 MR; DFG‐INST 216/793‐1 FUGG.

27 April 8.30–10.00 Disease and therapy

Session title LB01 EVs in Disease and Therapy

Cutting‐edge multiplexed ev biomarker detection: a game‐changer for predicting PD‐1/PD‐L1 inhibitor response

Presenter: Jessica Gobbo

INSERM1231, Dijon, France

Introduction : Extracellular vesicles (EV) are emerging as powerful biomarkers in immunotherapy, carrying tumour‐derived signals and immunosuppressive factors. Their minimally invasive and dynamic nature makes them valuable for predicting treatment response, monitoring therapeutic efficacy, and analysing the tumour microenvironment. This study aims to define a multiparametric signature to predict responses to PD‐(L)1 inhibitors before treatment initiation and develop a rapid, robust, and clinically feasible biomarker analysis method requiring minimal biological samples.

Methods : A total of 250 patients with metastatic melanoma, non‐small cell lung cancer, and lymphoma were included in this study ( NCT02662621 , NCT05744076 , NCT04417803 ). EVs were isolated from 500 µL of plasma using an optimized precipitation kit. Size and concentration were assessed via nanoparticle tracking analysis (NS300, Malvern) and transmission electron microscopy. EV markers (TSG101, CD9, CD63, CD81, Alix, and the negative control Grp94) were validated using capillary western blot (CWB). A multiplexed ELISA assay (MEA) was developed to establish a comprehensive protein signature, including PD‐L1.

Results : We optimized EV characterization by refining antibody concentration, blocking reagents, and sample concentration for CWB and MEA. Key advantages include ultra‐sensitivity (pg/mL), minimizing blood collection needs, enhanced standardization via automation, Optimized processing: in 3 h (CWB) and 1.5 h (MEA, five targets in triplicate). Circulating PD‐L1‐EV were identified as dynamic biomarkers for anti‐PD‐(L)1 therapy response. The lack of correlation between EV‐associated and free PD‐L1 suggests distinct biological origins. We showed that PD‐L1‐EV analysis could predict tumour response before medical imaging evidence. We further demonstrated that PD‐L1‐EV are biologically active, mimicking cancer cells by inhibiting CD8⁺ T‐cells. This highlights their immunosuppression role in resistance mechanisms (local or at the tumour distance). Finally, we identified a protein signature capable of predicting anti‐PD‐(L)1 therapy responses before treatment initiation.

Summary/Conclusion : We developed an ultra‐sensitive, rapid, and clinically adaptable EV biomarker detection method requiring minimal sample volume. This approach offers deeper insights into the tumour microenvironment from a simple blood test and could enhance patient selection in clinical practice. A national multicentre study is underway to validate this predictive signature in precision oncology.

Amyotrophic lateral sclerosis proteomic signature and treatment with mesenchymal stem cell‐derived extracellular vesicles

Presenter: Suzy Varderidou‐Minasian

UMC Utrecht, The Netherlands

Introduction : Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by the degeneration of both upper and lower motor neuronees. Despite the promise of new treatments, clinical trials face high failure rates. Mesenchymal stem cells (MSCs) and the extracellular vesicles (EVs) these cells release have gained significant interest due to their accessibility, capacity for migration to damaged tissues, and involvement in reparative processes. We utilize induced pluripotent stem cell (iPSC)‐derived motor neuronees, which provide a valuable method to study disease signatures derived from patient‐specific cells to get insight into the mechanisms underlying ALS and enable the testing of novel therapeutic agents. Several ALS iPSC‐derived motor neuronees were treated with MSC‐EVs and analysed with high‐throughput mass spectrometry (MS) approaches for the in‐depth characterization of relevant ALS disease pathways.

Methods : Thirty‐six motor neurone samples (9 ALS patients (carrying FUS, TDP‐43 or C9ORF72 mutations), 6 isogenic lines and 3 healthy controls) were analysed by MS for proteomic alterations. EVs were isolated from human bone marrow MSCs using ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot for EV markers. Motor neuronees were treated with EVs derived from 4x106 MSCs per well (6 wp) for 2 days. The isolated EVs and treated samples were analysed by MS and neurite length was measured using bright‐field microscopy.

Results : Principal component analysis across all lines and differentiation batches showed that individual variation is higher than technical variation. Global proteomic analysis revealed dysregulated ‘oxidative phosphorylation’ for C9ORF72‐ALS, ‘cytoplasmic translation’ for FUS‐ALS and ‘coding region instability determinant‐mediated mRNA stabilization’ for TDP‐43‐ALS mutation. Additionally, we identified universal proteomic signatures characteristic of ALS in general, including alterations in ‘ATP biosynthetic processes’ and ‘DNA replication.’ MSC‐EV treatment improved neurite outgrowth, with the observed proteomic signatures corresponding to recovery in both mutation‐specific and general ALS conditions.

Summary/Conclusion : By generating patient‐specific motor neuronee cultures, we highlight the proteomic variances observed during the differentiation and between distinct iPSC lines. In addition, we found both mutation‐specific and common ALS‐related alterations, such as dysregulation of oxidative phosphorylation and mRNA stabilization. MSC‐EV treatment restored protein expression, offering hope for future ALS therapies.

Exploring the immunomodulatory role of extracellular vesicles in stroma dense colorectal tumours

Anastasija Walsh 1,2 , Lei Lei 1,2 , Norashikin Zakaria 1,2 , Nial O'Reilly 1,2 , Roisin M Dwyer 6 , Aideen E Ryan 1,2

1 Discipline of Pharmacology and Therapeutics, School of Medicine, College of Medicine, Nursing and Health Science, University of Galway; 2 Lambe Institute for Translational Research, School of Medicine, College of Medicine, Nursing and Health Science, University of Galway; 3 Discipline of Pathology, School of Medicine, College of Medicine, Nursing and Health Sciences, Clinical Science Institute, University of Galway; 4 Division of Anatomical Pathology, Galway University Hospital; 5 Department of Colorectal Surgery, Galway University Hospital; 6 Discipline of Surgery, Lambe Institute for Translational Research, School of Medicine, College of Medicine, Nursing and Health Science, University of Galway

Introduction : CMS4 colorectal cancer (CRC), one of four consensus molecular subtypes (CMS), is marked by an immunosuppressive tumour microenvironment. Its characteristics include the presence of cancer‐associated fibroblasts (CAFs) and elevated TGF‐β production, together contributing to resistance against immune‐based therapies. Cancer cell EVs (T‐EVs) are essential mediators of cell communication within the tumour microenvironment. T‐EVs often contain immunomodulatory ligands. This study aims to investigate the expression of immunomodulatory ligands on CMS4 T‐EVs and examine how they influence immune cells and stroma within the tumour.

Methods : EVs were isolated from CMS4 cancer cells (HCT116 and SW480) TGF‐β1 treatment via SEC. Particle size and concentration were determined by NTA, TSG101 western blots were performed and TEM images were taken. T‐EVs were analysed for expression of cancer‐associated immunoinhibitory ligands by spectral flow cytometry. NK cells were incubated with SW480 secretome with/without EV depletion. Additionally, NK cells were treated with T‐EVs directly (5,000 EVs/cell). NK cells were then co‐cultured with SW480 cells to assess functional marker expression. Finally, to assess the effect of T‐EVs on CAFs, primary intestinal CAFs were treated with complete or EV‐depleted secretome, and immunomodulatory ligand expression was analysed.

Results : The addition of TGF‐β1 induced higher particle production in CMS4 cancer cells in the 100 nm range. These particles exhibited high positivity for immune‐inhibitory Siglec‐7, Siglec‐9, and Siglec‐10 ligands, PD‐L1, and CD24, with their expression remaining consistent regardless of TGF‐β1 addition. T‐EVs reduced expression of key activation marker NKG2D, but T‐EVs from TGF‐β‐treated cells were even more suppressive, further reducing expression. Similarly, the complete secretome upregulated Siglec‐9 and Siglec‐10 while downregulating the activation markers DNAM‐1 and NKG2D on NK cells. EV‐depleted secretome did not induce any of these effects, with expression of listed markers closer to baseline in this group. Furthermore, the induction of Siglec ligands & PD‐L1 observed previously in our lab as a result of secretome treatment was abrogated with EV depletion.

Summary/Conclusion : My findings demonstrate that EVs are key mediators of the immunomodulatory effects in the CMS4 tumour microenvironment. This highlights EVs as essential drivers of cancer‐mediated immune evasion.

Funding : Irish Cancer Society.

TNFα inhibitor thalidomide enhances osteosarcoma chemotherapy sensitivity

Manting Luo, Chong Zheng, Bin Yang, Jiwei Luo, Jianlong Li

Department of Orthopedic, Nanfang Hospital, Southern Medical University, Guangzhou, China

Introduction : Osteosarcoma‐derived extracellular vesicles and particles (EVPs) could induce liver metabolic dysregulation through Kupffer cells. Osteosarcoma chemotherapy faces significant challenges due to the severe systemic toxicity of chemotherapeutic agents like doxorubicin, which causes cardiac toxicity, hepatic dysfunction, and myelosuppression. These toxicities limit the dose and effectiveness of treatment. This study investigates a novel strategy involving TNFα inhibition, aimed at improving chemotherapy efficacy and mitigating the side effects associated with doxorubicin therapy.

Methods : An orthotopic osteosarcoma model was established using K7M2 cells implanted in the tibia of BALB/cJ mice. Mice were treated with doxorubicin (25 mg/kg, ip) either alone or in combination with thalidomide (50 mg/kg, ip, daily for 7 days). The study evaluated tumour growth, chemotherapy‐induced toxicity, and hepatic metabolic functions. Key assessments included Bodipy staining for hepatic lipid accumulation, qPCR for cytochrome P450 (CYP) enzyme expression, echocardiography for cardiac function, and haematological and biochemical analyses to assess systemic effects. TEM, NTA, and WB were used to identify EVPs.

Results : The osteosarcoma released EVPs, upregulated TNFα from Kupffer cells, causing hepatic lipid accumulation, and suppressed the expression of key CYP enzymes, disrupting normal drug metabolism. While doxorubicin alone hardly reduced tumour size, nor reduced the secretion of EVPs, it induced severe cardiotoxicity and metabolic dysfunction. In combination with the TNFα inhibitor, thalidomide restored CYP enzyme expression, alleviated hepatic lipid accumulation, and improved overall chemotherapy tolerance. This combination therapy resulted in enhanced tumour suppression and reduced toxicity, particularly in terms of heart function, compared to doxorubicin monotherapy.

Summary/Conclusion : This study introduces a novel therapeutic strategy by combining TNFα inhibition with doxorubicin to enhance osteosarcoma chemotherapy sensitivity. The mechanism involves restoring hepatic metabolic balance and reducing inflammation‐induced dysfunction, thereby improving chemotherapy tolerance and efficacy. This approach not only offers a promising new direction for osteosarcoma therapy but also has the potential for broader applications in other malignancies, particularly those with high chemotherapy‐associated toxicity.

Funding : [GuangDong Basic and Applied Basic Research Foundation (2024A1515030050) and Presidential Foundation of Nanfang Hospital, Southern Medical University (2023A031)].

Blood cell‐derived extracellular vesicles induce a pro‐inflammatory phenotype in tubular epithelial cells of proliferative lupus nephritis patients

Hilal N. Sensoy *1 , Michelle Tsang‐A‐Sjoe 2 , Niala Masoumi 1 , Monique A. J. van Eijndhoven 1 , Leontine Bosch 1 , Cristina Gomez‐Martin 1 , Joris J. T. H. Roelofs 3 , Yongsoo Kim 1 , Ekaterina S. Jordanova 4 , Kerstin M. Heutinck 5 , Kenny de Wildt 1 , Nils J. Groenewegen 1 , Ineke R. J. ten Berge 5 , Katrien Grunberg 6 , Raymond M. Schiffelers 7 , Jan R. T. van Weering 8 , Sandra A. W. M. Verkuijlen 1 , Irene E. Bultink 2 , Jaap M. Middeldorp 1 , Alexandre E. Voskuyl 2 , D. Michiel Pegtel 1 , and S. Rubina Baglio 1

* Presenting author, 1 Department of Pathology, Amsterdam UMC location Vrije Universiteit Amsterdam, 2 Department of Rheumatology and Clinical Immunology, Amsterdam UMC location Vrije Universiteit, 3 Department of Pathology, Academic Medical Center, University of Amsterdam, 4 Center for Gynaecologic Oncology Amsterdam, Department of Gynaecology, VU University Medical Center, 5 Department of Experimental Immunology, Academic Medical Center, 6 Department of Pathology, Radboudumc, 7 Department of Clinical Chemistry & Hematology, University Medical Center Utrecht, 8 Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University

Introduction : Proliferative lupus nephritis (LN) is a severe condition causing impaired kidney function that occurs in 40%–60% of systemic lupus erythematosus (SLE) patients. LN is characterized by a marked interferon (IFN) response in tubular epithelial cells (TEC), but the biological trigger has remained unclear. We previously showed that extracellular vesicles (EVs) released by B cells are enriched in polymerase III (Pol III) transcripts of endogenous and viral (EBV) origin. These transcripts carry pathogen‐associated molecular patterns (PAMPs) that can be recognized by innate RNA sensors. We hypothesize that in LN, due to glomerular damage, high amounts of inflammatory EVs reach the tubular epithelium, inducing an IFN response that exacerbates the disease.

Methods : Gene set enrichment analysis was used to identify key pathways and cell signatures in proliferative LN. RNA‐seq was used to define the pol‐III transcriptome of B cell EVs. In situ hybridization was employed to reveal the presence of EBV transcripts in human tissues. In vitro assays were used to uncover the mechanisms of internalization and the functional effect of B cell EVs on TEC cells.

Results : scRNA‐seq analysis of proliferative LN identified enrichment of blood cell transcripts, including (EBV‐infected) B lymphocyte RNAs in TEC cells. Notably, the same blood cell signatures were enriched in plasma EVs. SLE patient plasma and purified EVs contained the EBV‐encoded pol III‐transcript EBER1, which selectively accumulated in the cytoplasm of TEC in lupus but not IgA nephritis biopsies. Functional assays showed uptake of B cell EVs by primary TECs in a phosphatidylserine (PtdSer)‐dependent manner. Importantly, B cell EVs deliver EBERs into endosomes of primary TECs, driving IFN gamma, TNF alpha and IL6 production through TLR3 activation.

Summary/Conclusion : In SLE patients, blood cell‐derived inflammatory EVs target TEC cells via binding to PtdSer receptors, inducing an IFN‐mediated inflammatory response.

Circulating extracellular vesicles‐containing P. gingivalis OMVs from periodontitis patients trigger the release of inflammatory markers and the amyloid‐ß peptide

Presenter: Charles Ramassamy

INRS‐Centre Armand‐Frappier Santé‐Biotechnologie, Canada

Introduction : Periodontitis, a chronic inflammatory disease, and its key pathogen Porphyromonas gingivalis, have been implicated in several systemic disorders, including Alzheimer's disease (AD). However, the underlying mechanisms linking this oral pathogen to neurodegenerative pathology remain poorly understood. This study aims to investigate the systemic dissemination of Porphyromonas gingivalis and its contribution to Alzheimer's disease (AD) pathology through its outer membrane vesicles (OMVs). Specifically, we will identify P. gingivalis ‐derived OMVs (Pg‐OMVs) in the plasma of periodontitis patients and assess their impact on microglial and neuronal cells.

Methods : Circulating extracellular vesicles (cEVs) were isolated from the plasma of periodontitis patients using size‐exclusion chromatography. The presence of Pg‐OMVs in the cEVs sample was analysed by the detection of the Kgp protease marker using capillary western blot analysis and TEM immunogold labelling. Biodistribution and brain entry of PKH‐labelled cEVs were evaluated by their intravenous injection into the Tg(flk:EGFP) zebrafish model. The effects of cEVs and Pg‐OMVs on the secretion of inflammatory cytokines (IL‐4, IL‐6, IL‐8, IL‐10, IL‐1β, TNF‐α) by the human microglial HMC3 cells were determined using ELISA‐MSD. Their effects on the release of Aβ1‐42 by the N2a‐SW‐APP neuronal cells were measured using ELISA‐MSD.

Results : Our results show that cEVs contain Pg‐OMVs as confirmed by western blot and TEM immunogold labelling. cEVs injected into zebrafish traversed the blood‐brain barrier and were found in the brain 3 h after the injection. These cEVs triggered the secretion of inflammatory cytokines by HMC3 cells and Aβ1‐42 by N2a‐SW cells.

Summary/Conclusion : This study demonstrates that plasma derived‐cEVs from periodontitis patients can trigger inflammation and the production of Aβ1‐42. These results highlight the systemic spreading of Pg‐OMVs and the contribution of cEVs containing Pg‐OMVs to the development of neurodegenerative disorders. Thus, cEVs could be considered as critical tools to discover systemic markers of neurodegenerative disease in periodontitis patients.

Funding : Research Chair Louise & André Charron on Alzheimer's disease, NSERC.

27 April 8.30–10.00 EV sources and basic biology

Session title LB02 EV Sources and Basic Biology

Arabidopsis plants produce distinct subpopulations of extracellular vesicles that alter growth and infectivity of a fungal pathogen

Presenter: Benjamin Koch

Indiana University, USA

Introduction : Extracellular vesicles (EVs) secreted by mammalian cells are heterogenous in content, biogenesis, and function. Whether this is also true for EVs secreted by plant cells is not yet known. Though plant EVs have been observed and associated with immune response for 50 years, the precise role of plant EVs in immunity is unknown.

Methods : We used high‐resolution density gradient ultracentrifugation, protection assays, and total internal fluorescence microscopy (TIRF‐M) to separate, purify, and distinguish distinct subpopulations of Arabidopsis EVs. We characterized the response of plant EV subpopulations to biotic and abiotic stresses, including fungal infection, phytohormone treatment, and changes in temperature. We also analysed EV subpopulation‐specific defects of Arabidopsis mutants, including ESCRT, RAB, EXOCYST, and VAMP‐ASSOCIATED PROTEIN (VAP) family proteins. We studied the effect of plant EVs on the growth, development, and virulence of the fungal pathogen Colletotrichum higginsianum .

Results : The EV marker protein TETRASPANIN 8 (TET8) was detected specifically in medium‐density EVs and was uniquely not associated with cell wall polysaccharide nanofilaments that co‐pelleted with high‐density EVs. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted in separate EV populations using TIRF‐M, while PEN1 was co‐secreted with PENETRATION 3 (PEN3) much more often. RNA and ARGONAUTE proteins were not associated with EVs of healthy plants. Mutant analysis indicated a general role in EV secretion for EXO70 proteins, as well as a specific role of VAPs and RabA2a in the secretion of TET8+ and PEN1+ EV‐subpopulations respectively. In addition, exo70 family mutants were more susceptible to Colletotrichum infection. We found that EV subpopulations marked by TET8, PEN1, and RPM1‐INTERACTING PROTEIN 4 (RIN4) are secreted to the leaf extracellular space and the leaf surface in response to biotic and abiotic stress, while other EV subpopulations were less responsive. Treatment of Arabidopsis seedlings with plant EVs delayed the progression of Colletotrichum infection by altering fungal germ tube development and morphology.

Summary/Conclusion : Our data indicate that Arabidopsis EVs are highly heterogenous, that EV subpopulations have differing mechanisms of biogenesis, and that specific EV subpopulations contribute to plant immunity by interacting with fungi during early stages of infection on the leaf surface.

Natural EV vs. synthetic nanoparticle corona stability monitored by single‐particle spectral flow cytometry

Martin Wolf 1,2 , Nicole Maeding 1,2 , Antri Stefani 1,2 , Janine Zwicklhuber 1 , Negar Sedghi Aminabad 1 , Rodolphe Poupardin 1 , Sarah Hochmann 1 , Andre Görgens 2 , Samir el Andaloussi 2 , Dirk Strunk 1,2

1 Cell Therapy Institute, Paracelsus Medical University (PMU), Salzburg, Austria; 2 Austrian Red Cross Research, Vienna, Austria; 3 Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden

Introduction : Others and we have recently shown that the extracellular vesicle (EV) protein corona is crucial for their biological activity. The aim of this study was to compare corona formation and stability during purification between different nanoparticle entities.

Methods : Utilizing spectral flow cytometry (sFCM), we monitored nanoparticle corona formation and stability on reference EVs derived from the human embryonic kidney 293 cell line stably expressing an mNEON‐CD63 construct and purified in large‐scale from conditioned medium by tangential flow filtration. Silica and polystyrene nanoparticles (NPs) or synthetic lipids (LNPs) of 100 or 200 nm diameter were used for comparison. We established an artificial protein corona with BSA‐AF647 on all NPs. We monitored corona stability during the most commonly used EV isolation methods, tangential flow filtration (TFF), size exclusion chromatography (SEC) and ultracentrifugation (UCF), before and after purification, by sFCM.

Results : After 24 h, compared to reference EVs, we found 10× higher BSA‐AF647 fluorescence on silica NPs and 100× on LNPs. The EV‐BSA‐AF647 corona was significantly reduced but preserved by TFF and completely removed by SEC or UCF. In contrast, the corona on silica NPs and LNPs was not significantly reduced by all three purification methods.

Summary/Conclusion : Against our expectation that EVs establish a protein corona with similar properties to synthetic NPs, we found significant differences concerning corona formation capacity and stability. We speculate that EVs might only carry a soft corona formed by the interaction of corona proteins with EV membrane proteins, while synthetic NPs also form an inner hard corona by electrostatic binding.

Funding : This work was supported by funding from the European Union's Horizon Europe research and innovation program (grant agreement 101095635 PROTO, 101080267 NEXGEN‐PD and 101056712 HEAL to DS), by Land Salzburg (20102‐F2100572‐FPR ‘EV‐Quant’ and 20102‐F2001080‐FPR ‘Cancer Cluster II’ to DS.

Extracellular vesicle based transcriptomic investigation on age‐ and microgravity‐exposed 3D muscle organoid

Presenter: Samantha Ali

University of Florida, USA

Abstract unavailable

MBsomes and other EVs intercellular communication in the Skin

Presenter: Mariane Shouky

Institut Curie, France

Introduction : Orchestrated intercellular communication is essential to maintain skin homeostasis. Extracellular vesicles (EVs) play a crucial role in intercellular crosstalk in the skin, allowing exchange of proteins, lipids and genetic material. EVs are a heterogeneous group of cell‐derived membranous structures that are released by cells into the extracellular environment as exosomes and ectosomes, which originate from the endosomal system or which are shed from the plasma membrane, respectively. The mitotic midbody bridge at the final stage of cytokinesis could be a potential source of released midbody bridge‐derived ectosomes and MBsomes. Whether proliferating primary keratinocytes release MBsomes and midbody‐bridge ectosomes that could act as a means of intercellular communication in skin homeostasis, to our knowledge, has not been investigated yet. The aim of this project is to decipher the midbody bridge as a potential source of ectosomes as well as their signalling function in the epidermal/dermal crosstalk and skin homeostasis.

Methods : To study the biogenesis and the release of midbody bridge vesicles, we used electron microscopy and immunofluorescence approaches. In addition, purification of MBs and EVs from primary epidermal keratinocytes was performed by differential ultracentrifugation followed by sucrose cushion and size exclusion chromatography, respectively. The composition of MBs and EVs was investigated by proteomics and transcriptomics. In addition, to decipher MBs and EVs signalling function and potential role in skin epidermal/dermal crosstalk, migration and proliferation of recipient dermal fibroblasts were also assessed by Incucyte scratch‐wound assay and real‐time proliferation assay, respectively.

Results : Our results show that proliferating epidermal skin keratinocytes release MBsomes and midbody bridge vesicles carrying cytokinetic proteins. In addition, MBsomes and EVs, released by epidermal keratinocytes, enhance migration and proliferation of dermal fibroblasts.

Summary/Conclusion : This project highlights the midbody bridge as a potential source of a new subtype of large EV, MBsomes, in addition to other midbody bridge‐derived vesicles. It also shows the potential role of MBs in intercellular crosstalk in the skin.

Funding : NIH National Institute of Health.

Novel highly sensitive phosphatidylserine‐specific reagents allow measuring global EV turnover in vivo

Lavinia Flaskamp 1 , Annkathrin Scheck 1 , Monica Prechtl 1 , Christine Ried 1 , Jan Kranich 1 , Thomas Brocker 1

1 LMU Munich, Institute for Immunology, Munich, Germany

Introduction : Phosphatidylserine (PS) is a well‐established marker for apoptotic cells but is also present on extracellular vesicles (EVs). However, the prevalence of PS+ EVs remains debated, possibly due to variations in PS levels based on the EV source and differences in the sensitivity of PS‐binding reagents. To address these issues, we developed several PS‐detection reagents based on murine MFG‐E8 (including MFG‐E8‐EGFP and tetramerized MFG‐E8 C1 domains, known as C1‐tetramers) and evaluated their EV‐staining capabilities.

Methods : To compare the affinities of various PS‐binding proteins for small particles, we used liposomes with defined sizes and PS content, as well as EVs isolated from human and murine plasma using size exclusion columns. Liposomes and plasma EVs were stained with Annexin V or our MFG‐E8‐based reagents and analysed by imaging flow cytometry (IFC) or super‐resolution microscopy. C1‐Tetramers, the most sensitive reagent, were used to track EV in vivo turnover in blood by labelling EVs via intravenous injection of reagent. To determine the kinetics of EV clearance and EV in vivo half‐life, we measured the number of labelled EVs at different time intervals post‐injection using IFC.

Results : Our results show that PS+ EVs are highly prevalent in murine and human blood, but we observed significant variability depending on the detection reagent. The proportion of PS+ EVs ranged from 30% with Annexin V to over 90% with MFG‐E8 and C1‐Tetramers, which helps explain the conflicting reports on PS‐positivity in the literature. Using C1‐tetramers, we monitored circulating and cell‐bound EVs in vivo, finding that PS+ EVs in murine plasma were rapidly cleared from circulation (t1/2 = 29 min) but remained bound to specific cell types in the spleen.

Summary/Conclusion : We demonstrated that surface PS serves as a universal EV marker, present on nearly all EVs (approximately 90%) in blood. However, accurate analysis of externalized PS on EVs requires careful selection and titration of PS‐detection reagents, as prior studies have significantly underestimated PS‐positive EVs.

Funding : DFG‐CRC 1054 (B03, Z01, Z02).

ICAM5 as a novel marker for neurone‐derived extracellular vesicles

Presenter: Manish Bhomia

Uniformed Services University of the Health Sciences, USA

Introduction : Currently, markers reported in literature to isolate neuronal ‐derived extracellular vesicles (NEVs) lack specificity to the human central nervous system (CNS). Additionally, a proportion of these markers may exist as soluble proteins, further limiting their value. To address this research gap, we have identified a novel neuronal marker, intercellular cell adhesion molecule‐5 (ICAM5), that is highly specific to the CNS and can be used to isolate specific NEVs from human biofluids.

Methods : Mass spectrometry (MS) was performed using EVs isolated from primary human neuronal cell cultures. Novel EV markers identified from this screen were validated in control human serum and CSF (BioIVT Inc.). Size exclusion chromatography (SEC) was performed, and EVs were probed for CD63, CD81 and novel NEV markers. Electron microscopy (EM) was performed to confirm the NEVs in immunoprecipitated samples using a novel marker. Super‐resolution microscopy (SRM) was performed to demonstrate the surface presence of the NEV marker. Clinical TBI samples were analysed to quantify neurological injury markers in immunoprecipitated NEVs.

Results : The MS screen identified 63 proteins that were highly enriched and specific for the human brain, and ICAM‐5 was a potential NEV marker. Size exclusion chromatography confirmed the presence of ICAM‐5 in EVs in the same fractions as EV‐associated tetraspanins (CD‐63 and CD‐9) in human serum and cerebrospinal fluid samples. Further, EVs immunoprecipitated with ICAM‐5 antibody and confirmed with EM showed the presence of neuronal markers such as tau and neuronal specific enolase (NSE) and were negative for glial markers on a western blot. SRM analysis confirmed the presence of ICAM5 on the surface of EVs in both CSF and serum samples and colocalizing with tetraspanins. Finally, ICAM‐5 NEVs showed enhanced expression of UCH‐L1 in serum samples from patients with mild TBI.

Summary/Conclusion : Our data demonstrates that ICAM‐5 is present on the surface of EVs and is a novel and specific marker for enrichment of NEVs from human biofluid. Funding: Disclaimer The opinions expressed herein are those of the author(s) and are not necessarily representative of those of the Uniformed Services University of the Health Sciences, the Department of Defense, or the United States Army, Navy, or Air Force.

Funding : National Institutes of Health.

27 April 8.30–10.00 Physiology and Pathology

Session title LB03 EV Physiology and Pathology

Peripheral and central vascular inflammatory proteins after in vivo immune challenge in major depressive disorder

Presenter: Leandra K. Figueroa‐Hall

Laureate Institute for Brain Research, USA

Abstract unavailable

Omega‐3 deficiency is associated with changes in content and function of brain‐derived extracellular vesicles

Liam Barry‐Carroll 1 , Ivan Marniquet 1 , Claire Bruzaud 1 , Juliette Dupont 1 , Flavie Crespo 1 , Charlotte Madore 1 , Sophie Layé 1 , Jean‐Christophe Delpech 1

1 University Bordeaux, INRAE, Bordeaux INP, NutriNeuro, UMR 1286, F‐33000, Bordeaux, France—Bordeaux (France)

Introduction : A hallmark of the Western diet is a marked decrease in omega‐3 polyunsaturated fatty‐acids (n‐3 PUFA) consumption. This is a cause for concern, as reduced n‐3 PUFA consumption during development may cause long‐lasting cognitive abnormalities. Evidence from murine studies suggests the functions of microglial cells (brain immune cells) and neuronees are impacted in n‐3 PUFA deficient animals, resulting in cognitive changes; however, the exact mechanisms remain unclear. Therefore, we propose to study how reduced dietary n‐3 PUFAs can impact the content of brain‐derived EVs (BDEVs) and their resultant functions in recipient cells.

Methods : Male and female C57BL/6 mice were subjected to either a control diet (balanced) or a diet low in n‐3 PUFAs (deficient) from gestation until 3 months old. BDEVs were subsequently enriched from whole‐brain homogenate samples using a protocol of differential centrifugation and density‐based sucrose gradient. BDEVs were assessed by nanoscale flow cytometry, western immunoblot, TEM, and proteomic analysis. The functional impact of BDEVs was tested in vitro using BV2 microglial cells and human iNeurones treated with BDEVs using an Incucyte microscope.

Results : Deficiency in n‐3 PUFAs was associated with morphometric, synaptic, and proteomic changes in the brain. Specifically, the expression of proteins relating to EV biogenesis and release was altered in n‐3 PUFA deficient brains. Changes in the proteome were also reflected at the EV level in n‐3 PUFA deficient animals in a sex‐dependent manner, with a notably increased expression of proteins involved in cellular metabolism in males and synuclein processing and neurodegeneration in females. At the functional level, microglial phagocytosis was increased when subjected to deficient BDEVs compared to balanced BDEVs, while neurite length was reduced when iNeurones were treated with deficient BDEVs. Interestingly, only BDEVs from n‐3 PUFA balanced animals showed pro‐mitogenic capabilities, resulting in an increased percentage of Ki67+ microglia.

Summary/Conclusion : The results here reveal the capacity of specific nutrients to alter brain EV content and function, shedding light on novel mechanisms involved in the nutritional modulation of cognitive abilities.

Funding : Nouvelle‐Aquitaine

Placental extracellular vesicles as mediators in recruiting maternal microchimeric cells

Presenter: Isabel Graf

University Medical Center of Hamburg‐Eppendorf, Germany

Introduction : During pregnancy maternal (immune) cells are transferred to the developing foetus via the placenta. There is growing evidence that these cells, referred to as maternal microchimeric cells (MMc), can reduce the offspring's susceptibility to diseases later in life, as reflected, for example, by a lower risk for early life infections. However, there is limited understanding of how MMc overcome the placental barrier and which factors regulate their transfer. This study aims to reveal the mechanisms of transplacental MMc trafficking by analysing placenta‐specific extracellular vesicles (EVs) in maternal serum in normally progressing pregnancies and pregnancies affected by maternal infections.

Methods : EVs from third‐trimester serum samples of healthy and SARS‐CoV‐2‐infected pregnant women ( n = 60) were isolated via differential ultracentrifugation. Successful EV isolation was validated by nanoparticle tracking analysis, western blot analysis and transmission electron microscopy. Subsequently placental EVs were enriched by fluorescence activated vesicle sorting, and their proteome was analysed by Imaging Flow Cytometry and Mass Spectrometry. The EVs’ proteome was then correlated with the MMc frequencies detected in the newborns’ cord blood. Additionally, in order to evaluate the uptake of placental EVs by maternal immune cells, cell culture experiments were performed. Lastly, C57BL/6J (CD45.2, H‐2Db) and Balb/c CD45.1 (CD45.1, H‐2Dd) mice were mated allowing the identification of MMc in the foetus with subsequent validation of the identified proteins potentially driving MMc trafficking by qPCR.

Results : Following maternal SARS‐CoV‐2 infection, MMc transfer is significantly decreased. Paralleling decreased MMc transfer rates, the placenta comprises an altered EV messaging with increased EV secretion and an altered protein cargo after infection. This includes the significant downregulation of PSME1 (proteasome activator complex subunit 1), a component of the immunoproteasome, and upregulation of CACYBP (Calcyclin binding protein) after infection. Placental EVs are taken up by maternal immune cells. In a murine model we proof that the proteins which are significantly upregulated in placental EVs, are also significantly upregulated in those maternal cells which crossed the placenta (MMc).

Summary/Conclusion : Our data suggest that placental EV messaging plays a significant role for the recruitment of MMc to the foetus. These findings could potentially lead to the development of new strategies for improving foetal health.

Tanycyte‐derived extracellular vesicles containing Annexin A1 modulate microglia and neuronal functions to control energy balance

Presenter: Rafik Dali

University of Lausanne, Switzerland

Introduction : Energy balance requires precise communication between the periphery and the central nervous system. In the hypothalamus, tanycytes—specialized ependymoglial cells—line the walls and floor of the third ventricle and extend processes into the brain parenchyma. Thanks to their strategic location, tanycytes integrate peripheral metabolic signals and modulate neuronal function accordingly. However, the mechanisms by which tanycytes communicate with neural cells remain largely unknown.

Methods : Our study investigates Annexin A1 (ANXA1) as a tanycyte‐derived signalling molecule involved in neural modulation in response to energy imbalance. We analysed ANXA1 expression and localization along the third ventricle in vivo and in vitro, revealing its colocalization with the CD9 marker in the presence of glucose. Proteomic analysis of isolated tanycyte‐derived extracellular vesicles confirmed the presence of ANXA1 among classical vesicular proteins. Using publicly available single‐cell and single‐nucleus RNA sequencing data, we identified microglia and neuronees as potential targets of tanycyte‐derived ANXA1.

Results : Our findings show that ANXA1 is primarily expressed by dorsal tanycytes and classical ependymal cells, and its expression and localization are regulated by energy imbalance. In vitro, ANXA1 colocalizes with CD9 in the presence of glucose, suggesting its secretion via extracellular vesicles during positive energy balance. Functional experiments demonstrate that tanycyte‐derived ANXA1 modulates microglial number and morphology, as well as the activation of SF1‐expressing neuronees. These neural modifications ultimately influence brown adipose tissue thermogenesis in response to feeding.

Summary/Conclusion : Our study highlights a tripartite communication between tanycytes, microglia, and neuronees to regulate energy balance. These findings represent a first step toward understanding the complex hypothalamic networks involved in metabolic control.

Human milk extracellular vesicles drive GFAP expression and affect proliferation in differentiating neural stem cells in vitro

Marco Blasioli 1,2 , Marije Kleinjan 1 , Caroline de Theije 2 *, Marca Wauben 1 *

1 Utrecht University, The Netherlands; 2 Utrecht Medical Centre, The Netherlands

Introduction : Preterm infants are at high risk for long‐term neurodevelopmental impairments. Human milk feeding improves neurodevelopmental outcome in preterm infants, although the underlying mechanisms are unclear. Breast milk contains extracellular vesicles (mEVs) that contain immunomodulatory and neuroregenerative cargo. Neural stem cells (NSCs) are interesting therapeutic targets because of their potential to generate new neuronees, astrocytes, and oligodendrocytes. This study investigated the effect of mEVs on differentiating NSCs in vitro.

Methods : mEVs were isolated from healthy donors using a published protocol consisting of differential centrifugation, density‐gradient centrifugation and size‐exclusion chromatography. To control for the presence of co‐isolated milk components, a donor‐matched procedural (‘EV‐depleted’) control was generated. NSCs were differentiated in co‐culture with either mEVs (mEV‐NSCs) or EV‐depleted controls (mEVdep‐NSCs). Interaction with mEVs was assessed through flow cytometric analysis of NSCs exposed to fluorescently labelled mEVs. Proliferation of differentiating NSCs was tracked by fluorescent dye dilution and flow cytometry. Differentiation fate of NSCs was assessed after 5 days by immunocytochemistry (ICC) and flow cytometry (FC) targeted at well‐recognized markers of NSCs (nestin), neuronees (βIII‐tubulin), oligodendrocytes (Olig2), and astrocytes (GFAP).

Results : Flow cytometry revealed that NSCs interacted with mEVs already after 4 h of co‐culture. Exposure to mEV‐depleted controls resulted in lower numbers of differentiated NSC counts in ICC. Exposure to mEVs significantly increased NSC numbers compared to mEVdep‐NSCs, suggesting that mEVs may stimulate NSC proliferation. Generation tracking using FC corroborated this hypothesis, showing that mEV‐NSCs divide quicker over the differentiation period compared to mEVdep‐NSCs. After 5 days of NSC differentiation, mEV exposure did not affect nestin expression, βIII‐tubulin expression and neuronal morphology on ICC. However, both on FC and ICC, mEV exposure increased the generation of GFAP+ cells, which morphologically resembled astrocytes. The effect of mEVs on Olig2 expression is currently under investigation.

Summary/Conclusion : These results provide the first evidence that mEVs interact with NSCs in vitro, enhancing their proliferation rate compared to mEV‐depleted controls and promoting differentiation to an astroglial fate. These results highlight a potential role for mEVs in shaping neurodevelopment and warrant further investigation into their therapeutic potential for preterm brain injury.

Extracellular vesicle‐mediated antigen trafficking and innate immune modulation in bacterial infection

Jorge J. Canas, Samantha Enslow, Saloni Bhimani, Mariola J. Ferraro, Department of Microbiology and Cell Science, University of Florida, USA

Introduction : Extracellular vesicles (EVs) are emerging as key regulators of host immune responses, facilitating bacterial antigen trafficking and modulating innate immune activation. This study investigates the role of EVs in shaping innate immunity during Salmonella infection, focusing on dendritic cell (DC) and macrophage subset responses. Additionally, we employed a novel tetraspanin‐based pull‐down method to confirm the host origin of EVs and validate their immunomodulatory functions.

Methods : EVs were isolated from Salmonella‐infected and uninfected macrophages using ultracentrifugation. EVs were given to BALB/c mice intranasally for a period of 24 h. Flow cytometry was performed on lung, spleen, and gastrointestinal tissues from infected mice to assess DC and macrophage subset distribution, including cDC1, cDC2, monocyte‐derived DCs, plasmacytoid DCs, alveolar, and interstitial macrophages. In addition, we used a tetraspanin‐based ‐EV pull‐down approach to ensure specificity for host‐derived EVs, followed by western blotting or mass spectrometry. Western blotting confirmed EV marker expression, and one of the ubiquitinated Salmonella effectors was detected in EV as cargo.

Results : Flow cytometry analysis revealed that EV exposure significantly altered DC and macrophage subset distribution in infected tissues. cDC1 and cDC2 populations exhibited increased activation, while monocyte‐derived DCs and interstitial macrophages displayed enhanced antigen uptake following EV administration. Notably, ubiquitinated Salmonella effectors were enriched in EVs isolated early during infection, supporting a role for EVs in bacterial effector trafficking. The pull‐down approach confirmed that the immunomodulatory EVs were host‐derived, ruling out contamination with outer membrane vesicles (OMVs).

Summary/Conclusion : These findings highlight the immunoregulatory role of host‐derived EVs in modulating specific innate immune responses during Salmonella infection in BALB/c. The use of a tetraspanin‐based pull‐down method provides definitive evidence that these EVs originate from host cells and actively participate in immune signalling. These insights offer potential avenues for targeting EVs in host‐directed antimicrobial strategies.

Funding : R01 AI158749/AI/NIAID NIH HHS/USA.

27 April 8.30–10.00 Technologies and methods

Session title LB04 EV Technology and Methods

Label‐free sizing and multiplexed protein detection on single extracellular vesicles

Andreas Wallucks 1 , Molly Shen 1 , Philippe DeCorwin‐Martin 1 , Lucile Alexandre 1,2 , Hugues Martin 1 , Andy Ng 1 , David Juncker 1 ,

1 McGill University, Canada, 2 Université Paris Cité, France

Introduction : Just as single‐cell RNA sequencing has revolutionized studying cellular diversity, high multiplex single EV proteomics will be transformative to our understanding of EV heterogeneity by identifying distinct subpopulations and accurately mapping shifts in the EV proteome under various cellular states or applied stimuli. Current single EV proteomic technologies, such as flow cytometry, fall short of establishing single EV proteomic atlases due to high detection thresholds, limited protein multiplexing, or both. Here we introduce EV‐ID, a platform with a detection limit around 35 nm, capable of detecting dozens of proteins simultaneously in tens of thousands of single EVs from a 10 µL sample with fully automated data acquisition.

Methods : EV‐ID employs in‐house functionalized glass substrates forming both universal EV capture surfaces, and affinity surfaces for the enrichment of specific subpopulations. EVs are counted and sized using previously introduced EV size photometry (SP), enabled by label‐free interferometric scattering microscopy (iSCAT) on a commercial widefield epifluorescence microscope. EV protein expression is mapped via DNA exchange imaging, employing cyclic immunofluorescence imaging on DNA‐barcoded antibodies. Oligo labelling and erasure steps are performed fully automated in under 5 min each for multiple samples in parallel.

Results : The detection efficiencies of membrane and intraluminal EV proteins were optimized and validated on EVs from GFP reporter cell lines, and the absence of steric hindrance in single versus multiplex detection was confirmed by normalizing on total EV count via SP. Surface chemistries were developed for the capture of EVs from purified samples, as well as from raw cell supernatant, to eliminate purification biases. EV‐ID was employed to measure an initial panel of 20 proteins on tens of thousands of EVs from HUVEC, IMR90, HEK293T, HT29, and MDA‐MB‐231 cells, revealing cell‐specific and conserved co‐expression patterns.

Summary/Conclusion : EV‐ID enables high‐throughput, high‐sensitive, multiplexed profiling of single EVs with minimal sample requirements, generating protein distribution atlases with the potential to advance EV biology and diagnostics.

Engineering strategies based on skin structure for optimized exosome delivery

Ji Hoon Ha 1 , Sung Eun Kim 1 , Jin Young Kim 1 , Jin Mo Kim 1 , Sang‐Geun Han 1 , Yeon Gyun Jung 2 , Yoon Soo Cho 2†

1 Kolmar Korea, Republic of Korea; 2 Hanllim University, Hangang Sacred Heart Hospital, Republic of Korea

Introduction : The skin has a complex and diverse structure. The stratum corneum is exceptionally rigid—often compared to bricks and cement—and hydrophobic, while the epidermal and dermal layers beneath it are hydrophilic, exhibiting opposite characteristics. This structural complexity necessitates tailored exosome delivery strategies based on the target skin layer. Since the skin microbiome resides mainly in the stratum corneum, exosomes interacting with it must remain on the surface. Meanwhile, keratinocytes (forming the skin barrier) and melanocytes (involved in pigmentation) are located at the basement membrane, while fibroblasts in the dermis produce collagen at the skin's deepest layer. Thus, precise exosome delivery is essential to maximize therapeutic efficacy.

Methods : We optimized exosome delivery to specific skin layers by modifying the exosome surface, softening the skin barrier using a negative pressure mechanism, and enhancing skin retention with adhesive polymers. The localization of active ingredients within artificial skin was confirmed through fluorescence imaging using the Franz cell diffusion assay.

Results : This study engineered lactobacillus‐derived exosomes and developed a targeted delivery system using optimized formulations and negative pressure patches. A hybrid exosome system was designed by incorporating cationic modifications, an adhesive polymer network for exosome capture, and a negative pressure patch inspired by octopus suction cups for controlled distribution. Our results showed: For the skin surface, the cationic modification + adhesive polymer approach was most effective. For the epidermis, the negative pressure function + cationic modification + adhesive polymer method provided superior delivery. For the dermis, the negative pressure function + adhesive polymer approach was optimal.

Summary/Conclusion : This study underscores the need for integrated delivery strategies tailored to exosome function and target layer. The findings suggest that this hybrid exosome system holds promise for skin disease treatment and anti‐aging applications.

Funding : This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HP23C0061).

Exploring syntenin‐syndecan biosynthetic approaches for extracellular vesicle (EV)‐based delivery

Lukas Hyka 1 , Sofie Meeussen 1 , Sylvie Thuault 2 , Patrick Chames 3 , Guido David 1,2 , and Pascale Zimmermann 1,2

1 Laboratory for Extracellular Vesicle Research, Department of Human Genetics, Katholieke Universiteit Leuven, Leuven, Belgium. 2 Centre de Recherche en Cancérologie de Marseille (CRCM), Equipe Labellisée Ligue 2018, CNRS, Inserm, Institut Paoli Calmettes, Aix‐Marseille Université, Marseille, France. 3 Centre de Recherche en Cancérologie de Marseille (CRCM) CNRS, Inserm, Institut Paoli Calmettes, Aix‐Marseille Université, Marseille, France

Introduction : Modified EV‐based therapies are limited by the low efficiency of cargo loading and the large quantities of EVs necessary to observe desired effects. Here we investigate whether syntenin‐fusion to a cargo may help its efficient (i) loading inside of small (s)EVs and (ii) delivery to the cytosol of recipient cells. Additionally, we question whether coating the surface of such sEVs with a molecular address (e.g., fusion of a nanobody to Syndecan1‐C‐terminal fragment (SDC1‐CTF)) can improve uptake by target recipient cells.

Methods : EVs were collected from genetically modified HEK293 cells and fractionated by differential ultracentrifugation, size‐exclusion chromatography or collected from concentrated conditioned medium. MCF‐7 or Panc1 cells were used as EV recipient cells. Split GFP and split nanoluciferase assays were set up to quantify the cytosolic delivery of the syntenin‐fusion construct. EV loading and uptake were measured by Western blot, microscopy and chemiluminescence assays. Cas9 recombination efficiency was measured in T7 endonuclease assay.

Results : We show that syntenin‐fusion to Cas9 enhances sorting of Cas9 (internal sEV cargo) to sEVs and does not affect Cas9 functionality or specificity. We establish that syntenin loaded sEV uptake by recipient cells is saturable (time and dose). We document that syntenin supports efficient release of EV cargo into the cytoplasm of recipient cells (at least 30% of the total internalized dose). We document that SDC1‐CTF fusion supports efficient nanobody sorting into sEVs and that sEVs displaying αEGFR nanobody are better taken up by EGFR expressing recipient cells (at saturating dose).

Summary/Conclusion : In short, our study indicates that syntenin‐fusion supports efficient sEVs cargo loading and delivery. It also invites us to better understand how to increase the internalization of syntenin‐dependent sEVs for better EV‐engineering.

Funding : FWO and KU Leuven Internal Funds (Belgium)—Label Ligue contre le Cancer (France).

Nanophotonic liquid biopsy of circulating small extracellular vesicles for hepatocellular carcinoma detection

Presenter: Wei‐Chuan Shih

University of Houston, USA

Introduction : Recently, our research group has developed PlAsmonic NanO‐apeRture lAbel‐free iMAging (PANORAMA), a technique to detect individual nanoparticles as small as 25 nm using an array of gold nanodisks on an invisible substrate (AGNIS). (1) PANORAMA enables the counting, sizing, and localization of individual sEVs in both healthy individuals and cancer patients across various stages and types by utilizing AGNIS functionalized with antibodies against exosomal surface proteins CD9, CD63, and CD81. (2) By quantifying a portion of sEVs from 20 µL blood plasma, PANORAMA can differentiate 205 cancer patients and 106 healthy individuals with a sensitivity of 99.5% and a specificity of 97.3%. (3) In this study, we report new results towards a hepatocellular carcinoma (HCC) test.

Methods : We have analysed samples of three donor types: healthy plasma donors (HPD), non‐cancer disease patients (NCD) (including cirrhosis, fatty liver, etc.), and cancer patients (CP) (including liver cancer and cholangiocarcinoma). A test set of 76 samples was analysed, comprising 13 HPD, 21 NCD, and 42 CP. Among them, the sEV counts are 156±55 (HPD), 370 ± 130 (NCD), and 458 ± 100 (CP). Using the previously obtained sEV count threshold value of 280 results in a cancer detection sensitivity of 100% and a specificity of 55%. The moderate specificity is a direct consequence of 15 false positives from the NCD samples.

Results : To enhance specificity, we have next explored known molecular biomarkers such as cargo microRNAs (miRNAs): miR‐126‐3p, miR‐222‐3p, miR‐31, miR‐21, and miR‐221. Using a standard molecular beacon probe design (3′ BHQ2 quencher/5′ Cy3), we have analysed another sample set consisting of 80 samples (NCD and CP) in a blinded manner, yielding a sensitivity of 97%, a specificity of 95% (3 false positives and 1 false negative), a positive predictive value (PPV) of 91%, and a negative predictive value (NPV) of 98%. ROC analysis for the blinded set yielded an AUC of 0.94.

Summary/Conclusion : Our study underscores the potential of PANORAMA in quantifying and profiling sEVs for HCC detection. The test runs on 20 µL of plasma without any additional sample preparation or isolation and thus is highly suitable for routine tests and point‐of‐care applications.

Funding : National Institute of Health R01EB030623

Direct multi‐dimensional imaging of individual extracellular particles (EPs) enables the categorization of subtypes and the characterization of distinct biophysical properties and behaviours across different subtypes

Presenter: Simou Sun

Stony Brook University, USA

Introduction : Cells release extracellular particles (EPs) as part of their normal physiology and during acquired abnormalities. EPs are recently recognized as important mediators of non‐contact intercellular communication, and EP‐based therapeutic tools and diagnostic assays are considered to have high potential. The specific content of individual EPs depends on multiple factors, such as their biogenesis pathways and donor cell type and status. This significant intrinsic EP heterogeneity impedes analysis and understanding of their composition and functions.

Methods : We employ multidimensional fluorescent labelling on EPs, leveraging the robustness of newly developed small‐molecule fluorophores that can specifically target molecular features on EPs (such as the lipid bilayer, luminal metabolites, and surface receptors). We conduct total internal reflection fluorescence (TIRF) microscopy on either single‐EP arrays or on tracking diffusing single EPs at a two‐dimensional surface. The static single‐EP arrays were anchored to a soft material functionalized surface with little bias, and the diffusing EPs were interfaced with synthetic lipid membranes. We then develop an enhanced algorithm for colocalization analysis of the multiple labels on individual EPs. This analysis not only allows deep profiling of particle content but also performs quantitative characterization on the diffusive behaviour of individual EPs.

Results : We first categorize EPs derived from the same cell type into seven distinct subpopulations—some vesicular whereas others non‐vesicular, and we demonstrate that EPs from four cell types exhibit quantitatively distinguishable subpopulation distributions. Furthermore, we gain insights into specific particle features within each subpopulation, including CD63 counts, relative particle size, relative concentration of cargoes, and correlations among different cargoes. This high‐content analysis reveals common cargo sorting features in EP subpopulations across different cell types and suggests new statistics within the EP inherent heterogeneity that could differentiate EPs from two types of cancer cells and two types of normal cells. Additionally, we discuss how different EP subpopulations exhibit distinct biophysical properties and dynamic behaviour when interacting with synthetic lipid membranes.

Summary/Conclusion : Collectively, our study presents a robust imaging‐based single‐EP characterization platform, combining spatial and temporal imaging with high‐content analysis. This platform is poised to advance EP‐based theranostic assays and facilitate exploration into disease‐associated EP biogenesis and EP‐mediated intercellular communication.

‘Cassette‐like’ extracellular vesicle‐modification technology using membrane curvature‐sensing peptides for intracellular delivery

Presenter: Miho Nakabo

Osaka Metropolitan University, Japan

Introduction : Extracellular vesicles (EVs, exosomes) are secreted by almost all cells constituting a living organism. Due to their pharmacological advantages, EVs have been highly expected to be next‐generation drug carriers in DDS [1,2]. Meanwhile, there are urgent problems to be improved, especially in the low efficiency of cellular uptake of EVs [1,2]. As a bacterial vesicular marker, the curvature‐sensing peptide, nFAAV5 was developed with inspiration from the BAR protein [3]. nFAAV5, which has a helix structure, binds to vesicles less than 100 nm in diameter [3]. Because of the diameter ranges of EVs (approximately 30∼200 nm), nFAAV5 was considered to bind to the surface of EV membranes. We also synthesized a linked peptide of the nFAAV5 and octaarginine (R8) that is one of the representative cell‐penetrating peptides for macropinocytosis induction and increased cellular uptake (nFAAV5‐R8) [4].

Methods : All peptides were synthesized by Fmoc‐solid phase methods. Isolated EVs secreted from CD63‐GFP‐expressing cells were used for visualization of their cellular uptake after the peptide modification on the EVs.

Results : Internalization of EVs by cells was evaluated using a confocal laser microscope and a flow cytometer, and we confirmed increased cellular uptake of nFAAV5‐R8‐modified EVs. Meanwhile, in the case of the non‐crosslinked nFAAV5 and R8, cellular uptake of EVs was not significantly altered. nFAAV5‐R8‐modified EVs were observed using a cryo‐TEM system, and the vesicular morphology was maintained. Furthermore, the application of this technology to boron neutron capture therapy (BNCT) [5] demonstrated effective uptake of boron (10B) compounds and enhanced cancer cell killing activity by thermal neutron irradiation, suggesting its high potential for drug delivery in future therapy.

Summary/Conclusion : The developed technique for ‘cassette’‐like modification of functional peptides on EV membrane will be widely applicable in EV‐based molecular delivery system and controlled cellular communications. [1] I. Nakase, Processes, 9, 224 (2021), [2] I. Nakase, T. Takatani‐Nakase, Drug Metab., Pharmacokinet., 42, 110435 (2022), [3] K. Kawano, et al., Chem. Pharm. Bull., 69, 1075 (2021), [4] S. Futaki, I. Nakase, Acc. Chem. Res. 50, 2449 (2017), [5] S. Hirase, et al., Mol. Pharmaceutics, 19, 4, 1135 (2022)

ISEV2025 Poster Presentations

Poster Session 1 Disease and Therapy

Sub‐topic Biomarker 1

The unreferenced transcriptome as a source of robust biomarkers of prostate cancer in urinary extracellular vesicles

Anna Almeida 1 , Marc Gabriel 1 , Virginie Firlej 2,3 , Nicolas Vogt 1 , Lorena Martin‐Jaular 1 , Matthieu Lejars 1 , Marie‐Emmanuelle Legrier 1 , Katia Le Dudal 2 , Damien Destouches 3 , Francis Vacherot 3 , Raphaele Arrouasse 2 , Xavier Paoletti 1 , François Rozet 4 , Philippe Lecorvoisier 2 , Yves Allory 1 , Alexandre de la Taille 2 , Clotilde Théry 1 , Antonin Morillon 1

Anna Almeida and Marc Gabriel are the co‐first authors.

1 Institut Curie, France, 2 Henri Mondor Hospital, France, 3 University of Paris‐Est Créteil, France, 4 Institut Mutualiste Montsouris, France .

Introduction : Prostate cancer (PCa) is the second most common cancer in men worldwide nevertheless, current diagnostic tools for PCa are insufficient and invasive. In fact, only biopsies allow us to conclude that there is prostate cancer and not other diseases, such as prostatitis or benign prostatic hyperplasia (BPH). Early and non‐invasive diagnosis is therefore necessary. RNAs associated with extracellular vesicles (EVs) have attracted a lot of attention these last few years for being ideal candidates for cancer biomarkers in liquid biopsies. During palpation of the prostate by digital rectal examination (DRE), PCa cells and EVs, can be released from prostatic ducts into the urethra, and end up in urine. Urine containing EVs secreted from prostate tumour could be an excellent source of novel PCa biomarkers.

Methods : Following prostate massage during DRE, we collected urine from two independent cohorts of patients after informed consent was obtained. The discovery cohort named HOPE (70 patients) and the validation cohort (130 patients) were composed of prostate cancer, BHP and healthy patients. We performed stranded total RNA‐sequencing on uEVs from all samples. We applied reference‐free analysis of differential k‐mers, with a 5x5‐fold selection following machine learning, to detect all transcriptional events characterizing the PCa condition in contrast with healthy or BPH conditions. We compared this approach to standard annotated gene expression analysis.

Results : We revealed the distinct RNA cargo types present in uEVs of the three groups of patients. Several types of RNAs, including mRNA, circRNA, and lncRNA, were detected in each sample from urine. Additionally, using reference‐free analysis of differentially expressed k‐mer contigs, we could find a signature to discriminate PCa from healthy and BHP patients.

Summary/Conclusion : In summary, these results highlighted the advantage of the reference‐free analysis over the standard analysis in generating a transcriptomic signature that discriminated PCa patients from healthy and BPH patients, based on their urinary EV RNA cargo. These candidate RNA biomarkers could be potentially useful for non‐invasive prostate cancer diagnosis and active surveillance.

Funding : Institut Curie; ERC‐PoC; ICGEX; AFER; Malakoff‐Humanis; PSL

Subtopic Biological activity and therapeutic potency of EVs

Immunomodulatory potential of extracellular vesicles from Trichinella spiralis muscle larvae

Sofija Glamočlija 1 , Ljiljana Sabljić 1 , Anna Schmid 2 , Alisa Gruden‐Movsesijan 1 , Nataša Radulović 3 , Jelena Đokić 4 , Irma Schabussova 2 , Maja Kosanović 1

1 Institute for the Application of Nuclear Energy‐INEP, University of Belgrade, Serbia, 2 Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Austria, 3 Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia, 4 Institute of Molecular Genetics and Genetic Engineering, IMGGE, University of Belgrade, Serbia

Introduction : Helminth parasite Trichinella spiralis has evolved to modulate the immune system of the host by its larvae excretory‐secretory products (ES L1), that is, reduce hyperreactivity to both allergens and autoantigens. After discovering EVs in ES L1 (TsEVs), we aimed to investigate whether TsEVs exert immunomodulatory properties similar to whole ES L1.

Methods : TsEVs were enriched from ES L1 by a combination of ultracentrifugation and ultrananofiltration and characterized by electron microscopy (TEM), western blot and NTA. Immunomodulatory properties of TsEVs were tested on PBMC, dendritic cells (DCs) and in a murine model of ovalbumin (OVA)‐induced allergy. TsEVs interaction with target cells was investigated in DCs and TLR2/4‐transfected HEK293 cells. Signalling pathways were investigated by PCR.

Results : Enriched TsEVs were of 30‐80 nm and contained two glycoproteins characteristic of muscle larvae of the genus Trichinella. PBMCs treated with TsEVs had elevated production of IL‐10 and IL‐6, and decreased production of IL‐17. DCs treated with TsEVs exhibited a stabile tolerogenic phenotype and increased production of IL‐10 and TGF‐β. Such DCs polarized the immune response of T cells in co‐culture towards Th2 and the regulatory type. In the allergy model, intranasal administration of TsEVs caused a decrease in eosinophils, macrophages and NK cells in the lungs as well as OVA‐specific IgE in the sera. In lungs, there was an increase of CD103+ DCs, CD4+Foxp3+ Tregs and IL‐10‐producing Tregs, while CD11b+Ly6C+ cells were reduced. Cultivated immune cells from lungs and spleens of TsEVs‐treated mice exhibited lower production of the Th2 cytokines IL‐4, IL‐5 and IL‐13 and increased production of IL‐10. Pattern‐recognition receptors, DC‐SIGN and TLR2/4, were found to be involved in the interaction of TsEVs with target cells. TsEVs activated multiple signalling pathways in DCs.

Summary/Conclusion : TsEVs exhibit immunomodulatory properties which could be harnessed to design novel therapeutics for inflammatory diseases.

Funding : Funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia, Co. No. 451‐03‐66/2024‐03/200019 and No. 337‐00‐577/2021‐09/41; the Innovation Fund of the Republic of Serbia Grant No. 5906; the Government of Lower Austria project Danube‐Allergy Research Cluster (P17); the FWF project P 34867; and the OEAD projects CZ 04/2024, CZ 07/2023, CZ 15/2023, and RS 08/2022.

Delivery of apelin‐loaded extracellular vesicles for the treatment of pulmonary arterial hypertension

Presenter: Jihong Kim

Korea Institute of Science and Technology, Republic of Korea

Introduction : Pulmonary arterial hypertension (PAH) is a rare, life‐threatening disease characterized by vascular remodelling and increased pulmonary vascular resistance, leading to right heart hypertrophy. Despite its severity, currently approved drugs for PAH primarily provide symptomatic relief to patients. Apelin, an endogenous ligand for the APJ receptor, is a promising candidate for PAH due to its vasodilatory and pro‐angiogenic properties. However, clinical use of apelin in PAH is limited by its lack of selectivity for the pulmonary vasculature and its rapid degradation in blood plasma. To overcome this limitation, we engineered extracellular vesicles (EV) displaying apelin‐13 on their surface, which accumulate in the lungs and provide therapeutic effects against PAH.

Methods : EVs were collected from HEK293T cells and isolated following the MISEV2023 guidelines. The vasodilatory and pro‐angiogenic effects of Apelin EVs were evaluated in HUVECs. To track lung accumulation, apelin‐EVs labelled with Cy5.5 were injected into monocrotaline (MCT)‐induced PAH rats, and IVIS imaging was used to monitor EV distribution. The therapeutic potential of apelin‐EVs was tested in both the MCT‐PAH model and the Sugen5416/hypoxia (Su/Hx) PAH model.

Results : Results showed that apelin‐EVs activated endothelial nitric oxide synthase (eNOS) protein and enhanced endothelial cell proliferation and migration in scratch assay. Apelin‐EVs accumulated in lungs of MCT‐PAH and demonstrated significant therapeutic effects by reducing right ventricle hypertrophy, lowering vascular occlusion and decreasing heart fibrosis. Echocardiographic measurements, including PAT/PET, TAPSE, RV free wall thickness, and cardiac output, were improved as well. Additionally, Apelin EVs lowered mean pulmonary arterial pressure in Su/Hx‐PAH mice.

Summary/Conclusion : In conclusion, apelin‐loaded EVs successfully accumulated in the lungs and mitigated the effects of pulmonary arterial hypertension, highlighting their therapeutic potential in PAH treatment.

Extracellular vesicles from second trimester human amniotic fluid as candidate therapeutics against skeletal and cardiac muscle injury

Presenter: Laura Guerricchio

University of Genova, Italy

Abstract unavailable

Effect of EVs on systemic herpes infection in mice

Presenter: Vitalii Kordium

National Academy of Sciences of Ukraine, Ukraine

Introduction : Despite extensive research on EVs, their effect on viral diseases (except COVID‐19) remains unstudied. We have conducted pilot studies of the impact of EVs, secreted by human Wharton's jelly mesenchymal stem cells (MSCs), on systemic damage in mice of the BALB/c line by herpes simplex virus, type 1 (HSV‐1).

Methods : Virological, cytological, histological, and cellular methods were used. MSCs were isolated from the umbilical cord taken with the patient's informed consent. Work with animals complied with standards for conducting scientific experiments on animals, approved by the MESU.

Results : Experimental mice were infected by intracranial administration of HSV‐1. The next day, EVs were administered in the animals' tail veins in doses equivalent to the number of MSCs, ranging from 50 to 600 thousand per animal. The controls were: (1) mice infected with the HSV‐1, without administration of EV; (2) intact animals; (3) intact mice, injected with the maximum dose of EVs simultaneously with the experimental ones. As the disease progressed, the brains and internal organs of sick, dead, and recovered animals were studied. As a result, an inverse relationship was found between the administered EV dose and the mortality/survival of mice. The highest EVs doses led to a mortality increase (compared to the control). Mortality decreased as the EV doses decreased. Low doses showed an almost complete cure effect. In control group 1, the course of the disease and mortality were standard for the HSV‐1 infection. In control groups 2 and 3, there were no external negative signs. Virological studies have shown that the highest dose of EVs enhances the reproduction of the HSV‐1 in the diseased organism and increases the viral load compared to control 1 and to animals treated with lower doses of EVs. Histological studies have shown that systemic infection of an organism with HSV‐1 leads to damage to all internal organs. The administered EVs modify the degree and nature of damage. This is especially pronounced in the brain, lungs, liver, and spleen.

Summary/Conclusion : EVs administration for viral infection treatment causes dose‐dependent effects that differ from those in non‐infectious pathologies. The mechanisms that determine such differences require detailed study.

Extracellular vesicles from mesenchymal stem cells reverse neuroinflammation in the cerebellum and restore motor coordination in hyperammonemic rats

Paula Izquierdo‐Altarejos 1,2 , Victoria Moreno‐Manzano 3 , Carmina Montoliu 1,4 , Vicente Felipo 2

1 INCLIVA Biomedical Research Institute, Spain, 2 Laboratory of Neurobiology, Principe Felipe Research Centre, Spain, 3 Neuronal and Tissue Regeneration Laboratory, Príncipe Felipe Research Centre, Spain, 4 Pathology Department, University of Valencia, Spain

Introduction : Cirrhotic patients may show minimal hepatic encephalopathy (MHE), with mild cognitive impairment and motor deficits. Hyperammonemia and inflammation are the main contributors to the cognitive and motor alterations of MHE. Hyperammonemic rats reproduce these alterations. There are no specific treatments for the neurological alterations of MHE. Extracellular vesicles from mesenchymal stem cells (MSC‐EVs) are promising for treating inflammatory and immune diseases. We aimed to assess whether treatment of hyperammonaemic rats with MSC‐EVs reduced neuroinflammation in the cerebellum, restored motor coordination, and to study the mechanisms involved.

Methods : The effects of MSC‐EVs were studied in vivo by intravenous injection to hyperammonaemic rats and ex vivo in cerebellar slices. Motor coordination was analysed using the beam walking test. Effects on neuroinflammation were assessed by immunohistochemistry, immunofluorescence and Western blot.

Results : Injection of MSC‐EVs reduced microglia and astrocyte activation in the cerebellum and restored motor coordination in hyperammonaemic rats. Ex vivo experiments show that MSC‐EVs normalize pro‐inflammatory factors, including TNFα, NF‐kB activation and the activation of two key pathways leading to motor incoordination (TNFR1‐NF‐kB‐glutaminase‐GAT3 and TNFR1‐CCL2‐BDNF‐TrkB‐KCC2). TGFβ in the EVs was necessary for those beneficial effects.

Summary/Conclusion : In summary, MSC‐EVs treatment reverses neuroinflammation in the cerebellum of hyperammonaemic rats and the underlying mechanisms leading to motor incoordination. Therapy with MSC‐EVs may be useful to improve motor function in patients with MHE.

Funding : This work was supported in part by Ministerio de Ciencia e Innovación Spain (PID2020‐113388RB‐I00; AEI/10.13039/501100011033); Consellería de Educación Generalitat Valenciana (CIPROM 2021/082); and European Regional Development Funds/ERDF (PID2020‐113388RB‐I00 and CIPROM 2021/082). Part of the equipment employed in this work was funded by Generalitat Valenciana and co‐financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014‐2020).

Immunomodulation of mesenchymal stem cell‐derived EVs on B cells

Pamina Contreras‐Kallens 1 , Hannah Kimingi 1 , Yeyu Shen 1 , Hannah Aris 1, Dessi Malinova 2, Meadhbh Brennan 1

1 University of Galway, Ireland, 2 Queen's University, Belfast

Introduction : Autoimmune diseases (ADs) are characterized by B cell function dysregulation. Novel therapies such as antibody‐mediated B cell depletion have significant side effects. Mesenchymal stem cells (MSCs) have been proposed as an alternative therapy for ADs, as these cells bear potent immunomodulatory capacities, particularly when exposed to inflammatory conditions known as ‘licensing’. MSCs‐derived extracellular vesicles (EVs) have been proposed as a safer alternative to MSCs. The aim of this study was to evaluate the effect of EVs derived from licensed and unlicensed MSCs on B cell function and activation.

Methods : Bone marrow murine MSCs (BM‐MSCs) were cultured on tissue culture plastic in EV‐depleted media with IFN‐γ and TNF‐α to ‘license’ them, or in standard conditions. MSCs‐EVs were isolated from the conditioned media by size exclusion chromatography. EVs samples’ particle and protein concentrations were measured by NTA and BCA, respectively. For assessing the effect of EVs on B cells, primary B cells were isolated from an allogenic strain mouse and activated in vitro. The effect of licensed and unlicensed EVs on the expression of activation surface markers and proliferation was measured by spectral flow cytometry.

Results : The presence of pro‐inflammatory cytokines in the media increased the expression of both constitutive markers, such as CD73, and inducible markers, like MHCII, on the BM‐MSCs. These cells were also capable of secreting more EVs per cell, and these EVs contained a higher content of protein per particle compared to unlicensed EVs. BM‐MSCs‐derived EVs could modulate the expression activation markers CD69, CD24 and PD‐L1 on allogeneic murine activated B cells. The EVs capacity of modulating B cell proliferation was also studied.

Summary/Conclusion : Our findings demonstrate that BM‐MSC‐derived EVs can modulate B cell activation and function in vitro. Moreover, our study highlights the influence of MSCs' culture conditions on EV effects, suggesting potential therapeutic versatility in diseases characterized by immune dysregulation, such as ADs. Further elucidation of the underlying mechanisms is crucial to fully harness the clinical potential of MSCs‐derived EVs as immune therapies.

Funding : European Research Council (ERC) Starting Grant #852152 and Higher Education Authority (HEA) Ireland funding through the North‐South Research Program (ExoBcell project).

MSC‐EVs for Schwann cell modulation across multiple phases of peripheral nerve repair

Presenter: Maximilian Haertinger

Medical University of Vienna, Austria

Introduction : The intrinsic injury response of the peripheral nervous system allows for limited regeneration following axonal disruption. However, large‐scale nerve defects exceed these regenerative capabilities and remain a challenge in reconstructive surgery. At the cellular level, Schwann cells (SCs) were identified as the drivers of nerve repair and axonal guidance and are targeted by novel therapeutic applications. As SCs progress through the stages of nerve repair, they take on numerous roles. In this project, we investigate how adipose stromal cell EVs (ASC‐EVs) interact with and modulate SCs throughout the injury response.

Methods : EV interactions with the SCs were qualitatively and quantitatively assessed. Uptake dynamics were investigated with live cell imaging, imaging flow cytometry and pharmacological inhibitors of known internalization pathways. The intracellular fate of ASC‐EVs was visualized at a single molecule level using direct stochastic optical reconstruction microscopy (dSTORM). Protein and miRNA EV‐cargo were characterized and correlated to protein, miRNA and mRNA expression of EV‐stimulated SCs at various phases of nerve repair. Subsequently, validation experiments were performed to validate EV influence on modulated pathways and processes.

Results : ASC‐EVs were able to influence SC behaviour during all phases of peripheral nerve repair. In the initial phase, EVs mediate myelin clearance and aid in establishing a pro‐regenerative environment. This is followed by a time‐ and dose‐dependent effect on SC proliferation and migration. Lastly, the redifferentiation and remyelination of regrown axons may be influenced by ASC‐EV stimulation.

Summary/Conclusion : Our findings suggest that ASC‐EVs can influence SCs and aid in peripheral nerve regeneration; however, the level of support varied between phases. Hence, it is crucial to further test and verify the optimal timepoint, dose and route of administration. In addition, we identified various off‐target effects that need to be taken into consideration when further developing ASC‐EVs as a therapeutic option for peripheral nerve regeneration. While our results provide a promising step towards EV treatment in this challenging field of regenerative medicine, we also identified a further need for testing to ultimately decide if ASC‐EVs really are the Jack of all trades or masters of none.

Exploring the biodistribution of p53‐containing EVs for targeted cancer therapy

Ishai Luz 1 , Gil Shalev 2 , Doaa Massad 1 , Tomer Cooks 1 , Alexander Tendler 2

1 Ben‐Gurion University of the Negev, Israel; 2 AnserBio Ltd., Israel

Introduction : Extracellular vesicles (EVs) have emerged as promising tools for targeted delivery of therapeutic agents. EVs derived from corneal epithelial cells (CECs) are of particular interest due to their unique properties. These EVs contain the tumour suppressor protein p53 and play a crucial role in the local anti‐cancer defence mechanisms of the cornea. This study aims to investigate the biodistribution of CECs EVs containing wild‐type p53 in mice, and to validate their potential for targeting specific organs and tumours, laying the groundwork for future therapeutic applications.

Methods : EVs were harvested from CECs and purified using an IZON qEV10 35 nm column. Characterization was performed using nanoparticle‐tracking analysis (NTA). EVs were then fluorescently labelled with DiR and injected intravenously (iv) into athymic nude or C57Bl/6 mice (∼1 × 10 12 EVs/mL in 100 ul per animal). The biodistribution was monitored using in vivo imaging (NEWTON 7.0 real‐time imager) at several time points post‐injection (0.5, 1, 4, 24, and 48 h).

Results : We found that EVs predominantly accumulate in the liver, lungs, and spleen shortly after injection, with lower presence in the brain and the heart. This effect continued with substantial accumulation in those organs at 24 and 48 h. Injections of diluted amounts of EVs showed consistent biodistribution patterns, validating that EV staining is dose‐dependent and restricted to the blood circulatory system. Both the heart and brain exhibited a lower amount of staining across all experimental conditions, yet it was still higher than that of the dye‐only control. Preliminary results from intranasal delivery of EVs revealed accumulation in the lungs and brains of the treated mice 24 h post‐injection.

Summary/Conclusion : This study provides evidence that EVs derived from CECs can be efficiently delivered to specific organs in mice. The consistent biodistribution patterns and effective targeting highlight the potential of these EVs as a delivery vehicle of p53. Future research should focus on optimizing EV formulations and further exploring their therapeutic efficacy in various cancer models and tumour‐bearing mice. These findings pave the way for developing p53 replacement therapies targeting challenging cancer types harbouring inactivated or mutated p53, leveraging the natural targeting abilities of EVs for precision medicine. Funding: ExoProTher Medical Ltd.

Retinal transcriptome and vitreous extracellular vesicles delineating pathways involved in retinopathy of prematurity (ROP)

Presenter: Neha Sharma

L V Prasad Eye Institute, India

Introduction : Retinopathy of prematurity (ROP), a major ocular complication of preterm birth, is characterized by abnormal angiogenesis and formation of FVM (fibrovascular membrane) at the vitreo‐retinal junction, causing retinal detachment and blindness. Herein, we examined retinal gene profiling and extracellular vesicles, elucidating their involvement in disease pathogenesis.

Methods : Retinal transcriptome analysis was performed using the microarray platform, and pathway analysis was performed using GeneSpring GX (ROP = 3, controls = 3). EVs were isolated and characterised using NTA, TEM and western blotting. Further, small RNAseq was performed in vitreous humour (ROP = 8, control = 7), and validations were done by qPCR.

Results : 201 genes were significantly differentially regulated between the ROP retina and control retina (FC: ±1.5, p value < 0.05). Angiogenesis, retinal neural development, the complement pathway, macrophage activation, gliosis, and photoreceptor development were the top significant pathways. Interestingly, extracellular vesicle processes were seen among the significant ( p value = 1.429 × 10 −2 ) pathways in the retinal transcriptome. Earlier studies from our lab demonstrated the involvement of active microglia cells in ROP pathogenesis. EVs released from retinal cells are known to diffuse to vitreous humour. From our EVs small RNA sequencing data, 883 miRNAs were identified, of which 739 showed differential expression. Among these, 9 miRNAs exhibited significant upregulation, while another 9 were notably downregulated. Pathway analysis revealed an enrichment of inflammation, angiogenesis and development related pathways in the EVs of ROP subjects.

Summary/Conclusion : A significant involvement of inflammation, immune activation, and angiogenesis in ROP was seen in the retinal transcriptome and further confirmed by extracellular vesicle profiling. Further functional investigation could help in delineating their involvement in exacerbation/alleviation of ROP progression.

Funding : Department of Science and Technology Innovation in Science Pursuit for Inspired Research (INSPIRE), Government of India (IF190699).

Harnessing the angiogenic potential of stem cell‐derived EVs: therapeutic application in a preclinical model of neonatal pulmonary vascular disease

Presenter: Hala Saneh

University of Connecticut, USA

Introduction : Stem cells were shown to have a regenerative and angiogenic potential without engrafting in host tissues. It has been suggested that paracrine mechanisms mediate these therapeutic effects. We aim in this study to evaluate the angiogenic potential of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) and human induced pluripotent stem cells (hiPSCs). This evaluation is performed at a molecular level by using an in vitro angiogenesis assay and a preclinical model of neonatal pulmonary vascular disease (PVD).

Methods : MSC‐ and hiPSC‐EVs were isolated by ultrafiltration and size‐exclusion chromatography. EVs were then characterized using transmission electron microscopy, imaging flow cytometry and nanoparticle tracking analysis. Mass spectrometry and RNA sequencing were performed to evaluate their proteomic and genomic content and match it to pathways known to drive angiogenesis in early lung development. In vitro angiogenesis potential was assessed by adding EV preparations to human umbilical vein endothelial cells (HUVECs) cultured over basement membrane matrix. EVs were also administered intra‐tracheally to animal models of premature lung injury and PVD. The model was developed by exposing rats to antenatal inflammation (maternal lipopolysaccharide) and postnatal high oxygen levels (75% O 2 for 14 days). PVD was then assessed on day of life 28 using echocardiography to look for indirect signs of pulmonary hypertension, VEGF gene expression within digested lung tissues, and alpha smooth muscle actin staining of lung sections.

Results : MSC‐ and hiPSC‐EVs carry proteins involved in angiogenesis during early lung development. Identified pathways include VEGF, HIF1α and eNOS signalling pathways. RNA sequencing studies are ongoing. The HUVEC angiogenesis assay showed a significant improvement in the density of tube‐like structures with EV supplementation compared to control wells. A concentration of 1 × 10 11 particles per mL of media yielded the best result. The effects of EVs in animal models of preterm lung injury and associated PVD are currently being evaluated.

Summary/Conclusion : This study highlights the angiogenic potential of MSC‐ and hiPSC‐derived EVs and paves the way for the use of stem‐cell free translational therapeutic applications in pulmonary vascular disease associated with prematurity.

Funding : This study is funded by the Connecticut Children's Connection Grant awarded by the Connecticut Children's Foundation.

Assessment of endogenous procoagulant potential and extracellular vesicles (EVs) in thermally injured individuals

Presenter: Maria Cristina Bravo

University of Vermont, USA

Introduction : Severe thermal injury is associated with local and systemic responses, including the release of EVs from damaged or stressed cells and coagulopathy. Here we report on procoagulant potential and EVs in plasma from thermally injured individuals.

Methods : Patients ( n = 28) with varying burn severity (total body surface area, %TBSA: 39% ± 18%) were enrolled prospectively in an IRB approved study upon admission. Blood was collected, platelet poor plasma prepared within 30 min, and aliquots frozen at −80°C. For all analyses plasma was thawed in the presence of corn trypsin inhibitor to prevent contact pathway activation. Endogenous thrombin generation (TG) potential was assessed using the standardized calibrated automated thrombogram protocol, reagents (MP‐phospholipid only), and instruments (Stago); parameters of lag time and peak thrombin were extracted. To isolate EVs, freshly thawed plasma was centrifuged at 1500 × g for 15 min and 0.5 mL of supernate plasma was loaded onto an Izon Gen2 8.5 mL 70 nm agarose gel column and gravity eluted with HBS. EV isolates were defined by a purified collection volume of 2.9–4.9 mL after sample loading. Plasma concentrations of functional tissue factor (TF) and surfaces expressing phosphatidylserine (PS) were established using kinetic assays measuring the capacity of isolates to support extrinsic tenase and prothrombinase assembly and function, respectively. Quantification was based on standard curves using titrations of commercial sources of TF or PS‐bearing vesicles. Total EV (TotEV) and Intermediate sized EV (100–500 nm, IntEV) concentrations were determined in EV isolates using an Izon qNano Gold instrument. Data are presented as mean ± SD; measures were compared using Pearson correlations (significance: p ≤ 0.05).

Results : 27 of 28 individuals had endogenous TG, with an average lag time of 16.8 ± 7.1 min and peak thrombin of 124 ± 66 nM. Samples had a TotEV concentration of 1015 ± 1074 × 10 9 /mL, with approximately 20% being IntEV (148 ± 176 × 10 9 /mL). Functional assays measured 60.9 ± 64.0 fM TF and 669 ± 539 nM PS in collected samples. TBSA (%) correlated with TF ( r = 0.454) and IntEV ( r = 0.409) levels but not with TG parameters or PS levels. TF correlated with IntEV ( r = 0.507) and strongly with PS ( r = 0.722).

Summary/Conclusion : Thermal burn severity was associated with increased levels of both TF‐bearing vesicles and EVs of intermediate size, but not endogenous TG potential.

Extracellular vesicles derived from the probiotic Propionibacterium freudenreichii modulate inflammatory response via activation of C‐Jun and NFκB transcription factors in host cells

Presenter: Juliana Guimaraes Laguna

INRAE, France

Introduction : Propionibacterium freudenreichii is a Gram‐positive dairy bacterial species with beneficial traits, including anti‐pathogenic activity, anticancer potential, gut epithelial barrier protection, and immunomodulatory properties. These probiotic traits have been consistently associated with surface proteins and secreted metabolites, including short‐chain fatty acids and bifidogenic factors. Recently, we showed that extracellular vesicles (EVs) produced by the probiotic P. freudenreichii strain CIRM‐BIA 129 (EV129) mimic the immunomodulatory properties of parent cells in vitro (i.e., modulating NFκB transcription factor activity and LPS‐induced IL‐8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The objective of this study is to investigating the mechanisms of interaction of EV129 and their effects on the immune system modulation through signal transduction on intestinal epithelial HT‐29 cells.

Methods : EV129 was purified from bacterial culture supernatants by size‐exclusion chromatography (SEC) and characterized by NTA and TEM. Their ability to interact with HT‐29 cells and to be internalized was evaluated by flow cytometry and confocal microscopy. The impact of various incubation times, EV doses and inhibitors of the cellular internalization was evaluated. The level of mRNA transcripts involved in the TLR4‐NFκB pathway was determined by RT‐qPCR and interleukin‐8 (IL‐8) release by ELISA. The protein expression was analysed by western blot.

Results : Our findings point out that EV129 is internalized by HT‐29 cells mainly through a dynamin‐dependent endocytosis mechanism. EV129 downregulated TLR4, C‐JUN and NFkB gene expression, leading to a reduction in the expression of the pro‐inflammatory cytokines IL8, IL1b and TNF in inflamed HT‐29 cells. EV129 also downregulated IRF3 and IRF5 gene expression in inflamed HT‐29 cells and upregulated MAPK1 gene expression in the non‐inflamed HT‐29 cells. EV129 seems to activate the transcription factors C‐Jun and NFkB via two different intracellular routes, but how they trigger the activation of these pathways remains unknown.

Summary/Conclusion : This work contributes to demonstrating the promising future of probiotic‐derived EVs such as EV129 as a biotherapeutic tool against inflammatory bowel diseases.

Funding : This research was funded by Universidade Federal de Minas Gerais, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the C16910 /SAD‐Bretagne region fellowship for Accueil de chercheuses et chercheurs internationaux en post‐doctorat and the BACTER‐EV‐BOOSTER project.

Engineering the next generation of biologically active vascular grafts

Justine Clarke 1 , Mara Boumpouli 2 , Manuel Salmeron‐Sanchez 1 , Niall Paterson 2 , Catherine Berry 1 .

1 University of Glasgow, UK; 2 Terumo Aortic, UK

Introduction : Cardiovascular diseases (CVDs) are the leading cause of death globally. The use of vascular grafts has shown great promise in treating CVDs; however, they are still at risk of developing acute thrombosis and intimal hyperplasia over time. Promotion of endothelialisation at the internal wall of the blood vessel will enhance the natural repair process and subsequently limit the formation of scar tissue and restenosis. In collabouration with Terumo Aortic, using their synthetic Polyethylene Terephthalate (PET) endovascular grafts for treatment of abdominal aortic aneurysms (AAAs), mesenchymal stromal cell (MSC)‐derived extracellular vesicles (EVs) will be investigated as a biologically active graft coating.

Methods : MSCs were primed in normal (21% O 2 ) and hypoxic (1% and 5% O 2 ) culture to generate MSC‐EVs with enhanced angiogenic factors. The EVs were isolated (ultrafiltration and size exclusion chromatography) and characterised by size, concentration and cytokine cargo. EVs were then assessed for potential angiogenic effects; human umbilical vein endothelial cells (HUVECs) were cultured on Matrigel with (i) basal media, (ii) vascular endothelial growth factor (VEGF), (iii) endothelial growth media, (iv) normoxic EVs and (v) hypoxic EVs. Following overnight incubation, HUVECs were stained with Calcein AM and analysed for endothelial cell tube formation to determine MSC‐EV angiogenic properties. Finally, PET graft samples were prepared with a proprietary coating to facilitate EV binding via integrins. EVs were then fluorescently stained with CFSE (green) and imaged using confocal microscopy.

Results : Normal and hypoxic MSC‐EVs had a similar size and concentration. Five percent hypoxic EVs promoted angiogenesis (as positive controls), and 1% hypoxia produced some tube formation, whilst normoxic EVs mirrored the negative control. MSC‐EVs successfully adhered to the graft material, with its woven topography manipulating EV distribution across the surface.

Summary/Conclusion : Hypoxic conditions appeared to promote EVs with angiogenic factors, which could be immobilised onto the PET graft material. The angiogenic potential will be further assessed by (i) analysing EV metabolites, proteomics and miRNA and also (ii) analysing HUVEC and peripheral blood mononuclear cell (PBMC) interaction with immobilised MSC‐EVs on graft material.

Funding : EPSRC, LifETIME CDT, Terumo Aortic.

Efficacy of ginger‐derived extracellular vesicles as a novel therapeutic approach for rheumatoid arthritis

Hiroki Kaneta 1 , Tomoyuki Nakasa 2 , Yimiti Dilimulati 1 , Dan Moriwaki 1 , Shigeru Miyaki 1, 3 , Nobuo Adachi 1

1 Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University; 2 Department of Artificial Joints and Biomaterials, Graduate School of Biomedical and Health Sciences, Hiroshima University; 3 Medical Center for Translational and Clinical Research, Hiroshima University Hospital

Introduction : Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint destruction due to inflammation. Despite advancements in pharmacotherapy, complications and inadequate disease control highlight the need for new treatments. Extracellular vesicles (EVs), including exosomes and microvesicles, play a vital role in intercellular communication, carrying proteins, miRNAs, and mRNAs that influence cell function. Plant‐derived EVs offer potential advantages like stability, cost‐effectiveness, and mass production capabilities. Ginger, known for its anti‐inflammatory effects, has demonstrated efficacy in RA by inhibiting the proliferation and migration of synovial fibroblasts. This study aimed to investigate the therapeutic potential of ginger‐derived EVs in RA treatment.

Methods : Ginger‐derived EVs were isolated using ultracentrifugation and characterized for miRNA content. In vitro, the anti‐inflammatory effects were assessed on TNFα/IL‐1β‐stimulated RA synovial fibroblasts (RASFs) by measuring MMP3 and IL‐6 gene expression, cell proliferation (MTT assay), and migration (scratch assay). In vivo, collagen antibody‐induced arthritis (CAIA) was induced in DBA/1J mice, and the effect of orally administered ginger‐derived EVs was evaluated based on arthritis scores, histological assessments (HE, Safranin‐O, TRAP staining), and miRNA profiling. All animal experiments were approved by the relevant animal ethics committee. Statistical significance was set at p < 0.05.

Results : Ginger‐derived EVs significantly suppressed MMP3 and IL‐6 expression, reduced RASF proliferation, and inhibited migration in vitro. In vivo, ginger‐derived EVs markedly reduced arthritis scores, decreased synovitis, inhibited cartilage degradation, and reduced osteoclast activity without adverse effects. Microarray analysis revealed that ginger‐derived EVs contained miRNAs identical to human miRNAs with reported anti‐inflammatory properties.

Summary/Conclusion : This study provides the evidence of ginger‐derived EVs effectively suppressing inflammation and proliferation in RA models, highlighting their potential as a novel, cost‐effective, and scalable therapeutic approach. Our findings suggest that ginger‐derived EVs could represent a new avenue for RA treatment that minimizes adverse effects while leveraging natural anti‐inflammatory properties. Further studies are warranted to evaluate their long‐term stability and to optimize extraction methods for broader clinical application.

Funding : The JCR Grant for Promoting Research for Early RA.

Enhancing the therapeutic potential of Hoffa MSC‐derived extracellular vesicles through differential pre‐conditioning in a vertical wheel bioreactor

Eva Pecháčková 1 , Alexander Otahal 1 , Karina Kramer 1 , Andrea de Luna 1 , Stefan Nehrer 1

1 Center for Regenerative Medicine, University for Continuing Education Krems, Austria

Introduction : Osteoarthritis (OA) is a degenerative joint disease that leads to cartilage breakdown, pain, and reduced mobility. Advances in regenerative medicine have highlighted extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) as a potential therapeutic approach for OA treatment due to their anti‐inflammatory and regenerative properties. However, MSC‐EV efficacy depends on the MSC source and conditioning, underscoring the need for standardized protocols. Different stimuli, such as inflammation and hypoxia, can modify EVs properties. This study investigates the biological potency of EVs derived from Hoffa's fat pad MSCs (HFP‐MSCs) under pre‐conditioning conditions (IL‐1β, TGF‐β3, and hypoxia) in vertical wheel bioreactors to generate EVs with the best regenerative and anti‐inflammatory effects on OA‐affected cells.

Methods : HFP‐MSCs were cultured in vertical wheel bioreactors and pre‐conditioned with IL‐1β, TGF‐β3, or hypoxia until metabolic activity plateaued. The metabolic activity and cell concentration were measured throughout the incubation period. The characterization of MSCs was evaluated by flow cytometry and trilineage differentiation. EVs were isolated from conditioned media using ultrafiltration and characterized for size and concentration by NTA. Surface markers such as CD9, CD63, and CD81 were analysed by flow cytometry.

Results : Pre‐conditioning with TGF‐β3, IL‐1β, or hypoxia did not alter the MSC phenotype; key markers (CD90, CD44, CD73, CD105) remained consistent across conditions. Results from NTA showed that TGF‐β3‐conditioned EVs are of a higher concentration and slightly larger size, while IL‐1β‐conditioned EVs had a narrower size range and lower concentration rates. Hypoxia‐conditioned EVs displayed a greater concentration with a varied size distribution, reflecting enhanced vesicle shedding under hypoxic stress. Consistent expression of EV markers CD9, CD63, and CD81 was observed.

Summary/Conclusion : This study underscores the importance of pre‐conditioning HFP‐MSCs in optimizing the therapeutic potential of their EVs for OA treatment. By selectively applying pre‐conditioning stimuli—IL‐1β, TGF‐β3, or hypoxia—the study successfully demonstrates how these conditions distinctly influence EVs characteristics, including size, concentration, and marker expression, which are critical for enhancing regenerative and anti‐inflammatory effects. These findings pave the way for further investigation in ex vivo models and set a foundation for potential clinical applications in OA therapy.

Funding : This study is funded by the Austrian Science Fund (FWF), grant number P37289.

Anti‐inflammatory and anti‐catabolic effect of EVs isolated from Hoffa´s fat pad mesenchymal stem cells on osteoarthritic chondrocytes and synovial fibroblasts

Slavomira Gulova 1 , Alexander Otahal 2 , Karina Kramer 2 , Markus Rothammer 2 , Zsombor Lacza 3 , Denisa Harvanova 1 , Stefan Nehrer 2 , Andrea De Luna 2

1 Associated Tissue Bank, Faculty of Medicine, P. J. Safarik University and L. Pasteur University Hospital in Kosice, Kosice, Slovakia; 2 Center for Regenerative Medicine, Department for Health Sciences, Medicine and Research, University for Continuing Education Krems, Krems, Austria; 3 Department of Sport Physiology, University of Physical Education, Inst. Clinical Experimental Research, Semmelweis University, Budapest, Hungary

Introduction : Osteoarthritis (OA) is the most common form of degenerative joint disease in adults. Effective OA treatment should confer regenerative and immunomodulatory properties. The therapeutic effect of mesenchymal stem cells (MSCs) is mediated through secretome rich in soluble factors and extracellular vesicles (EVs). The aim of this study was to determine how Hoffa's fat pad MSC (HFP‐MSC)‐derived EVs could be applied as a cell‐free therapy for OA.

Methods : Primary HFP‐MSCs were isolated from Hoffa tissue of 3 patients undergoing total knee replacement after informed consent and ethical approval. HFP‐MSCs were cultured in vertical wheel bioreactors on microcarriers in xeno‐free media supplemented with human blood products (citrate‐anticoagulated platelet‐rich plasma or hyperacute serum). EVs were isolated from serum‐free supernatant via ultrafiltration (100 kDa cut‐off) and characterized via nanoparticle tracking analysis (NTA) in scatter and fluorescence mode. Western blot was used to assay the presence of EV marker proteins. To assess the therapeutic potential of HFP‐MSC‐EVs, patient‐derived chondrocytes or synovial fibroblasts were co‐cultured with M1 macrophages in an inflammation model. Treatment effects of HFP‐MSC EVs were determined via gene expression analysis using RT‐qPCR. Cytokine release was assayed via ELISA. Uptake of HFP‐MSC‐EVs was analysed via fluorescent labelling and confocal microscopy.

Results : CD73 and CD9 were colocalised on HFP‐MSC‐EVs, indicating their MSC origin. Labelled HFP‐MSC‐EVs were internalised by both cell types. RT‐qPCR analysis showed that HFP‐MSC‐EVs decreased the expression of inflammatory genes (interleukin‐6, interleukin‐8, cyclooxygenase‐2 (COX‐2), chemokine (C‐C motif) ligand 2 (CCL‐2) and 5 (CCL‐5)) in synovial fibroblasts and catabolic enzyme genes (matrix‐metalloproteinases 3 (MMP‐3) and 13 (MMP‐13)) in chondrocytes. HFP‐MSC‐EVs modulated the release of IL‐6, TNF‐α, IL‐4 and IL‐10 by chondrocytes and synovial fibroblasts.

Summary/Conclusion : HFP‐MSCs cultured in vertical wheel bioreactors on microcarriers in xeno‐free conditions yield EVs capable of modulating inflammatory and catabolic gene expression in cells central to OA progression. Owing to their potential disease‐modifying effects, HFP‐MSC‐EVs offer a promising cell‐free therapy approach for OA.

Funding : BMBWF (No. SK11/2024), VUaVP35 UPJS (No. vvgs‐2023‐2730), APVV (No. SK‐AT‐23‐0005).

Sub‐topic Cancer Initiation and Promotion

Early pancreatic cancer detection via extracellular vesicle‐based classifier: a blinded cohort analysis

Presenter: Juan P. Hinestrosa

Xzom USA

Abstract unavailable

Tribbles pseudokinase 3 regulates CD276 loading into extracellular vesicles in oral squamous cell carcinoma

Wen‐Wei Chang 1 , Yu‐Hao Huang 1 , Yen‐Min Huang 2 , Hsueh‐Te Lee 3

1 Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan; 2 School of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan; 3 Institute of Anatomy & Cell Biology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan

Introduction : Extracellular vesicles (EVs) are key mediators in cancer progression, largely due to the cargo they carry. CD276, an immune checkpoint molecule, plays a significant role in cancer behaviour, contributing to immune evasion, proliferation, and therapy resistance. Tribbles pseudokinase 3 (TRIB3) is a pseudokinase with a scaffolding function, and its role in regulating the EV loading of CD276 in oral squamous cell carcinoma (OSCC) remains unclear. This study aims to investigate the function of TRIB3 in the EV loading process of CD276 and its potential as a biomarker in OSCC.

Methods : The Cancer Genome Atlas (TCGA) datasets were used to assess correlations between TRIB3, CD276, and ESCRT‐associated proteins ALIX/TSG101. EVs were collected from OSCC cells by ultrafiltration. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot. A CD276‐EGFP cDNA plasmid was used for studying the intracellular trafficking of CD276 and its escape from lysosomal degradation. Plasma EVs from OSCC patients and healthy donors were obtained to analyse CD276 levels by western blot and ELISA.

Results : Analysis of TCGA datasets revealed a positive correlation between TRIB3 or CD276 expression and ALIX/TSG101. TRIB3 knockdown did not significantly affect EV size or particle concentration, but reduced TSG101, ALIX, and CD9 protein levels in EVs, with no change in whole cell lysate levels. Structural modelling suggested interactions between TRIB3 and ESCRT proteins, indicating TRIB3's role in regulating EV cargo loading. TRIB3‐silenced OSCC cells exhibited reduced CD276 on cell membranes and in EVs, despite unchanged total CD276 levels, indicating TRIB3's involvement in intracellular trafficking. Transfection with CD276‐EGFP showed CD276 escaping lysosomal degradation, suggesting that TRIB3 might prevent lysosomal fusion. Plasma EVs from OSCC patients showed detectable CD276 in three out of four samples, with the highest levels in a recurrent case, while a relatively low level of CD276 was detected in healthy donors.

Summary/Conclusion : TRIB3 regulates CD276 loading onto EVs and prevents its lysosomal degradation in OSCC cells. These findings suggest that TRIB3 is a promising therapeutic target and CD276 in EVs could serve as a potential biomarker for OSCC diagnosis, especially in recurrent cases.

Funding : National Science and Technology Council in Taiwan.

Cancer‐associated circulating bacterial tsRNAs identified by large‐scale serum RNA‐seq analysis

Presenter: Kazuki Oshima

Keio University, Japan

Introduction : Recent advances in RNA sequencing technology revealed the presence of circulating RNA fragments that are not derived from the human genome (Max et al. PNAS 115: E5334, 2018). Since bacterial RNAs are encapsulated in EVs released from bacteria (bacterial EV; bEV), we investigated whether bacterial RNAs can be identified in human blood and their levels can be associated with the host disease condition, such as cancer.

Methods : Serum samples obtained from cancer patients ( n = 1406) and non‐cancer individuals ( n = 383) were prospectively collected and subjected to small RNA‐seq analysis. The obtained data were mapped to the genomes of bacteria reported to be associated with cancer. We collected bEVs released from the identified bacteria by ultracentrifugation of bacterial culture supernatants followed by sucrose density gradient centrifugation. Collected bEVs were then administered into the tail vein of STAM mice, a MASH‐based hepatocellular carcinoma (HCC) model. Functional analysis of the identified bacterial RNAs was performed using an immortalized human hepatic stellate cell line (LX‐2).

Results : We identified transfer RNA‐derived small RNAs (tsRNAs) derived from bacteria, the levels of which were associated with the presence of HCC with AUROC > 0.75 compared to non‐cancer individuals. We focused on the tsRNAs from Klebsiella pneumoniae ( K. pneumoniae ), which were detectable in bEVs purified from K. pneumoniae culture supernatants (Kp‐bEVs). Tail vein administration of Kp‐bEVs to STAM mice resulted in increased tumour growth in the liver and increased expression of the inflammatory cytokines, such as IL‐1β and IL‐6. The expressions of these cytokines were increased by transfection of K. pneumoniae tsRNAs into LX‐2.

Summary/Conclusion : Several bacterial tsRNAs were found in the human blood and associated with HCC. The bEVs released from K. pneumoniae accelerated liver carcinogenesis, which can be explained by the increased expression of the inflammatory cytokines in hepatic stellate cells. Thus, measuring bacterial tsRNAs could be useful for predicting the risk of HCC.

Time‐dependent modulation of signalling pathways in melanoma spheroids by platelet‐derived extracellular vesicles

Zeynep Tavukcuoglu 1 , Zhilin Qiu 1 , Alessandra V. de Sousa Faria 1 , Saara Laitinen 2 , Mari Palviainen 1 , Pia R‐M Siljander 1,2

1 University of Helsinki, Finland, 2 Finnish Red Cross Blood Service, Finland

Introduction : A dynamic crosstalk between platelets and cancer cells promotes haematogenous metastasis. Depending on the activating receptors, platelets generate different extracellular vesicles (PEVs) that may educate cancer cells in the circulation or within the tumour microenvironment. We engaged multiple receptors to generate a spectrum of PEV types and investigated how they altered cell transcriptomes in a melanoma model at different time‐points.

Methods : Human melanoma cell lines (MV3 and A2058) were studied in 3D spheroid cultures. Human platelets were activated with collagen related peptide (CRP), fucoidan from Fucus vesiculosus (FFV), thrombin and collagen co‐stimulus (TC) and Ca2+ ionophore (Ca2+). PEVs were isolated by size‐exclusion chromatography and characterized according to the MISEV guidelines. Spheroids were treated twice with PEVs for 5 days. On Day 5, the treatment lasted either 6 or 24 h before RNA extraction and sequencing.

Results : The number of differentially expressed genes (DEGs) was higher at the 6 h time‐point in A2058 spheroids and at the 24 h time‐point in MV3 spheroids, and time point was a main factor for clustering. The most significant differences in DEGs were induced by CRP and FFV PEVs, whereas TC and particularly Ca2+ PEVs, altered far fewer DEGs. In A2058 spheroids, both CRP and FFV PEVs enriched mitochondrial metabolism‐related pathways at 6 h. The PI3K‐Akt and MAPK signalling pathways were commonly activated by both CRP and FFV PEVs; however, they displayed distinct temporal dynamics that were also cell‐type dependent. Finally, CRP and FFV PEVs boosted different immunomodulatory pathways (TGF‐β and type‐I interferon, respectively), which also had cell‐type and time‐dependent patterns.

Summary/Conclusion : The impact of PEVs on the melanoma transcriptome depends on multiple factors: time, cell type and PEV induction. Understanding the shared and unique signalling pathways could help the development of antiplatelet strategies in cancer therapy.

Wnt mutations change the secretome of colorectal cancer cell lines

Nadine Winkler 1 , Antonia Schubert 1,2,3 , Lukas Schik 1 , Oksana Voloshanenko 1,2 , Dominic Helm 1 , Michael Boutros 1,2

1 German Cancer Research Center (DKFZ), Germany; 2 Heidelberg University, Germany; 3 National Center for Tumor Diseases (NCT), University Hospital Heidelberg, Germany

Introduction : Wnt signalling is pivotal for stem cell behaviour and cell differentiation. While mutations in Wnt pathway components are found in several cancer types, for example, colorectal cancer (CRC), little is known about the different modes of Wnt ligand trafficking and their effect on cancer pathophysiology. To travel extracellularly, Wnt ligands are secreted on carriers, such as extracellular vesicles (EVs). EV‐bound Wnt ligands have been shown to promote recipient cells' proliferation and invasiveness. However, it remains unclear how oncogenic Wnt mutations affect the CRC secretome, particularly EV secretion and cargo, and which mechanisms are mediated through EV‐bound versus non‐EV‐bound Wnt ligands.

Methods : Using CRISPR/Cas9, we generated two cell lines from the CRC line HCT116: APCtrunc with a clinically relevant truncation of APC, causing constitutive Wnt pathway activation, and EVIKO, a knock‐out of the Wnt cargo receptor EVI/Wntless, rendering the pathway inactive. Wnt ligand secretion from HCT116 wild‐type, APCtrunc, and EVIKO cells was characterized by western blots. EV fractions were enriched from the HCT116 lines, the CRC line SW480, and healthy colon fibroblasts with differential centrifugation. The effect of Wnt mutations on particle quantity and size was investigated using nanoparticle tracking analysis and electron microscopy. The proteomic profiles of EV fractions and cell lysates were identified by mass spectrometry.

Results : We established and characterized cell lines with Wnt‐specific mutations, HCT116 APCtrunc and EVIKO, observing a differential secretion of selected Wnt ligands. Two distinct particle fractions with vesicle morphology were reproducibly enriched from cell supernatants. All fractions contained classical EV markers, while different Wnt ligands and Wnt pathway receptors were detected on HCT116 APCtrunc‐ and EVIKO‐derived large and small EVs. Furthermore, the Wnt mutational background altered the number of particles secreted per mL of conditioned medium. We will further assess the influence of the isolated EV and non‐EV fractions on cell migration and tumour invasiveness.

Summary/Conclusion : By investigating the role of Wnt signalling mutations in EV biogenesis, we will improve our understanding of how Wnt signalling modulates oncogenic signal distribution via EVs in CRC.

Funding : This work is funded by the DKFZ‐MOST Cooperation Program in Cancer Research of the German Cancer Research Center and the Else Kröner‐Fresenius‐Stiftung (2022_EKEA.189).

GABARAPL2‐Alix mediates dynamic crosstalk between autophagy and exosome pathways

Naveen Soni, Megha Chaudhary, Bhawana Bissa

Department of Biochemistry, Central University of Rajasthan, Ajmer, India

Introduction : Autophagy, in addition to its primary function of nutrient recycling, has been identified as a growth regulator for both healthy and tumour cells. On the other hand, exosomes are small extracellular vesicles secreted by most cells that carry genetic information such as DNA, RNA, and proteins from host cells. Multivesicular bodies (MVBs) facilitate the crosstalk between both pathways through the formation of a hybrid amphisome and facilitate tumour cell homeostasis.

Methods : We used ultracentrifugation to isolate exosomes from control and chloroquine‐treated MDA‐MB‐231 cells. Exosomes were characterized by DLS, NTA, TEM and western blotting. We analysed the change in autophagy and exosome pathway genes after inhibition of autophagy and exosome pathways with CQ and GW4869, respectively. We also performed knockdown of GABARAP, GABARAPL1 and GABARAPL2 and analysed the change in exosome gene expression. Additionally, we also performed confocal microscopy to evaluate the localization of Alix and CD63 inside the MDA‐MB‐231 cells after inhibition of autophagy.

Results : Inhibition of exosome biogenesis led to an increase in breast cancer cell proliferation. However, we observed a significant reduction in cancer cell proliferation when both pathways are inhibited simultaneously. To assess the reciprocal regulation of two pathways, we blocked the autophagy pathway and observed an increase in the secretion of exosomes from MDA‐MB‐231 cells, along with decreased expression of Alix and CD63. On the contrary, inhibition of exosome biogenesis led to an increase in the expression of ATG5 and ATG16L1, which caused a significant decrease in the expression of GABARAPL2. Interestingly, the knockdown of GABARAPL2 rescues the decrease in Alix expression upon autophagy inhibition, thus highlighting the essential role of GABARAPL2 in Alix secretion. Our study highlights for the first time the synergistic effects of autophagy and exosome pathway inhibition in restricting cancer cell growth as well as the involvement of GABARAPL2 in the regulation of exosome secretion via modulating the Alix expression.

Summary/Conclusion : We conclude that combined inhibition of autophagy and exosome pathways promotes cancer cell death. Furthermore, inhibition of exosome secretion enhances tumour cell proliferation. Moreover, autophagy protein GABARAPL2 interacts with Alix and regulates its secretion.

Funding : SERB‐SRG (F.30‐528/2020(BSR)), SERB‐POWER Grant (SPG/2021/002833), ICMR extra‐mural research grant (52/27/2020‐BIO/BMS).

Annexin A2‐mediated distinct small EV proteomic landscape modulates recipient cell signalling in triple‐negative breast cancer

Presenter: Jamboor K. Vishwanatha

University of North Texas Health Science Center, USA

Introduction : Tumour‐derived extracellular vesicles (TEVs) promote cancer metastasis by interacting with distant recipient cells. In metastatic triple‐negative breast cancer (TNBC), the lungs are frequently affected, with metastasis occurring in 20‐30% of cases. Annexin A2 (AnxA2), a membrane‐associated protein, is notably elevated in TNBC tissues and EVs, leading to poor patient outcomes. It ranks among the top EV proteins and recruits cargo like microRNAs and proteins. Our lab's research shows reduced lung and brain metastasis with lowered small EV (sEV) AnxA2 levels in vivo. We aim to evaluate the functional role of AnxA2 in driving TNBC lung metastasis via sEVs.

Methods : The lung metastatic MDA‐MB‐4175 (LM2) cells were used to stably downregulate AnxA2, with their metastatic potential evaluated via migration, invasion, and proliferation assays. sEVs were isolated using optimized differential ultracentrifugation and filtration method and characterized per MISEV2023 guidelines. The protein cargo of sEV was analysed through quantitative proteomics. Recipient cell uptake and downstream signalling modulation were also assessed.

Results : AnxA2 downregulation reduced the metastatic potential of LM2 cells, as evidenced by decreased migratory, invasive, proliferative, and plasmin‐generating abilities. sEVs isolated from conditioned cell culture media were positive for ESCRT proteins, heat shock proteins, and tetraspanins (CD9, CD81), while negative for GM130, calnexin, and cytochrome c. Their size distribution ranged from 30 to 300 nm, displaying distinct double‐membraned vesicles under cryo‐electron microscopy. Proteomic profiling revealed a unique sEV proteomic landscape among groups, differentially impacting biological processes like tumour cell proliferation, cell viability, and migration of tumour cells and fibroblasts. Uptake studies of sEVs by non‐metastatic TNBC epithelial cells and lung fibroblasts showed altered uptake and changes in downstream functional processes.

Summary/Conclusion : Lowering AnxA2 expression in TNBC cells reduces their metastatic potential by changing the sEV proteomic cargo, affecting their interactions with recipient cells at local and distant sites. Our findings reveal novel functionality of AnxA2 in the progression of TNBC metastasis through sEVs.

Extracellular vesicles from temozolomide‐treated glioblastoma multiforme cells: insight into the morphological features and the role of bioactive cargo in the spread of cell death signals

Mariana Karimova 1 , Diana Vardanyan 2 , Stefano Tacconi 1 , Luciana Dini 1

1 The Sapienza University of Rome, Italy, 2 University of Salento, Italy

Introduction : Glioblastoma multiforme (GBM) is a highly malignant and heterogeneous type of brain tumour. A significant role of extracellular vesicles (EVs) in shaping the tumour microenvironment and promoting cell survival and resistance to chemotherapy in GBMs is supported by emerging data. Formerly we reported significant changes in GBM‐EV composition and morphology after exposure to the commonly used chemotherapeutic drug temozolomide (TMZ). Further, we extended our research to identify the effect of isolated EVs on recipient GBM cells and to determine to which extent this influence is related to different classes of molecules in EV cargo.

Methods : EVs were isolated by UF/SEC from a sensitive (U87MG) and a resistant (U251MG) to chemotherapy GBM cell line. EVs from untreated or TMZ‐treated cells were characterized by mass spectrometry, TEM, cryo‐EM, and NTA. The contribution of bioactive molecules carried by GBM‐EVs in promoting or decreasing the expression of cell death markers in recipient GBM cells was analysed by single or simultaneous physical or enzymatic elimination of the protein and/or nucleic acid cargo of the EVs. Cell response to native or treated EVs was assessed by western blot.

Results : Treatment of GBM cells with TMZ promotes EV release with a larger diameter and a relevant percentage of multilamellar EVs. Proteomic data highlights the differential impact of TMZ treatment on GBM‐EVs cargo, which further affects the physiological response of GBM recipient cells. EVs from TMZ‐untreated U87MG cells increase the expression of apoptotic (caspases 3/8/9) and ferroptotic (CD71 and GPX4) markers in recipient U251MG cells, while promoting a decrease in cell death markers in U87MG. The inactivation and/or elimination of protein and/or RNA content results in changes in the response of the recipient cells, the intensity of which depends on the GBM type.

Summary/Conclusion : The current research provides an insight into the heterogeneity of signalling carried out by GBM‐derived EVs and the influence of TMZ treatment. The cargo of EVs (i.e., proteins and RNA) contributes differently to the signalling. Thus, its modification leads to changes in the response of the recipient cells, with an intensity dependent on the cell type.

Funding : 000301_RICER CA_SAPIENZA_2022_Grande Progetto di Ateneo to LD.

Sub‐topic Cancer progression and metastasis 1

Microglia derived extracellular vesicles play a key role in the recruitment of myeloid cells in glioblastoma

Miguel Quiralte Pulido, Isabela Albuja Ron, Irene Bertolini

Molecular and Oncogenesis Program, The Wistar Institute, Philadelphia

Abstract unavailable

Macrophage‐mediated CD63 upregulation and extracellular vesicle secretion facilitate pancreatic cancer progression

Chieh Shen 1 and Chu‐An Wang 2

1 Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan

Introduction : Chronic inflammation is a key risk factor in pancreatic cancer development, with inflammatory responses playing a significant role in cancer progression. Extracellular vesicles (EVs) are essential mediators of cell communication. This study examines the role of immune cells in regulating EV biogenesis in pancreatic cancer cells and the consequent promotion of cancer malignancy.

Methods : We conducted single‐cell RNA sequencing on human pancreatic ductal adenocarcinoma (PDAC) tumours. We performed co‐culture experiments to investigate molecular changes in pancreatic cancer cells interacting with macrophages. EVs were isolated by size exclusion chromatography; particle numbers were determined by nanoparticle tracking analysis. Immunofluorescence staining and light‐sheet microscopy were utilized to observe the membrane network and track intracellular particles.

Results : Single‐cell RNA sequencing of pancreatic tumours revealed increased macrophage populations from early to advanced cancer stages. Macrophages maintained a high level of ERK status and promoted metastatic ability in pancreatic cancer cells through the secretion of TIMP metallopeptidase inhibitor 1 (TIMP‐1). Concurrently, the expression of CD63, the cell‐surface receptor for TIMP‐1, was increased in pancreatic cancer cells during interactions with macrophages. Knockdown of CD63 not only resulted in the loss of responsiveness to macrophage induction but also significantly affected cell adhesion and the vesicle network in pancreatic cancer cells. CD63 knockdown in pancreatic cancer cells led to notable membrane aggregation, suggesting defects in the vesicle biogenesis. Nanoparticle tracking analysis demonstrated that EV secretion in pancreatic cancer cells increased following treatment with macrophage‐conditioned medium (MCM), while CD63 knockdown resulted in decreased EV secretion and altered composition of particle size compared to control cells. Co‐culturing PKH67‐labelled PANC‐1 cells with pancreatic stellate cells (PSCs) indicated that EV uptake may be mediated by CD63 expression. Pancreatic cancer cell‐derived EVs were able to promote collagen expression in PSCs. Last, analysis of TIMP‐1/CD63 expression levels in clinical samples revealed an association with poorer outcomes in pancreatic tumours.

Summary/Conclusion : Our study revealed that the interactions between macrophages and pancreatic cancer cells lead to the upregulation of CD63 and promote EV secretion, facilitating the transition from the early to advanced stages of pancreatic cancer.

The invadopodia associated protease MT1‐MMP is secreted on sEVs via the syntenin‐syndecan pathway

Marie Huber 1 , Emilie Connen 1 , Rania Ghossoub 1 , Raphaël Leblanc 1 , Guido David 2 , Pascale Zimmermann 1,2 , and Sylvie Thuault 1

1 Centre de Recherche en Cancérologie de Marseille (CRCM), Equipe Labellisée Ligue 2024, CNRS, INSERM, Institut Paoli Calmettes, Aix‐Marseille Université, Marseille, France; 2 Department of Human Genetics, KU Leuven, University of Leuven, Leuven, Belgium

Introduction : Matrix metalloproteinases (MMPs) control the degradation and activity of extracellular matrix components (ECM). Overexpression of MT1‐MMP, a transmembrane MMP, predicts poor prognosis in triple negative breast cancer (TNBC). Cellular studies largely document that MT1‐MMP is delivered to invadopodia, cancer cell structures allowing localized ECM degradation. MT1‐MMP is also present at the membrane of small extracellular vesicles (sEVs). However, the mechanisms supporting MT1‐MMP loading into sEVs and the role of sEV MT1‐MMP in invadopodia activity are obscure. The syntenin PDZ protein, in association with the syndecan heparan sulphate proteoglycans (SDC), is a major player controlling sEV biogenesis.

Methods : The interaction between syntenin and MT1‐MMP was examined by immunofluorescence experiments and surface plasmon resonance technology (BIAcore) using wild‐type and mutant proteins. MT1‐MMP surface levels and endocytosis were analysed by biotinylation of cell surface protein, after depletion of syntenin or SDC using siRNA. In addition, collection of sEVs by series of ultracentrifugation of culture media from MDA‐MB‐231 TBNC cells after depletion of syntenin or SDC allowed us to assess the effect of the syntenin‐SDC sEVs pathway on MT1‐MMP loading into sEVs. Finally, the gelatin degradation assay revealed the influence of syntenin on the matrix degradation potential of MDA‐MB‐231 cells.

Results : Here, we show that MT1‐MMP and syntenin colocalize mainly in the late endosome compartment. We demonstrate that the syntenin C‐terminal region comprising the PDZ domains interacts with the intracellular domain of MT1‐MMP. Surprisingly, the interaction is independent of the MT1‐MMP PDZ binding motif. We identified the PRR sequence as the main motif required for syntenin‐MT1‐MMP interaction. We observed that the depletion of syntenin does not impact MT1‐MMP cell surface levels, nor its endocytosis. However, the depletion of syntenin and SDC impairs the loading of MT1‐MMP in sEVs. In addition, syntenin depletion impairs the matrix degradative potential of MDA‐MB‐231 cells.

Summary/Conclusion : These results suggest that the syntenin‐SDC sEVs may regulate the invasive potential of cancer cells through sorting of MT1‐MMP in sEVs at invadopodia, as sEVs and invadopodia might function in a coordinated manner. Disrupting syntenin‐SDC may thus be of therapeutic benefit for TNBC treatment. Additionally, this work suggests that syntenin‐SDC sEVs may have matrix penetrating properties.

Reciprocal tumour‐endothelial interactions mediated by extracellular vesicles during glioblastoma progression—impact on tumour invasion, vascular morphogenesis and mobilization of immune effector cells

Presenter: Janusz Rak

McGill University, Canada

Introduction : Glioblastoma (GBM) remains incurable in spite of decades of intensive research. Florid neovascularization, a morphological hallmark of GBM, is still poorly understood in terms of mechanisms and function. While oncogenic pathways and tumour microenvironments are recognized as forces driving vascular responses in GBM, the identity of mediators that control endothelial growth, regulatory effects, barrier function and immunomodulation are still to be fully elucidated. Here we present evidence suggesting that extracellular vesicle (EV) pathways play a major role in vascular growth, patterning, paracrine (angiocrine) effects of endothelial cells and immunoregulation.

Methods : Small EVs from conditioned media of human glioma stem cells (GSC) were isolated by ultracentrifugation and size exclusion chromatography and tested for endothelial cell responses in vitro (migration) and in vivo (xenografts). Similarly, EVs from endothelial cells were assayed for impact on GSCs. We also examined vascular properties of gliomas in the context of genetically disrupted vesiculation pathways.

Results : We observed that while proneural GSCs produced mainly soluble angiogenic factors (VEGF), their mesenchymal counterparts shed EVs enriched for oncogenic epidermal growth factor receptor (EGFRvIII) while engaging several different pathways of vesiculation. These EGFRvIII‐EVs triggered a non‐angiogenic vascular growth process (vasectasia) with distinct molecular features. Endothelial EVs promoted a switch from a proneural to a mesenchymal phenotype in GSCs. Genetic disruption of vesiculation pathways altered the interactions between intracranial tumours, vasculature and immune effector cells. Targeting EV‐mediated vascular effects resulted in prolonged survival of mice with GBM.

Summary/Conclusion : EVs contribute to interactive networks that couple cancer progression and vascular and immune responses in GBM.

Funding : Fondation Charles Bruneau and CIBC, Canadian Institutes of Health Research.

Extracellular vesicle‐packaged PD‐L1 impedes macrophage‐mediated antibacterial immunity in preexisting malignancy

Presenter: He‐Jing Zhang

Wuhan University, China (People's Republic)

Introduction : Malignancies can compromise systemic innate immunity, but the underlying mechanisms are largely unknown. The innate immune cells, such as macrophages, have been identified as primary cells targeted by tumour‐derived extracellular vesicles (TEVs), prompting us to investigate the roles of TEVs in altering host innate antibacterial immunity.

Methods : Based on the murine bacteraemia model, the bacterial phagocytosis of liver macrophages was detected by immunofluorescence on frozen sections, and the circulating level of Escherichia coli ( E. coli ) was detected by a fluorescent microplate reader. The phagocytosis level of macrophages was detected by flow cytometer, confocal fluorescence microscopy, and fluorescent microplate reader. The bactericidal capacity of macrophages was evaluated by CFU counting from culture lysis. The murine sepsis score and bacterial burden in vital organs were assessed to evaluate sepsis progression. Additionally, RNA sequencing analysis was applied to screen potentially impaired phagocytosis‐related pathways. Peripheral blood samples were collected from preoperative patients with oral squamous cell carcinoma (OSCC) to isolate plasma‐derived sEVs, and the sEV PD‐L1 level was assessed through ELISA.

Results : Here we find that TEVs deliver PD‐L1 to host macrophages, thereby impeding antibacterial immunity. Mice implanted with Rab27a‐knockdown tumour are more resistant to bacterial infection than wild‐type controls. Injection of TEVs into mice impairs macrophage‐mediated bacterial clearance, increases systemic bacterial dissemination, and enhances sepsis score in a PD‐L1‐dependent manner. Mechanistically, TEV‐packaged PD‐L1 inhibits Bruton's tyrosine kinase/PLCγ2 signalling‐mediated cytoskeleton reorganization and ROS generation, impacting bacterial phagocytosis and killing by macrophages. Neutralizing PD‐L1 markedly normalizes macrophage‐mediated bacterial clearance in tumour‐bearing mice. Importantly, circulating sEV PD‐L1 levels in tumour patients can predict bacterial infection susceptibility, while tumour patients treated with αPD‐1 exhibit fewer postoperative infections.

Summary/Conclusion : Through the PD‐L1/PD‐1 axis, TEV‐packaged PD‐L1 could downregulate the BTK/PLCγ2 pathway to inhibit cytoskeleton reorganization and ROS production of macrophages. Thus, it remotely suppresses macrophage‐driven bacterial clearance, leading to increasing infection susceptibility in tumour hosts.

Funding : This work was funded the National Natural Science Foundation of China (82341023, 81922038), the Medical Science Advancement Program (TFJC2018005), the Innovative Research Team of High‐level Local Universities in Shanghai (SHSMU‐ZLCX20212300) and the Fundamental Research Funds for the Central Universities (2042022dx0003).

Horizontally intercellular delivery of TXNDC5 mRNA‐containing small extracellular vesicles contributes to tumour‐associated macrophage‐mediated prostate cancer metastasis

Cong Hu 1 , Tianyang Wu 1 , Yinjie Zhu 1 , Xinxing Du 1 , Xinrui Wu 1 , Yanhao Dong 1 , Zehong Peng 1 , Kenneth J. Pienta 2 , Jiahua Pan 1 , Liang Dong 1 , Wei Xue 1

Kenneth J. Pienta, Jiahua Pan, Liang Dong, and Wei Xue are co‐senior authors.

1 Department of Urology, Ren Ji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 The Cancer Ecology Center, The Brady Urological Institute, Johns Hopkins School of Medicine, Baltimore, USA

Introduction : Prostate cancer (PCa) incidence is increasing rapidly, and metastatic PCa remains incurable. The tumour microenvironment exerts a multifaceted and important influence on tumour metastasis. Tumour‐associated macrophages (TAM) are important components of the tumour microenvironment, most of which favour the M2 phenotype that facilitates PCa metastasis. However, the precise mechanisms underlying this process remain inadequately understood. Extracellular vesicles (EVs) are pivotal mediators of intercellular communication, with the capability to transmit molecular signals; however, their involvement in TAM‐mediated PCa metastasis remains to be elucidated.

Methods : Single‐cell RNA sequencing was employed to investigate the infiltration of M2 macrophages in metastatic and non‐metastatic PCa (mPCa, nmPCa) primary foci. M2 macrophages were induced in vitro from THP1. Collected cell culture medium (CCM) from M2 macrophages CCM‐isolated EVs were employed for co‐incubation with PCa, and the effects of M2 macrophage CCM and the EVs in it on PCa metastasis were explored by transwell and scratch assays. To further investigate what components of EVs could mediate the above effects, we focused on co‐altered mRNA molecules after treatment of PCa cell lines with M2 EVs by RNA sequencing. The horizontal cell‐to‐cell delivery of mRNAs was further explored by detecting constructed fluorescent‐tagged mRNAs in EVs with nanoflow cytometry.

Results : Compared with nmPCa, mPCa primary foci exhibited greater infiltration of M2 macrophages, and M2 macrophage‐derived EVs accounted for a higher proportion of total tissue EVs. M2 EVs were able to alter the mesenchymal phenotype of PCa cells and promote metastasis of PCa. We found that TXNDC5 mRNA was co‐elevated in two DU145 and PC3 cell lines after treatment with M2 EVs. We subsequently further demonstrated that TXNDC5, as well as TXNDC5 in M2 EVs, promotes PCa metastasis. By exogenously transfecting Cy3‐tagged TXNDC5‐Flag fused mRNA into M2 macrophages, we demonstrated the presence of TXNDC5 mRNA in M2 EVs by nanoflow cytometry and confocal microscopy to validate cellular horizontal transfer.

Summary/Conclusion : We demonstrated for the first time that horizontally intercellular delivery of TXNDC5 mRNA‐containing small extracellular vesicles contributes to TAM‐mediated PCa metastasis.

Multi‐omics profiling demonstrates dynamic changes of cancer‐associated fibroblasts and their derived extracellular vesicles during prostate cancer progression

Cong Hu 1 , Yingchao Zhang 1,2 , Xinxing Du 1 , Tianyang Wu 1 , Helen H. Zhu 1,2 , Liang Dong 1 , Wei Xue 1

Helen H. Zhu, Liang Dong, and Wei Xue are co‐senior authors.

1 Department of Urology, Ren Ji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 State Key Laboratory of Oncogenes and Related Genes, Renji‐Med‐X Stem Cell Research Center, Shanghai Cancer Institute, Shanghai, China

Introduction : Cancer‐associated fibroblasts (CAF) are widely present in prostate cancer (PCa) stroma, and they exert a critical role in tumour progression. Extracellular vesicles (EVs) are one of the modes of intercellular communication, which can reflect the state of the disease and likewise contribute to its progression. The research of CAFs and their derived EVs in different stages of PCa can provide us with information about the state of disease contributing to subsequent treatment.

Methods : Normal prostate fibroblasts (NPF) derived from WT C57/B6 mice, hormone‐sensitive PCa CAF (HSPC CAF) derived from the transgenic model (Pb‐Cre4: Ptenf/f; Hi‐myc), castration‐resistant PCa CAF (CRPC CAF) derived from the transgenic model (Pb‐Cre4: Ptenf/f; Trp53f/f), and neuroendocrine PCa CAF derived from the transgenic model (Pb‐Cre4: Trp53f/f; Rb1f/f) were isolated and identified by immunofluorescence. RNA sequencing was performed on four groups. EVs separated from the above CAFs were identified according to minimal information for studies of extracellular vesicles 2018 (MISEV2018), then proteomics and metabolomics analyses were performed to search for differences at the EV level.

Results : RNA sequencing demonstrated that CAFs derived from HSPC had enhanced oncological properties (HSPC CAF vs. NPF). Functions such as angiogenesis and cell migration were improved, and the PI3K‐Akt and MAPK signalling pathways were significantly enriched. When the disease progressed (CRPC CAF vs. HSPC CAF), the above differences were further enriched. Proteomics of CAF EVs further demonstrated PI3K‐Akt signalling pathway was enriched in the early and late stages of the disease (HSPC CAF vs. NPF, NEPC CAF vs. CRPC CAF). EVs metabolomics revealed 30, 18, and 145 significant differences in three comparison groups, respectively (HSPC CAF vs. NPF, CRPC CAF vs. HSPC CAF, NEPC CAF vs. CRPC CAF). Tryptophan metabolism, glycerophospholipid metabolism and ABC transporters ranked in the top three (CRPC CAF vs. HSPC CAF).

Summary/Conclusion : CAF‐derived EVs may be able to reflect the disease state represented by the CAF. The PI3K‐Akt signalling pathway may be the key pathway in CAF‐mediated disease progression, and it may be able to regulate tryptophan metabolism to adjust PCa properties during treatment.

Role of kindlin‐2 and small extracellular vesicles in the invasion of triple‐negative breast cancer tumours

Neelum Yousaf Zai 1 , Lucia Languino 2 , Mark F. Santos 3 , Aurelio Lorico 3 , Khalid Sossey‐Alaoui 1

1 The MetroHealth System, Case Western Reserve University School of Medicine, USA; 2 Thomas Jefferson University, USA. 3 Turo University Nevada, USA

Introduction : We identified the kindlin‐2 (K2) oncogenic axis as a major driver of the metastatic process in BC. Given that we have shown that K2 is enriched in cancer cell‐secreted sEVs, we hypothesized that K2‐containing sEVs deliver K2 and its associated cargo to stromal/immune cells as well as cancer cells, in both an autocrine and a paracrine manner, and play a major role in the activation of the metastatic process.

Methods : We used sEVs isolated from TNBC cancer cell lines to investigate the effect on tumoursphere invasion in a 3‐dimensional model of tumourspheres grown in Matrigel. sEVs were isolated from cultures of parental and Kindlin‐2‐KO derivatives to investigate the autocrine effect of sEVs. We also used fibroblast‐cancer cell co‐cultures to investigate the paracrine effect of sEVs.

Results : Here we show that the invasion potential of tumourspheres derived from MDA‐MB‐231 and 4T1 cells was significantly enhanced in the presence of sEVs isolated from parental BC cells, compared to their K2‐KO derivatives. Thus, we confirmed the autocrine effect of sEVs on cancer cells. We also determined a paracrine effect of the K2 + ‐sEVs by showing that treatment of normal mouse 3T3 fibroblasts with CM from mouse 4T1 BC cell cultures significantly induced expression of α‐SMA, a marker of the myofibroblast‐activated phenotype of CAFs, a result that we replicated in human WI‐38 fibroblasts treated with CM from MDA‐MB‐231 BC cells, therefore supporting the role of BC‐secreted factors in the acquisition of the CAF phenotype by normal fibroblasts. Treatment of 3T3 or WI‐38 fibroblasts with sEVs derived from mouse 4T1 or MD‐MB‐231 BC cells, respectively, significantly induced expression of α‐SMA. We further show that MDA‐231‐derived tumourspheres treated with CM of Wi‐38 fibroblasts that were activated (CAFs) with MDA‐231‐derived sEVs exhibited enhanced invasion compared to the tumourspheres derived from treatment with CM of naïve Wi‐38 fibroblasts. This invasion phenotype was further inhibited when fibroblasts were pretreated with K2‐sEVs isolated from K2‐KO cancer cells.

Summary/Conclusion : Together, our data support the provocative hypothesis that K2 present in the sEVs secreted by BC tumours coordinates the systemic activities that drive the metastatic behaviour of BC tumours and warrants further investigations.

Funding : MetroHealth Startup Funds

Education of the bone marrow microenvironment by B‐cell acute lymphoblastic leukaemia‐derived large oncocosomes

Presenter: Valentina R. Minciacchi

Universitätsmedizin Mainz, Germany

Introduction : Within the bone marrow microenvironment (BMM), leukaemia cells have been shown to hijack some of the intercellular communication routes usually employed by benign cells of the haematopoietic system. This results in the establishment of an environment permissive to leukaemia growth and protection from the action of drugs. A major adverse communication route of leukaemia cells is the release of extracellular vesicles (EVs) that are capable of reprogramming the BMM. Highly motile and aggressive tumour cells are known to shed large oncosomes (LO). These have been shown to condition the tumour microenvironment and contribute to disease progression. Given the high deformability of leukaemia cells, this study therefore focused on the role of LO in the communication between leukaemia cells and the BMM.

Methods : Leukaemia mouse models; differential and density gradient centrifugation; flow cytometry analysis and sorting; bulk RNAseq; fluorescent microscope imaging.

Results : We show that LO isolated from mice with BCR‐ABL1+ B‐cell acute lymphoblastic leukaemia (B‐ALL) alter the phenotype of target primary mesenchymal stromal cells (MSC). Treatment of MSC with purified B‐ALL‐derived LO results in an increased expression of peroxisome proliferator‐activated receptor gamma (PPAR‐g) and fatty acid‐binding protein 4 (FABP4), which are indicators of increased adipocyte differentiation potential. Consistently, these MSCs show an augmented ability to differentiate into adipocytes following stimulation. Furthermore, LO‐treated MSC specifically support the growth of BCR‐ABL1+ BA/F3 B‐LL cells but do not increase proliferation of the empty vector control cells. Differentiation of LO‐treated MSC into adipocytes not only increases their ability to sustain leukaemia cell growth but also further stimulates the colony forming potential of leukaemia cells. Finally, transcriptome analysis together with targeted secretome profiling indicate that conditioned by B‐ALL‐derived LO contributes to the recently identified role of ECM remodelling and growth factor availability in B‐ALL progression.

Summary/Conclusion : This study shows that LO released by B‐ALL cells contributes to the establishment of a tumour‐permissive environment which supports leukaemia progression.

Funding : DFG grant MI 2669/2‐1.

The role of platelet‐derived extracellular vesicles in the formation of the lung cancer pre‐metastatic niche

Presenter: Camilla Locatelli

Fondazione IRCCS Istituto Nazionale dei Tumori, Italy

Introduction : Tumour growth and wound healing share common characteristics such as modulation of immune response, cell proliferation/death and tissue remodelling. A major step in wounding is activation of platelets and the subsequent release of their cargo in blood. Interactions between platelets and cancer cells control the different phases of metastasis formation, like intravasation, protection of circulating tumour cells from share stress, extravasation and grounding of metastatic niche. Via extracellular vesicles (EVs), platelets traffic circulating nucleic acids and proteins within the tumour microenvironment. EVs from platelets are the most abundant EV's subpopulation in blood, suggesting their potential use as biomarkers in liquid biopsy. However, the role of lung cancer educated platelets‐EVs (LCEP‐EVs) and their cargo in cancer progression remains unknown.

Methods : Lung cancer patients’ blood was collected at surgery, and LCEP‐EVs were isolated from conditioned medium (CM) by ultracentrifugation and characterized following MISEV guidelines. Functional experiments were carried out in vitro by treating cells with LCEP‐EVs or healthy donor‐EVs (HDEP‐EVs).

Results : Using CM of lung cancer cells or primary tumours, we showed that tumour enhanced the secretion of PLT‐EVs compared to CM of normal epithelial cells (HBEC). PLT‐EVs displayed high levels of pro‐tumourigenic miRNAs (miR‐126 and miR‐29a) and released several cytokines (IL‐6, CXCL1, IL‐1β, and CCL2) known to be implicated in metastatic dissemination. LCEP‐EVs at two different time points (8 weeks) displayed very small variation in terms of surface markers and expression of miR‐29 and miR‐126, previously detected also in PLTs. LCEP‐EVs induced endothelial activation (upregulation of CXCL1, CCL2, CXCR4 and VCAM1) and enhanced neutrophil adhesion and migration compared to HDEP‐EVs, without affecting angiogenesis. Interestingly, our data confirmed the extrusion of neutrophils extracellular traps (NETs) from neutrophils exposed to LCEP‐EVs compared to HDEP‐EV. LCEP‐EVs modulated the inflammatory switch of lung fibroblasts. dPCR and ELISA analysis showed that LCEP‐EVs contained higher levels of miR‐126, miR‐29a and the complement protein C4a than HDEP‐EVs. Simultaneous modulation of endothelial cells with miR‐29a and C4a recapitulates changes observed with LCEP‐EVs treatment.

Summary/Conclusion : Our data indicated that lung cancer educates platelets towards a pro‐tumourigenic phenotype that is involved in endothelial and immune cell activation.

Plasma‐derived sEVs from HNSCC patients modulate macrophage function through NF‐κB signalling

Presenter: Diana Huber

Ulm University Medical Center, Germany

Introduction : As one of the most immunosuppressive cancers, head and neck squamous cell carcinomas (HNSCC) show an increased NF‐κB activation with downstream production of immunosuppressive and tumour‐promoting factors. Plasma‐derived small EVs (sEVs) from HNSCC patients contain molecules, which can contribute to systemic immunosuppression. Here, we investigate the influence of plasma‐derived sEVs from patients with HPV‐positive and HPV‐negative HNSCC on macrophage function.

Methods : sEVs were isolated from plasma of HNSCC patients and healthy donors by size‐exclusion chromatography (EV‐Track: EV200068 ). All donors provided written informed consent. Monocytes from buffy coats were used to generate primary macrophage cultures, which were incubated with plasma‐derived sEVs to investigate their effects on the NF‐κB signalling by translocation assay, macrophage activation by flow cytometry and proteome alterations by mass spectrometry (MS), using HPV‐positive and ‐negative sEVs. Also, uptake dynamics of different sEVs were investigated by using several uptake inhibitors, and the effects of HNSCC‐sEV‐treated macrophages on tumour cells were evaluated.

Results : Activation of p65‐dependent NF‐κB signalling was transiently increased by HNSCC‐sEVs, which was confirmed by p65 translocation assays. The HPV status of sEV donors affected levels of the NF‐κB activation and uptake of sEVs, indicating an HPV‐dependent modulation of NF‐κB pathway. HNSCC‐sEVs induced a mixed phenotype, rather than M1/M2 polarization of macrophages, and MS analysis revealed time‐dependent changes in the proteome of sEV‐treated macrophages. Furthermore, HNSCC‐sEV‐treated macrophages, but not sEVs alone, increased tumour cell migration and reversed EMT marker expression, supporting metastasis formation. Pre‐incubation with sEVs affected phagocytic activity of macrophages.

Summary/Conclusion : Plasma‐derived sEVs from HNSCC patients alter immunosuppressive properties of macrophages and promote metastasis formation. Importantly, the HPV status of the patients must be considered. The sEV‐dependent NF‐κB activation may be useful for future therapeutic strategies on modulation of tumour‐associated macrophages through targeting sEVs or suitable molecules within this pathway.

Funding : Deutsche Krebshilfe.

Circulating serum extracellular vesicle proteins for early prediction of platinum resistance in ovarian cancer

Kalpana Deepa Priya Dorayappan 1 , Vincent Wagner 2 , Molly Morton 1 Anna Gonzalez 1 , Lianbo Yu 2 , Takahiko Sakaue 3 , Shyam Sundaram 1 , Colin Hisey 1 , Thomas Conrads 4 , Larry Maxwell 3 , Casey Cosgrove 1 , Floor Backes 1 , Qi‐En Wang 1 , David O'Malley 1 , David E. Cohn 1 Karuppaiyah Selvendiran 1

1 Ohio State University, USA; 2 University of Iowa, USA; 3 Kurume Medical University, Japan; 4 Inova Health system, USA

Introduction : Platinum resistance in high‐grade serous ovarian carcinoma (HGSOC) is associated with a poor prognosis. While the initial response to platinum‐based chemotherapy is often high, 15%–20% of patients exhibit primary resistance, and nearly all patients develop resistance in the recurrent setting. Currently, no predictive or diagnostic biomarkers specific to platinum resistance exist. This study aims to identify clinically relevant extracellular vesicle (EV) protein signatures as biomarkers for detecting platinum resistance in HGSOC.

Methods : Serum EVs were isolated using differential centrifugation and size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis (NTA) and ImageStream flow cytometry. Differentially expressed EV proteins in primary vs. recurrent ovarian cancer (OC) patients were identified using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), with their potential to predict OC recurrence assessed. Identified proteins were validated in preclinical mouse models of primary and recurrent HGSOC. Candidate proteins were further validated using ELISA in whole serum and EV samples and immunohistochemistry (IHC) in tissues.

Results : We identified distinct EV proteins, including TMEM205 and CFH, in patients with platinum‐resistant (PR) HGSOC compared to platinum‐sensitive (PS) patients. These proteins were validated in patient‐derived PR cell lines and clinically relevant mouse models post‐platinum therapy. Serum samples from PS and PR‐HGSOC patients corroborated these findings. EV CFH and EV TMEM205 demonstrated excellent diagnostic potential for platinum resistance, with area under the curve (AUC) values of 0.95 and 0.84, respectively. Both proteins showed greater sensitivity and specificity for PR disease than the established biomarker Ca125 (AUC 0.516). The combined biomarker panel further improved diagnostic performance, with a true positive rate (TPR) of 0.938, a false positive rate (FPR) of 0.062, and a high Matthew's correlation coefficient (MCC). The Youden index for the optimal cut‐off was 0.877.

Summary/Conclusion : Our study highlights the potential of EV proteins, particularly CFH and TMEM205, as novel biomarkers for predicting platinum resistance in HGSOC. This discovery opens new pathways for prediction of clinical behaviour and therapeutic strategies, providing opportunities to better understand and target mechanisms of chemoresistance for patients with HGSOC.

Funding : DOD‐ OCRP Clinical Translational Award 2022.

The influence of Jagged ligands on the extracellular vesicle‐mediated tumourigenesis in multiple myeloma

Domenica Giannandrea 1 , Valentina Citro 1 , Natalia Platonova 1 , Giulia Sauro 1 , Lara Paonessa 1 , Nazanin Abazari 1 , Silvia Ancona 1 , Anna Pistocchi 1 , Elisabetta Calzavara 2 , Mauro Turrini 2 , Lavinia Casati 1 , Elena Lesma 1 , Raffaella Chiaramonte 1

1 University of Milano, Italy, 2 Valduce Hospital, Como, Italy

Introduction : Multiple myeloma (MM) is an incurable haematological malignancy characterized by the aggressive interaction between MM cells and bone marrow (BM) cells. Notch pathway members are overexpressed in MM, leading to the activation of Notch signalling within the BM population and promoting their pro‐tumourigenic activities. Additionally, extracellular vesicles (EVs) play a crucial role in the pathological communication between MM and BM. Indeed, we already reported the role of the Notch2 receptor in the tumourigenic effect of MM‐derived EVs (MM‐EVs). This study investigates the role of Jagged 1 and 2 ligands in the impact of MM‐EVs on enhancing the promoting effect of the BM populations on MM progression, specifically focusing on osteoclastogenesis and angiogenesis as key steps in MM progression.

Methods : MM cells were constitutively inhibited for the expression of Jagged1 and 2 (J1/2KD), with a scramble control (SCR). EVs were isolated from MM cells (MMSCR‐EVs) and knockdown cells (MMJ1/2KD‐EVs) and characterized for the protein expression via western blot, size and number using nanoparticle tracking analysis and electron microscopy, and the uptake in recipient cells by flow cytometry. Notch pathway activation was measured through Notch reporter assays both in vitro (HeLa cells) and in vivo (transgenic zebrafish embryos). The pro‐tumourigenic effects of MMSCR‐EVs and MMJ1/2KD‐EVs were evaluated based on their osteoclastogenic and angiogenic potential. The osteoclastogenic and angiogenic role of vesicular Notch pathway members was confirmed by examining the effect of the Notch activation inhibitor, the γ‐secretase inhibitor (GSI), on EVs from MM cells as well as from the BM of MM patients (informed consent and IRB approval from Insubria, n. 1/2018).

Results : MM‐EVs were found to carry Jagged1 and 2, enhancing Notch signalling in recipient cells. Notably, MM‐EVs induced the tumourigenic activity of osteoclasts and endothelial cells in a Notch‐dependent way. Indeed, GSI effectively reduced the osteoclastogenic and angiogenic potential of MM‐EVs in vitro as well as the angiogenic effects of EVs from the BM of MM patients.

Summary/Conclusion : These findings suggest that targeting the Jagged ligands may inhibit the pro‐tumourigenic activities of MM‐EVs in the BM.

Funding : Investigator Grant to RC (20614) from Associazione Italiana Ricerca sul Cancro.

Platelet‐derived extracellular vesicles promote melanoma progression by functional changes

Zeynep Tavukcuoglu 1 , Umar Butt 1 , Alessandra V. de Sousa Faria 1 , Johannes Oesterreicher 2 , Wolfgang Holnthoner 2 , Saara Laitinen 3 , Mari Palviainen 1 , Pia R‐M Siljander 1,3

Umar Butt and Alessandra V. de Sousa Faria contributed equally . 1 University of Helsinki, Finland; 2 Ludwig Boltzmann Institute for Traumatology, Austria; 3 Finnish Red Cross Blood Service, Finland

Introduction : Platelets promote tumour progression and haematogenous metastasis by interacting with cancer cells. Upon activation, platelets generate different platelet‐derived extracellular vesicles (PEVs) that may educate cancer cells. We activated platelets through multiple receptors, including glycoprotein VI and C‐type lectin‐like receptor 2, to generate different PEV types and investigated their effects on cancer hallmarks such as proliferation, invasion, and pro‐angiogenic potential using melanoma spheroids as a cancer model. Additionally, we analysed PEV‐induced changes in the cancer EV profiles.

Methods : MV3 and A2058 metastatic melanoma cell lines were studied in the 3D spheroid cultures. Human platelets were activated with collagen‐related peptide (CRP), fucoidan from Fucus vesiculosus (FFV), thrombin and collagen co‐stimulus (TC) and Ca2+ ionophore. PEVs were isolated by size‐exclusion chromatography and characterized according to the MISEV guidelines. Spheroids or cells were treated with PEVs and used in functional assays of proliferation, invasion, and endothelial tube formation, as well as for analysing melanoma EV production and their tetraspanin profiles by SP‐IRIS.

Results : CRP and FFV PEVs had the most profound effects on altering cancer hallmark functions compared to the TC and Ca2+ PEVs. Specifically, CRP and FFV PEVs increased proliferation, invasion, and cancer EV production in both spheroid types. In contrast, endothelial tube length increased only with TC PEVs in A2058 cells, although RNA‐seq data showed that CRP, FFV, and TC PEVs all activated angiogenesis‐related genes in both cell types. All PEV types induced distinct tetraspanin signatures of melanoma EVs, and the most EVs were produced by CRP and FFV PEVs.

Summary/Conclusion : Our study revealed both shared and distinct cancer‐promoting functions of PEVs, which contributed to the metastatic capabilities of the melanoma spheroids. Inhibiting platelet receptors that drive PEVs’ cancer‐promoting effects could reveal new targets for cancer therapy.

Tumour extracellular vesicles promote metastasis through neutrophil mobilization and NETs formation

Presenter: Louis Bochler

UMR_S1109 Tumor Biomechanics, France

Introduction : Metastasis accounts for most of the cancer‐related deaths, when tumour cells disseminate from the primary site to form secondary tumours. Metastatic progression is facilitated by pre‐metastatic niches (PMN) induced by tumour‐secreted factors, including extracellular vesicles (EVs). Tumour‐derived EVs (TEVs) carry cargos that can induce microenvironmental changes favouring metastatic seeding. TEVs are known to shape the immune system, yet their precise mode of action and consequences on metastatic growth remain unclear, especially during the early phase of the disease.

Methods : We used a murine model of highly metastatic breast cancer cells (4T1‐HM), and its low metastatic counterpart (4T1‐LM) where the Ral GTPase‐dependent EV secretion is silenced. We analysed the immune composition of PMNs in an orthotopic model and in a TEVs priming model by flow cytometry and confocal imaging. We performed experimental lung metastasis assays to follow metastasis formation with bioluminescence and an ex vivo imaging approach.

Results : One week after orthotopic grafting of 4T1‐HM in the mammary gland, metastatic cells are not detected in the lungs, yet we observed a drastic increase in the number of neutrophils in these PMNs. TEVs are directly contributing to this phenotype since intravenous injection of 4T1‐HM‐derived EVs leads to a potent recruitment of neutrophils. Strikingly, most of the recruited neutrophils produce neutrophil extracellular traps (NETs) upon 4T1‐HM TEVs injection, while injection of the soluble 4T1‐HM secretome only results in neutrophil recruitment. NET depositions have been shown to favour metastasis progression and have been identified as PMN landmarks in human patients. Importantly, EVs isolated from 4T1‐LM do not recruit neutrophils nor trigger NETosis in the lungs. In vitro treatment of isolated neutrophils confirmed that only 4T1‐HM TEVs triggered NETosis, whereas neither the soluble secretome nor 4T1‐LM secreted factors induced this response. Experimental metastasis assays revealed that 4T1‐HM TEVs priming of mice prior to tumour cell injection increases metastasis formation, yet neutrophil depletion abrogates this effect. Currently, we investigate how neutrophils impact early metastasis formation using ex vivo lung imaging.

Summary/Conclusion : Altogether, our results show that TEVs from highly metastatic cells induce a fast recruitment and activation of neutrophils in the PMN and thereby facilitate early metastatic seeding.

Exploring extracellular vesicle dynamics in ex vivo breast cancer metastasis models

Nao Nishida‐Aoki 1* , Moe Saito 2 , Yoshiki Hatashita 2 , Takafumi Inoue 2

1 Waseda Institute for Advanced Study; 2 Dept Life Sci & Med Biosci, Department of Life Science and Medical Bioscience

Introduction : Organ colonization is one of the rate‐limiting steps during tumour metastasis, but cancer cells facilitate the process by modulating the surrounding tissue environment by transferring extracellular vesicles (EVs). Understanding the target cells and the spatial reach within the complex and densely packed tissue architecture is essential for elucidating the roles of cancer EVs in metastasis establishment. However, conventional in vitro culture models fail to replicate tissue complexity, and in vivo studies remain technically challenging for analysing nanoscale EVs. In this study, we aimed to investigate the dynamics of EV transfer at the early metastasis phase in a physiologically relevant tissue context.

Methods : We developed ex vivo brain and lung metastasis models using tissue slice culture. Human breast cancer cells were sparsely seeded onto 250 µm‐thick mouse brain and lung tissue slices, cultured in an air‐liquid interface. To label EVs sensitively, CD63‐HaloTag fusion protein was stably expressed in the breast cancer cells. The EV transfer was analysed using 2‐photon live imaging or confocal microscopy. To determine the cell type preference of EV uptake, cancer EVs were isolated from 0.22 µm‐filtered breast cancer supernatant by ultracentrifugation, stained with PKH67, and supplemented to the organ tissue slices. The recipient cell types were identified through the cell surface marker staining by IHC or flow cytometry.

Results : The seeded cancer cells were attached and integrated into organ tissue slices. The live imaging successfully demonstrated real‐time EV secretion from cancer cells to the surrounding tissue environment. The distribution of EVs supplemented on organ slices was uneven, suggesting selective uptake by specific cell types. In the brain metastatic models, microglia was one of the recipient cells of cancer EVs, while in the lung tissues, epithelial cells exhibited higher EV uptake compared to immune cells.

Summary/Conclusion : This study established ex vivo metastasis models that allow spatiotemporal analysis of cancer EV secretion at metastatic organs. Our findings highlight selective incorporation of cancer EVs by different cell types in the metastatic organs, providing insights into how cancer EVs will contribute to metastasis establishment.

Funding : JSPS KAKENHI Grant‐in‐Aid for Research Activity Start‐up, JSPS KAKENHI Grant‐in‐Aid for Scientific Research (C), Mitsubishi Fund, The Noguchi Institute Research Grant.

M2‐macrophage extracellular vesicles facilitate metastasis in colorectal cancer cells by upregulating a proto‐oncogene FAM83A

Melis Isik, Burak Derkus

Department of Chemistry, Faculty of Science, Ankara University, 06560 Ankara, Turkiye

Introduction : The crosstalk between cancer and immune cells adds a layer of complexity to our understanding of metastasis formation. This study explores the intricate dynamics between immune cells and colon cancer cells (HT‐29) during the post‐tumourigenic phase. The hypothesis is based on the premise that M2 macrophages, known to promote cancer metastasis, exert their effects through extracellular vesicles (EVs). To test this hypothesis, EVs were obtained from M0, M1, and M2 macrophages (M0‐EVs, M1‐EVs, and M2‐EVs), and their effects on colon cancer metastasis were studied in vitro.

Methods : THP‐1 cells were polarized into M1 and M2 phenotypes, induced by GM‐CSF and M‐CSF, respectively. The success of polarization was examined for CD80 (M1) and CD206 (M2) markers using flow cytometry (FC) and immunoblotting. EVs were isolated from M0, M1, and M2 macrophages through serial ultracentrifugation. Briefly, a gradual centrifugation was applied for cell culture conditioned media (CCM, 400 mL), and CCM was ultracentrifuged (Himac CP100WX) for 90 min at 120,000 × g to obtain an EV pellet. The obtained EVs were characterized by immunoblotting for CD63 and CD81, nanoparticle tracking analysis, and transmission electron microscopy. To test our hypothesis, we induced HT‐29 cells by M0‐EVs, M1‐EVs, and M2‐EVs (10, 50, and 100 µg) to investigate their role in cancer metastasis. For this purpose, we performed transcriptome analysis in addition to microscopic and immunophenotypic evaluations.

Results : The findings revealed a significant decrease in epithelial gene/protein expression (Ecad) and an increase in mesenchymal gene/protein expression (NCad) in cells exposed to M2‐EVs, indicating that M2‐EVs facilitate epithelial‐mesenchymal transition (EMT). Comprehensive transcriptomic analyses of M2‐EVs‐treated HT29 cells identified a subset of genes, including FAM83A, which is upregulated upon M2‐EVs induction, but not in the case of co‐culture with M2 macrophages, indicating that EVs play a direct role in EMT. Strikingly, targeted silencing of FAM83A via siRNA abrogated M2‐EVs‐mediated FAM83A overexpression and inhibited EMT activation, highlighting its potential as a therapeutic target.

Summary/Conclusion : The findings in this work elucidate the immunological mechanisms underpinning cancer metastasis through EV‐mediated interactions. Identifying immune cell functions and key molecular targets may advance the development of immunotherapies aimed at inhibiting metastasis.

Funding : TUBITAK, Grant No: 123S856.

Extracellular vesicles from highly invasive melanoma subpopulations increase the invasive capacity of less invasive melanoma cells through mir‐1246‐mediated inhibition of CCNG2

Tim Kingreen 1 , Stefanie Kewitz‐Hempel 1 , Christian Rhode 2 , Gerd Hause 3 , Cord Sunderkötter 1 , and Dennis Gerloff 1

1 Department of Dermatology and Venereology, Martin‐Luther‐University Halle‐Wittenberg, Halle (Saale), Germany; 2 Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany; 3 Biocenter, Martin‐Luther‐University Halle‐Wittenberg, Halle (Saale), Germany

Introduction : Invasive growth is a critical process in tumour progression, requiring the activation of various molecular processes in tumour cells at the invasive front. Intercellular communication between heterogeneous tumour cells enhances cellular activation and adaptation to specific microenvironments. One mechanism of intercellular communication is the delivery of miRNAs through tumour cell‐derived extracellular vesicles (EVs).

Methods : A highly invasive melanoma cell subpopulation was established through the use of 2D Boyden chamber invasion assays. The separation and characterization of EVs were conducted in accordance with the MISEV guidelines. RNA analyses were performed using next‐generation sequencing. Functional analyses were conducted in a 3D Matrigel‐embedded spheroid system.

Results : We observed that conditioned media from a highly invasive cell subpopulation (BLM‐HI) enhances the invasive capacity of the parental cell line (BLM). The treatment of BLM cells with EVs derived from BLM‐HI cells resulted in a significantly enhanced invasive capacity, as observed in Matrigel‐embedded spheroids and in 2D Boyden chamber assays, with a dose‐dependent effect. Conversely, the invasive capacity of BLM cells was reduced when secretion of EVs was inhibited by a sphingomyelinase inhibitor. To investigate the molecular mechanisms behind this effect, we performed next‐generation sequencing and identified an enrichment of miR‐1246 in these EVs. In functional analyses we demonstrated that both the EV‐mediated delivery of miR‐1246 and overexpression contribute to the enhanced invasiveness of BLM cells. We identified a binding site of miR‐1246 in the 3’UTR of cyclin G2 (CCNG2) and demonstrated direct binding by a luciferase reporter assay. Increased expression of CCNG2 has been associated with cancer metastasis and poor patient outcomes in other malignancies.

Summary/Conclusion : Our study demonstrates that intercellular communication between heterogeneous melanoma cells contributes to an increased invasive capacity via EV‐transported miRNAs. Our findings indicate that miR‐1246 plays a role in the pro‐invasive phenotype of melanoma cells by targeting CCNG2, which is associated with metastasis and unfavourable patient outcomes.

Funding : This study was supported by Hiege‐Stiftung Die Deutsche Hautkrebsstiftung (FKZ 2023/2) and the Wilhelm Roux Funding Program NAUTICA: Non‐Autonomous Mechanisms in Cancer (FKZ 32/19) by the medical faculty of the Martin‐Luther‐University Halle‐Wittenberg.

Extracellular vesicle‐mediated crosstalk in 2D and 3D cancer microenvironment

Presenter: Saroj Saroj

Shiv Nadar University, India

Introduction : Extracellular vesicles (EVs) are vital mediators of intercellular communication in tumour microenvironments (TME). The interactions between the structural components of the TME and cancer cells enable these cells to adopt an invasive phenotype, allowing them to spread from the primary tumour to distant sites through a complex, multistep metastatic process. In this study, we utilized 2D and 3D culture to understand the cell interactions and cell‐ECM interactions. The main objective of the project is to analyse the crosstalk mediated via EVs in tumour microenvironment employing several spectroscopies as well as biochemical techniques.

Methods : In this project, 2D cell culture as well as tumour spheroids using 3D cell culture were established. Spheroids mimic the tumour features observed in the in vivo system closer to the original tumours. Spheroids will be generated using the MCF 7, MCF 10A and HEK 293 cell lines. The cancer EVs from 2D and 3D systems were isolated and characterised using AFM, FE‐SEM, DLS, and FACS. Followed by cancer cell‐derived EVs transfected/co‐incubated to normal cell lines. After transfection, EVs were isolated from modified normal cells and cells as well as EVs characterised via FT‐IR, AFM, DLS, and immunofluorescence imaging.

Results : Transfection/co‐incubation will help us to understand how efficiently cancer derived EVs influence the phenotype and behaviour of non‐cancerous cells, potentially shedding light on the mechanisms that underlie metastatic processes. Taken together, 2D system datasets revealed that cancer EV delivery to normal cells had a profound effect on normal cells' physical properties, as evident from IR spectroscopy (surface functionality), DLS (EV count), flow cytometry (CD‐63 biomarker expression), AFM measurements (diameter), and confocal imaging analysis (shape and size), as well as increased proliferation of the modified normal cells.

Summary/Conclusion : 2D datasets reveal that normal cells take up cancer cell EVs, then modified normal cells become more active to secrete a large number of EVs. 3D culture datasets generation is on the way. In the future, mass spectrometry and PCR analysis will be performed to elucidate further insights into cancer EV‐based cellular crosstalk.

Tumoural hypoxic extracellular vesicles protect the triple‐negative breast tumour microenvironment from macrophage phagoptosis

Bianca C. Pachane 1,2 , Pedro H. T. Bottaro 1 , Aline M. Machado 1 , Cynthia A. de Castro 3 , Gabriela Guerra 1 , Larissa T. Gozzer 1,4 , Marina M. Grigoli 5 , Artur D. Zutião 5 , Angelina M. Fuzer 5 , Marcia R. Cominetti 5 , Wanessa F. Altei 2,6 , Heloisa S. Selistre‐de‐Araujo 1

1 Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Universidade Federal de São Carlos—UFSCar, São Carlos, SP, Brazil; 2 Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP, Brazil; 3 Pathology and Biocompatibility Laboratory, Department of Morphology and Pathology, Universidade Federal de São Carlos—UFSCar, São Carlos, SP, Brazil; 4 Universidade de Aveiro, Aveiro, Portugal; 5 Department of Gerontology, Universidade Federal de São Carlos—UFSCar, São Carlos, SP, Brazil; 6 Radiation Oncology Department, Barretos Cancer Hospital, Barretos, SP, Brazil

Introduction : The highly metastatic triple‐negative breast cancer (TNBC) relies on its tumour microenvironment (TME) to maintain phenotypic heterogeneity and progression. Extracellular vesicles from hypoxic TNBC were previously shown to aid tumoural invasion, but their function in the tumour microenvironment is still unclear. In this study, we sought to understand the effect of TNBC‐derived hypoxic EVs (EVh) in the TME in vitro.

Methods : EVh were separated from hypoxic (1% O 2 ) MDA‐MB‐231 conditioned media using ultracentrifugation and filtration, and characterized following MISEV2023 guidelines. Using direct, indirect and multicellular circulating co‐culture (MC‐CC) models, we observed how EVh interfered with tumoural and endothelial cells, fibroblasts, monocytes and macrophages.

Results : EVh promoted monocyte differentiation to M2‐like macrophages and inhibited macrophage‐derived phagocytosis in endothelial and tumoural cells. The protection of endothelial, tumoural and stromal cellular integrity by EVh increased pro‐tumoural and pro‐angiogenic signalling, collagen matrix synthesis and potential differentiation to cancer‐associated fibroblasts. The multicellular circulating co‐culture method was corroborated by other co‐culture protocols, leading to an adequate system with potential for investigating other tumour‐related processes, including circulating tumour cells and metastasis.

Summary/Conclusion : Overall, our results suggest that tumoural EVs from hypoxic cells are essential for maintaining a cohesive TME and contribute to understanding tumour basic biology.

Funding : This work was funded by the São Paulo Research Foundation through grants 2019/05149‐9, 2021/01983‐4, and 2022/04146‐9; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), code 001; and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Investigating lipid raft composition of extracellular vesicles secreted by triple‐negative breast cancer and non‐cancerous breast cells to develop an optimal cancer‐targeting EV‐mimetic formulation

Oliwia Majchrzak 1,2 , Carolina Lopes Silva 1,2 , Isabel Meister 1,2,3 , Stoyan Tankov 1 , Julien Boccard 1,2,3 , Olivier Jordan 1,2 , Paul Walker 1 , Serge Rudaz 1,2,3 , Gerrit Borchard 1,2

1 University of Geneva, Switzerland; 2 Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), Switzerland; 3 Swiss Center of Applied Human Toxicology, Switzerlandjlv

Introduction : Extracellular vesicles (EVs) play a crucial role in intercellular communication between donor and recipient cells [1, 2]. Numerous studies have reported that the lipid raft composition within EV membranes varies significantly depending on the originating cell type [3]. However, little is known about the correlation between EV lipid profiles and selective affinity for target cells. In this study, we investigated differences in the lipid raft composition of EVs released from breast cancer and healthy cells using untargeted lipidomic profiling. This work aims to determine how specific lipid content in EV membranes may be leveraged in the design of cancer‐specific delivery nanosystems.

Methods : We investigated the EVs secreted by triple‐negative breast cancer (TNBC) cell lines (MDA‐MB‐231, MDA‐MB‐453, MDA‐MB‐468) and a healthy breast cell line (MCF10A). EV isolation was performed using a double ultracentrifugation. Characterization of the EVs was conducted according to [1], focusing on the quantification of particle number concentration and particle size using NanoTracking Analysis. Morphology was assessed via transmission electron microscopy. Protein composition was confirmed through western blot. Lipidomic profiling was conducted with an untargeted reversed‐phase liquid chromatography hyphenated with high resolution mass spectrometry method.

Results : Fundamental characterization defined the EVs' size range between 150 and 300 nm, with a zeta potential of −30 mV at a sample pH of 7.2 and a particle concentration of 5 × 10 9 particles/mL consistent across all cell sources from which the EVs were isolated. TEM imaging highlighted lipid raft folding. EVs were positive for CD9, Hsp70, and TSG101 markers. Lipid annotation was conducted on bond type level based on fragmentation spectra and retention time behaviour. This approach allowed the monitoring of 567 lipid species after quality filtering and curation. The first axis of principal component analysis (PCA) clearly discriminated EVs derived from cancerous versus healthy cells, mainly through hexosylceramides. PC2 highlighted differences among EVs secreted by TNBC lines driven by phosphatidylcholines and ether phosphatidylethanolamines.

Summary/Conclusion : Lipidomic data support the biological hypothesis that the secretome of TNBC cells diverges markedly from that of healthy cells. These findings will contribute to developing EV‐mimetic formulations envisioned as drug delivery vehicles targeted to cancer cells.

Funding : Swiss_Government_Excellence_Scholarship_2022.0159 [1] DOI:10.1002/jev2.12404 [2] DOI:10.1172/JCI87316 [3] DOI:10.3389/fmolb.2023.1198044.

Subtopic Disease/therapy‐relevant mechanism(s) of action 1

Cerebellar mitovesicle secretion is altered in spinocerebellar ataxia and regulated by the activity of the potassium channel Kv3

Pasquale D'Acunzo 1,2 , Yalan Zhang 3 , Leonard K. Kaczmarek 3 , Efrat Levy 1,2

1 Nathan S. Kline Institute for Psychiatric Research, NY, USA; 2 NYU Grossman School of Medicine, NY, USA; 3 Yale University School of Medicine, CT, USA

Introduction : Kv3 voltage‐gated potassium channels mediate the repolarization of fast‐firing neuronees, including Purkinje cells. Dominant negative mutations in the Kv3.3 subunit trigger uncontrolled duration and frequency of action potentials and cause spinocerebellar ataxia type 13 (SCA13), a neurodegenerative disorder characterized by Purkinje cell loss and ataxia of gait. We have previously shown that Purkinje neuronees in the cerebellum of a mouse model of SCA13 bearing the disease‐causing mutation Kv3.3‐G592R are characterized by several endocytic abnormalities, including lysosome impairments, accumulation of undigested inclusions, and a higher number of multivesicular bodies. These abnormalities are associated with higher secretion of exosomes, without affecting ectosomes. We hypothesized that in Kv3.3‐G592R mice the concomitant presence of ion imbalance caused by the lower activation of Kv3 channels and downstream mitophagy block caused by endolysosomal abnormalities leads to alterations in mitochondria that reverberate to changes in mitovesicles, the mitochondria‐derived extracellular vesicles (EVs). Therefore, we investigated changes in mitochondria and mitovesicles in the brain of male and female Kv3.3‐G592R and Kv3.3 knockout (KO) mice, as compared with wild‐type (WT) littermate controls.

Methods : EVs were isolated from cerebella and right hemibrains of Kv3.3‐G592R, Kv3.3‐KO, and WT mice using a high‐resolution iodixanol gradient and analysed by transmission electron microscopy (TEM), NTA, total protein content, and Western blotting. Mitochondria were studied in the left hemibrains and cerebella by Western blotting and TEM.

Results : The number of mitovesicles in the cerebellar extracellular space was inversely correlated with the Kv3 channel activity: lowest in WT, higher in Kv3.3‐G592R (impaired activity), and highest in Kv3.3‐KO (no activity) mice. Mitochondrial fragmentation markers (phospho‐Drp1 Ser616, Ser637) followed the same trends, consistent with a higher production and secretion of mitovesicles. Under TEM, Kv3.3 channels were found to be enriched at the contact sites between the plasma membrane and mitochondria, both in WT and Kv3.3‐G592R mice, suggesting a direct regulation of the mitochondrial ion metabolism mediated by Kv3.3.

Summary/Conclusion : Mitovesicles are a previously unidentified player altered in SCA13 cerebella. Mechanistically, we describe a novel pathway in vivo linking mitochondria and mitovesicle homeostasis to plasma membrane depolarization, shedding new light on the still obscure biology that regulates mitovesicle secretion in the brain.

Funding : NIA ( AG086510 ).

Exploring the role of mesenchymal stem cell‐derived extracellular vesicles in Alzheimer's disease: the impact of MSC preconditioning on targeted therapeutic efficacy

Eva Costanzi 1 , Marco Grisetti 1 , Maddalena Bettucci 1 , Ramona Meanti 1 , Elena Lonati 1 , Giulia Morello 3 , Valeria Vasciaveo 3 , Michela Guglielmotto 3 , Antonio Torsello 1 , Alessandra Bulbarelli 1 , Elena Tamagno 3 , Mario Buffelli 2 , and Silvia Coco 1

1 University of Milano‐Bicocca, Italy; 2 University of Verona, Italy 3 University of Turin, Italy

Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) hold promise as therapeutic agents for neurodegenerative disorders, including Alzheimer's disease (AD), by carrying bioactive molecules across the blood‐brain barrier. MSC‐EVs modulate the neural environment by reducing inflammation and preventing neuronal death. Recent studies indicate that MSC‐preconditioning may influence EV cargo, promoting specific therapeutic effects, particularly anti‐inflammatory and anti‐apoptotic responses. Our study explores how preconditioning with oxygen‐glucose deprivation (OGD) or a cytokine cocktail (TNF‐α and IFN‐γ) affects the therapeutic efficacy of MSC‐EVs in both in vitro and in vivo models.

Methods : Human bone marrow‐derived MSCs were preconditioned with proinflammatory cytokines TNFα (20 ng/mL) and IFNγ (25 ng/mL) for 24 h or with OGD for 3 h. EVs were isolated from conditioned MSC culture medium using ultracentrifugation and characterized by immunoblotting, scanning electron microscopy (SEM) and nanoparticle tracking analysis (NTA) to assess EV marker presence, morphology, size and concentration. For in vitro assays, N2A neuroblastoma cells underwent 4 h of OGD insult, followed by the administration of MSC‐EVs obtained from the two preconditioning methods and a 24‐h recovery period. Apoptotic gene expression (casp3, casp7, bax, bcl‐2) was analysed by qRT‐PCR. In 3xTg and 5xFAD AD mouse models, EVs from preconditioned MSCs were administered intranasally over 2 days, and behavioural tests were conducted 15 and 30 days post‐treatment to assess possible cognitive improvements.

Results : MSC preconditioning using two distinct methods led to the release of EVs with different characteristics. Cytokine preconditioning produced smaller EVs (< 110 nm diameter), while OGD preconditioning yielded larger EVs (> 110 nm diameter) with enhanced anti‐apoptotic properties. Preliminary findings in 3xTg and 5xFAD mouse models suggest overall improvement in cognitive impairments: cytokine‐preconditioned EVs more effectively reduced anxiety and improved short‐term memory, while OGD showed a stronger effect in enhancing spatial memory.

Summary/Conclusion : These findings suggest that MSC‐EVs preconditioned with different stimuli offer unique therapeutic profiles, underscoring the potential to tailor EV‐based therapies for AD‐associated neuroinflammation.

Funding : PRIN: PROGETTI DI RICERCA DI RILEVANTE INTERESSE NAZIONALE—Bando 2022 Prot. 202229X8HW PRIN: PROGETTI DI RICERCA DI RILEVANTE INTERESSE NAZIONALE—Bando 2022 PNRR Prot. P2022LR49L.

A novel extracellular vesicle‐based approach for targeted ferroptosis to overcome chemoresistance in lung cancer

Presenter: Anjugam Paramanantham

University of Missouri, USA

Introduction : Lung cancer (LC) remains a leading cause of cancer mortality, with cisplatin (CDDP) and its analogues as standard treatments. However, chemoresistance, due to evasion of apoptosis, limits its effectiveness, resulting in a 5‐year survival rate below 10% for advanced LC. To address this challenge, ferroptosis, an iron‐dependent non‐apoptotic cell death mechanism, offers a promising approach to bypass traditional cell death pathways and overcome resistance. In this study, we developed ExoFeRTF, a novel delivery system utilizing extracellular vesicles (EVs) to encapsulate iron oxide nanoparticles (IONPs), functionalized with transferrin (TF). These EVs specifically target LC cells with transferrin receptor overexpression, promoting ferroptosis and reversing chemoresistance.

Methods : ExoFeRTF was engineered by loading IONPs into TF‐decorated EVs derived from MRC‐9 lung fibroblast cells. The EVs were characterized following the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, ensuring rigorous validation of their size, purity, and functionality. This physicochemical characterization included nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, and western blotting for the detection of EV‐specific markers. The therapeutic potential of ExoFeRTF was tested in CDDP‐resistant LC cell lines, patient‐derived tumouroids (PDTs), circulating tumour cell‐derived cells, and xenograft models. Ferroptosis induction was assessed by cell viability, mitochondrial disruption, glutathione (GSH) depletion, and changes in ferroptosis markers (xCT/SLC7A11, GPX4). Tumour targeting and ferroptosis induction were confirmed using magnetic resonance imaging (MRI) and Prussian Blue staining.

Results : ExoFeRTF demonstrated structural stability, tumour specificity, and ferroptosis induction in CDDP‐resistant LC cells, PDTs, and xenograft models. Significant mitochondrial damage, GSH depletion, and downregulation of ferroptosis markers were observed. Additionally, ExoFeRTF inhibited Nrf2 signalling, enhancing susceptibility to cisplatin.

Summary/Conclusion : This study demonstrates the potential of ExoFeRTF, an EV‐based system, to overcome LC chemoresistance by inducing ferroptosis. Following MISEV guidelines, ExoFeRTF offers targeted, efficient strategy for addressing treatment‐resistant LC.

Funding : This study was supported, in part, by the University of Missouri School of Medicine (UM‐SoM) startup funds (A.S.) and the Department of Defense (DoD) through the Lung Cancer Research Program (LCRP) award W81XWH‐22‐1‐0016 (A.S.), the National Institutes of Health R01HL140684 (H.Y.), and the Department of Veterans Affairs (VA) Clinical Science Research & Development (CSR&D) Merit Award CX002498 ‐01A2 (J.K.).

Exosomal miR‐10b‐3p deriving from umbilical mesenchymal stem cells alleviate ischemia‐reperfusion acute kidney injury by targeting and inhibiting renal tubular CH25H/25OHC

Presenter: Qin Yang

Southeast University, China (People's Republic)

Introduction : Acute kidney injury (AKI) is a common critical condition in clinical practice, and there is no recognized targeted therapy. Mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) have shown promising therapeutic effects in various AKI animal models, but their mechanisms are not yet fully understood. Therefore, this study aims to investigate the therapeutic effects of extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hucMSCs) on AKI induced by bilateral renal ischaemia‐reperfusion (I/R) models and their potential mechanisms.

Methods : HucMSCs‐EVs were harvested via xenogenic‐free (human platelet lysate) medium culture. The biodistribution of hucMSCs‐EVs was imaged using the IVIS imaging system. The therapeutic effects of hucMSCs‐EVs in renal I/R injury were assessed, and transcriptome changes in mouse kidney tissue were analysed through RNA sequencing (RNA‐seq) datasets. Multi‐omics analysis of EVs identified potential miRNAs associated with changes in 25‐hydroxycholesterol (25OHC).

Results : Ex vivo imaging showed that hucMSCs‐EVs effectively homed to ischaemic kidneys and alleviated tubular interstitial inflammation and cell death following I/R‐AKI. RNA‐seq analysis revealed that hucMSCs‐EVs regulated tubular lipid metabolic disturbances after AKI, while kidney‐targeted lipidomics revealed abnormal accumulation of cholesterol metabolites after I/R. Differential gene expression analysis demonstrated that hucMSCs‐EVs suppressed the expression of cholesterol 25‐hydroxylase (CH25H). ELISA results suggested that hucMSCs‐EVs primarily mitigate the abnormal accumulation of 25OHC after AKI by downregulating CH25H expression. Multi‐omics analysis of EVs showed that the miRNAs within EVs may be involved in the regulation of CH25H, and miRNA prediction analysis further identified potential miRNAs targeting CH25H. In vitro experiments showed that miR‐10a‐3p derived from hucMSCs‐EVs inhibited CH25H expression in tubular cells, thereby improving the abnormal accumulation of 25OHC and reducing tubular interstitial inflammation and cell death. Finally, overexpression or inhibition of miR‐10a‐3p enhanced or diminished the protective effects of hucMSCs‐EVs in I/R mice, indicating that hucMSCs‐EVs alleviate I/R‐AKI by carrying miR‐10a‐3p to target and inhibit renal tubular CH25H/25OHC.

Summary/Conclusion : We demonstrated that hucMSCs‐EVs can target damaged kidneys and reduce the accumulation of 25OHC after I/R‐AKI by carrying miR‐10a‐3p to target the key enzyme CH25H, thereby alleviating inflammation and death in tubular cells, improving ischaemic AKI, and promoting renal tubular repair.

Decoding the miRNA cargo of bone marrow‐derived MSC‐EVs and their mRNA targets: A transcriptomic approach to improve the management of corneal disorders

Manon Jammes 1,† , Yedizza Rautavaara 2,† , Seyedmohammad Moosavizadeh 1,3,† , Petros Moustardas 2 , Trung Bach 1 , Abbas Tabasi 1 , Jiemin Wang 1 , Neil Lagali 2,* , Thomas Ritter 1,3,*

1 Regenerative Medicine Institute, School of Medicine, University of Galway, Galway, Ireland; 2 Department of Biomedical and Clinical Sciences, Faculty of Medicine, Linköping University, Linköping, Sweden; 3 CURAM Centre for Research in Medical Devices, University of Galway, Galway, Ireland †/* These authors contributed equally to this work .

Introduction : Corneal transparency, avascularity and structural organization are essential in conducting the light to deeper structures, and the disruption of this integrity unfailingly leads to impaired eyesight. Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs; MSC‐EVs) are known to mediate the anti‐inflammatory, immunomodulatory and regenerative properties of their parental cells, emerging as a promising approach to address corneal diseases. In the last years, licensing MSCs with cytokines revealed considerable potential in enhancing their therapeutic properties. Despite undeniable accumulating evidence of licensing efficacy, the mechanisms underlying these effects remain to be understood. Latest investigation efforts identified miRNA signalling as a promising effector in mediating MSC‐EV therapeutic potential.

Methods : Human bone marrow (BM)‐MSCs derived from three donors were cultured for 72 h with or without 50 ng/mL IFN‐γ and 50 ng/mL TGF‐β1, and EVs were isolated through size exclusion chromatography. The miRNA content of MSC‐EVs was sequenced and quantified before determining the differential expression profile using EVs derived from naïve MSCs as a reference control. Wound closure of immortalised human corneal epithelial cells (HCE‐T) treated with naïve and licensed EVs derived from one MSC donor was monitored over 11 h, followed by total RNA extraction and transcriptomic analysis of mRNA expression.

Results : miRNA sequencing revealed that EV content was highly donor dependent. Differential expression analysis enabled the determination of the most up‐ and down‐regulated miRNA for each donor in EVs derived from licensed MSCs compared with naïve MSCs. Additionally, the HCE‐T wound closure rate was significantly enhanced when treated with EVs derived from licensed MSCs, and mRNA profiling allowed the identification of mRNA whose expression was significantly up‐ and down‐regulated. The combination of mRNA profiling results and the miRNA targets predicted using online databases enabled us to draw up a list of the most promising miRNA‐mRNA interactions possibly mediating the MSC‐EV therapeutic effect.

Summary/Conclusion : EVs derived from licensed the BM‐MSC improved the proliferation and migration potential of HCE‐T. Despite major donor‐dependent variations, the miRNA profiling of MSC‐EVs revealed promising candidates aligning with the HCE‐T transcriptomic analysis. Altogether, these findings provide a solid foundation for further exploring miRNA signalling in MSC‐EVs as a treatment for corneal epithelial disorders.

Cargo delivery via tumour‐derived extracellular vesicle enhances T cell stemness via enhancing the cGAS‐STING pathway

Presenter: Seong‐A Kim

Korea Institute of Science and Technology, Republic of Korea

Introduction : The cGAS‐STING pathway responds to cytosolic DNA within cells in the context of tumour immunity, infection, and various inflammatory diseases, inducing the recruitment and activation of immune cells. DNA damage accumulation leads to the formation of micronuclei and cytosolic material, activating cGAS‐STING. In tumours, this cytosolic material is engulfed by macrophages and dendritic cells, promoting their maturation and activation. Additionally, cGAS‐STING activation in non‐phagocytic cells enhances antitumour immunity. Intrinsic activation of STING within CD8 T cells is essential for generating and sustaining CD8 T cells with a stem‐like phenotype, contributing to durable immune responses. Here, we developed the cargo‐loading strategy into tumour‐derived EVs and investigated how they affected an antitumour effect.

Methods : Tumour‐derived extracellular vesicles (EVs) were isolated to study the impact of tumour‐derived EVs on T cell activation and stemness following the MISEV 2023 guidelines. The content within secreted tumour‐derived EVs was quantified after EV isolation. Flow cytometry was conducted to evaluate the activation of the cGAS‐STING pathway in tumour cells and T cells in response to cargo‐loaded tumour‐derived EV both in vitro and in vivo. Modulation of EV secretion in vivo, achieved through blocking EV generation, tumour EV injection, or synthetic cargo complexes, was analysed to determine the impact of EVs on enhancing T cell stemness in vivo.

Results : We observed an increase in the cargo following the induction of cargo loading in tumour cells. The cargo‐loaded EVs activated the cGAS‐STING pathway and promoted immune response in tumour cells, an effect not observed with EVs without inducing cargo loading. The cargo‐loaded tumour‐derived EVs enhanced the proportion of cGAS‐STING‐activated TCF1+ CD8 T cells in tumour microenvironment. Inhibition of EV secretion reduced immune cell uptake of tumour‐derived EVs, particularly by CD8 T cells, and decreased the proportion of TCF1+ CD8 T cells in vivo. Injection of cargo‐loaded tumour EVs promoted cGAS‐STING activation and increased TCF1+ CD8 T cells within the tumour.

Summary/Conclusion : The induction of cargo loading in tumour increased the cargo loaded into tumour‐derived EVs. The cargo‐loaded tumour‐derived EVs enhanced antitumour immune response by activating the cGAS‐STING pathway and upregulating TCF1 expression in CD8 T cells.

Harnessing the power of dental pulp stem cells: exosomes as a promising therapy for keloid inhibition

Presenter: Ming Yan

Universitätsklinikum Hamburg‐Eppendorf, Germany

Introduction : Dental pulp stem cells (DPSCs), discovered in 2000, are valuable for regenerative medicine due to their low immunogenicity, high availability, and multidirectional differentiation. However, risks like immune responses and tumour formation limit traditional stem cell therapies, increasing interest in DPSCs‐derived exosomes (DPSCs‐Exos) as a safer, cell‐free alternative. This study explores the effects of DPSCs‐Exos on keloid fibroblasts, assessing their potential to inhibit fibrosis without affecting normal fibroblast behaviour.

Methods : Dental pulp stem cells (DPSCs) were isolated by the tissue block culture method, and keloid fibroblasts (KFs) and normal skin fibroblasts (NFs) were isolated by enzymatic digestion. DPSCs‐Exos were obtained by ultracentrifugation, and KFs and NFs were treated with different concentrations of DPSCs‐Exos (blank control, 25, 50, 100 µg/mL). The CCK‐8 assay was used to detect its effect on cell proliferation; cell migration ability was detected by cell scratch and Transwell assay; synthesis of extracellular matrix was detected by hydroxyproline content assay; and the expression level of fibrotic disease‐related genes in cells was detected by PCR assay.

Results : DPSCs‐Exos added to the culture medium were able to be taken up by fibroblasts, and DPSCs‐Exos affected the biological behaviours of NFs and KFs. DPSCs‐Exos promoted the proliferation of NFs but had no significant effect on KFs. They inhibited the migration of KFs and the synthesis of extracellular matrix but did not affect NFs ( p < 0.05). Additionally, DPSCs‐Exos inhibited the expression of genes related to the pro‐fibrotic phenotype of KFs, such as CTGF, TGF‐β1, and TGF‐β2, while having no significant effect on NFs ( p < 0.05).

Summary/Conclusion : Our study confirms that under in vitro conditions, DPSCs‐Exos can inhibit the biological behaviours of KFs associated with fibrosis without adversely affecting the normal biological behaviours of NFs. This finding is of great significance for the treatment of keloids, as well as the inhibition of keloid formation, and suggests to us the great potential that DPSCs‐Exos can be used as a cell‐free regenerative drug.

Mesenchymal stem cell‐derived extracellular vesicles delivered let‐7a‐5p and miR‐16‐5p to ameliorate inflammation and glycolysis by inhibition of the TLR4/NF‐κB pathway in sepsis‐acute kidney injury

Presenter: Zhi‐qing Chen

Southeast University, China (People's Republic)

Introduction : Human umbilical cord mesenchymal stem cell‐derived extracellular vesicles (hucMSC‐EVs) have shown great therapeutic potential as a cell‐free therapy in various kidney diseases. However, the role and underlying mechanisms of hucMSC‐EVs in sepsis‐acute kidney injury (S‐AKI) remain largely unclear.

Methods : hucMSC‐EVs were purified and characterized. Animal and cell models of S‐AKI were established by CLP and LPS, respectively. The biodistribution of hucMSC‐EVs in mice and cellular uptake in HK‐2 cells were detected by the IVIS system and flow cytometry, respectively. The effects of hucMSC‐EVs on survival rate, renal function and histopathological changes of S‐AKI mice were also determined. Then, the mRNA and protein expression levels of inflammation and glycolysis related genes were detected in both renal tissues and HK‐2 cells after hucMSC‐EVs administration. miRNA profiles in hucMSC‐EVs were analysed by high‐throughput miRNA sequencing. The differentially expressed genes after hucMSC‐EVs intervention were identified by tissue RNA‐seq, and the potential regulatory mechanisms were further verified using a series of experiments.

Results : hucMSC‐EVs homed to the injured kidneys and, in a dose‐dependent manner, reduced body weight loss, increased survival and improved renal function in S‐AKI mice. Similarly, flow cytometry revealed that LPS‐induced injury in HK‐2 cells increased the uptake of hucMSC‐EVs. Furthermore, hucMSC‐EVs significantly inhibited inflammation and glycolysis both in vivo and in vitro. Mechanically, let‐7a‐5p and miR‐16‐5p, which are highly enriched in hucMSC‐EVs, co‐target and inhibit the TLR4/NF‐κB pathway in tubular epithelial cells. Finally, inhibition of let‐7a‐5p and miR‐16‐5p attenuated the renoprotective effect of hucMSC‐EVs against S‐AKI.

Summary/Conclusion : We demonstrate that let‐7a‐5p and miR‐16‐5p carried in hucMSC‐EVs ameliorate S‐AKI by alleviating inflammation and glycolysis through targeted inhibition of the TLR4/NF‐κB pathway. This study provides new theoretical and experimental foundations for the application of hucMSC‐EVs in the treatment of S‐AKI.

Over 6 years and 2500 procedures using single‐source MSC exosomes in patients

Presenter: Edward H. Park

Recharge Biomedical, USA

Introduction : Because of the lack of specific MHC self‐identity antigens on MSC exosomes, there is reason to believe that exogenously‐sourced EVs would work similarly to endogenously‐produced ones and potentially help repair and restore function in vivo.

Methods : After informed consent, including the clear understanding that the use of MSC exosomes was not FDA‐approved, nor the standard of care, over 500 patients, in over 1000 encounters, underwent over 2500 procedures.

Results : The presenter will review the procedures, complications, general response rates, and possible mechanisms of action for these powerful and naturally regenerative EVs. The presentation will include a cursory review of the omics of the EV product that was used.

Summary/Conclusion : With multiple trials in the USA and Europe of MSC exosomes for various indications, reviewing the common reactions, complications, and effects of a therapeutic modality that may soon be available for off‐label use in the clinic is valuable. The presenter's clinical experience will demonstrate high safety, efficacy, and predictable benefits from the use of exogenous MSC exosomes.

Detection of integrin α6β4 on CD9‐enriched extracellular vesicles derived from tumour cells informs the treatment with CY001 to optimize precision medicine strategies for pancreatic ductal adenocarcinoma

Wei‐Ni Tsai 1 , Tai‐Shan Cheng 1 , Peter Mu‐Hsin Chang 1 , Hsueh‐Ju Lu 2 , Ly James Lee 1 , Chi‐Ying F. Huang 1

1 National Yang Ming Chiao Tung University, Taiwan; 2 Chung Shan Medical University, Taiwan Ly James Lee and Chi‐Ying F. Huang are co‐senior authors .

Abstract unavailable

Epimedium brevicornu Maxim‐derived extracellular vesicle‐like particles reverse postmenopausal osteoporosis by promoting angiogenesis in human umbilical vein endothelial cells

Presenter: Xiuping Cai

The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, China (People's Republic)

Introduction : Postmenopausal osteoporosis (PMOP) is a common and multiple systemic bone disease. Epimedium brevicornu Maxim (EP), the sovereign herb used for the treatment of PMOP among traditional Chinese medicines, has active ingredients and mechanisms that are still unclear. Herein, Epimedium brevicornu Maxim‐derived extracellular vesicle‐like particles (EP‐EVLPs) were naturally isolated from fresh EP leaves and strongly demonstrated the potential targeted treatment of EP‐EVLPs for PMOP.

Methods : EP‐EVLP was isolated from fresh Epimedium brevicornu Maxim leaves by differential ultracentrifugation. They were characterized and identified by transmission electron microscopy (TEM), flow nano analyser, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), agarose gel electrophoresis, thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). In vivo experiments were conducted to explore the bone‐targeting, anti‐osteoporosis activity and biocompatibility of EV‐EVLP. In vitro, alizarin red staining (ARS) and qRT‐PCR were used to detect osteogenic differentiation of hBMSCs and MC3T3‐E1. In addition, Human Umbilical Vein Endothelial Cells (HUVECs) activity and apoptosis were evaluated by flow cytometry and CCK‐8, while angiogenesis of HUVECs was verified by wound healing assay, Transwell migration assay and tube formation assay.

Results : Epimedium brevicornu Maxim‐derived extracellular vesicle‐like particles (EP‐EVLPs) were naturally isolated from fresh EP leaves and strongly demonstrated the potential targeted treatment of EP‐EVLPs for PMOP. EP‐EVLPs had a cup membrane structure and contained a variety of proteins, small‐molecule compounds, lipids, and nucleic acids. In vivo EP‐EVLPs were able to accumulate and act on bone tissue to exert an anti‐osteoporotic effect without toxicity. It was interesting that the results of metabolomics and network pharmacology analyses combined with cellular experiments revealed that the anti‐osteoporotic effect of EP‐EVLPs was achieved by promoting angiogenesis and then improving the bone microenvironment in HUVECs instead of promoting osteogenic differentiation.

Summary/Conclusion : In summary, our study showed that EP‐EVLPs may be the effective components of EP with the bone‐targeting activity and anti‐osteoporosis effect, which may represent a new clinically promising nano platform for the prevention and treatment of PMOP.

Tissue‐derived EV transcriptomic profile reveals prostate cancer evolutionary response and molecular signature following neoadjuvant therapy

Yanhao Dong 1 , Donglei Yang 2 , Xinxing Du 1 , Pengfei Hou 2 , Cong Hu 1 , Zehong Peng 1 , Zhicheng Zheng 1 , Wei Zhang 1 , Penghui Liao 1 , Tianyang Wu 1 , Kedi Yuan 1 , Lihan Lai 1 , Yingjie Zhu 1 , Jiahua Pan 1 , Liang Dong 1 , Pengfei Wang 2 , Wei Xue 1

1 Renji Hospital Shanghai Jiao Tong University School of Medicine, Department of Urology, Shanghai, China; 2 Renji Hospital, Shanghai Jiao Tong University School of Medicine, Institute of Molecular Medicine, Dept. of Laboratory Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Shanghai, China Liang Dong, Pengfei Wang, and Wei Xue are co‐senior authors .

Introduction : Prostate cancer (PCa) continues to be a growing health concern. Understanding the evolutionary and molecular changes driving these responses is of great importance. However, identifying materials that accurately reflect both tumour‐specific molecular characteristics and tumour microenvironment (TME) remains challenging. Tissue‐derived extracellular vesicles (EVs) are lipid‐bilayer particles (30–150 nm in diameter) carrying nucleic acids and other cargo, playing a significant role in cellular communication and influencing cancer progression. This study investigates tissue‐derived EVs as potential indicators of tumour adaptation to therapeutic pressures in PCa, providing new insights into their role in cancer TME and its evolutionary response to neoadjuvant treatment.

Methods : Tissue‐derived EVs were collected from prostatectomy samples of 18 high‐risk PCa patients, including 6 treated with neoadjuvant hormone therapy (NHT), 6 with neoadjuvant chemohormonal therapy (NCHT), and 6 who were treatment‐naïve. Tissue‐derived EVs were isolated, sequenced, and aligned to a reference genome for RNA quantification and differential expression analysis. Differentially expressed genes were further annotated and analysed for pathway enrichment. Clinical follow‐up was conducted to assess therapeutic impact.

Results : We successfully characterized tissue‐derived EVs from the samples. With a fold change > 1.5 and p < 0.05, we identified 219 and 288 upregulated genes in the NHT and NCHT groups, respectively, compared to the treatment‐naïve group. Comparing NHT with NCHT, we found 124 upregulated and 18 downregulated genes in NHT. Gene ontology analysis revealed enriched pathways related to tumour growth and invasion in treatment‐naïve patients, while systemic treatment appeared to reduce these processes. Chemotherapy was associated with enhanced antigen processing and presentation pathways, as indicated by KEGG analysis. Immune profiling showed increased T‐cell infiltration and dendritic cell stimulation in the NCHT group compared to NHT and untreated groups, suggesting that systemic therapy may promote a more immunoresponsive TME.

Summary/Conclusion : This study is the first to identify evolutionary changes in tissue‐derived EVs in response to systemic treatment in PCa, providing insights into the molecular adaptations within the TME. Further analysis will deepen our understanding of the role of tissue‐derived EVs in TME modulation and their potential as therapeutic targets in PCa.

Funding : This research was funded by the National Natural Science Foundation of China (82227801, 82472142).

Extracellular vesicle characteristics are indicative of extracorporeal photopheresis response for graft versus host disease

Presenter: Rachel E. Crossland

Newcastle University, UK

Introduction : Graft versus host disease (GvHD) is a serious complication of allogeneic haematopoietic stem cell transplantation (HSCT), affecting 40‐70% of patients. Steroid treatment can cause adverse effects, and lead to refractory disease. Extracorporeal photopheresis (ECP) therapy is frequently used to treat steroid‐refractory disease; however, the exact mechanism of action is unknown, and biomarkers to determine ECP response are lacking. This study investigates the role of circulatory extracellular vesicles (EVs) during ECP therapy for GvHD.

Methods : Serum EVs were isolated and characterised from n = 12 paediatric ECP patients ( n = 6 complete response (CR), n = 6 partial/non‐responders (PR)) at sequential timepoints (TP). EV microRNA cargo, surface marker and proteomic profiling were performed using nCounter, flow/imaging cytometry and data‐independent acquisition mass spectrometry, respectively. Expression of serum immuno‐oncological cytokines was assessed by electrochemiluminescence.

Results : EV modal size was significantly larger for patients who reached CR vs. PR. Analysing EV microRNA cargo showed three principle component analysis (PCA) clusters. Eight microRNAs were significantly differentially expressed in CR versus PR patients ( p < 0.05). MicroRNA target analysis showed enrichment of GvHD and/or ECP‐related pathways, such as allograft rejection, interleukin and TLR‐signalling. The most highly expressed EV‐microRNA in all patients was anti‐inflammatory miR‐451a. Focusing on bulk EV surface markers, there was a significant interaction between the ECP cycle and response for markers associated with antigen presentation and effector immune cells. Assessing surface markers on a single EV level, there was differential expression of markers associated with NK cells, platelets, MHC‐II and PDL‐1. Gene ontology enrichment analysis of proteomics data revealed significantly differentially expressed proteins between CR versus PR. Namely, proteins associated with proteasomal degradation and complement activation were upregulated in PR, indicative of an altered immunomodulatory response. Serum soluble factors were associated with ECP response, including altered expression of inflammation, Treg and IFN‐g markers known to be targeted by microRNAs altered in CR versus PR EVs.

Summary/Conclusion : EV surface markers and cargo may contribute to, and help further elucidate, the mechanisms of ECP action. EV characteristics during ECP show potential as biomarkers of treatment response.

Funding : The Pathological Society of Great Britain, the UK Photopheresis Society, Newcastle Hospitals Charity, and Mallinckrodt Pharmaceuticals.

The potential mechanisms of pulmonary fibrosis management via mesenchymal stromal cell extracellular vesicles include alveolar macrophage polarization regulation

U. Dyachkova, M. Vigovskii, N. Basalova, V. Popov, E. Lagereva, R. Lazarev, A. Efimenko, O. Grigorieva

Medical Research and Educational Institute, Lomonosov Moscow State University, Moscow, Russia

Introduction : Extracellular vesicles secreted by mesenchymal stromal cells (EV‐MSC) are considered as possible drugs for the pulmonary fibrosis treatment. EV‐MSCs transfer specific non‐coding miRNAs that are able to inhibit fibroblast differentiation into myofibroblasts, which is a key process in the fibrosis development and progression [doi:10.3390/cells9051272]. EV‐MSC administration to mice with bleomycin‐induced pulmonary fibrosis led to decreased fibrosis severity due to miR‐29c and miR‐129 transfer [doi:10.1038/s12276‐023‐01017‐w].

Methods : Using in vivo imaging of fluorescent signal accumulation (IVIS) we found that PKH26/DiR labelled EV‐MSC could be detected in the lungs for at least 3 h after intratracheal administration to mice with bleomycin‐induced pulmonary fibrosis, which is not observed after EV‐MSC injection into the jugular vein. EV‐MSC can be considered as a potential drug, and to determine its mechanism of action, we evaluated the co‐localization of CD68+ lung macrophages, FAPa+ activated cells, aSMA+ myofibroblasts and labelled EV‐MSC after their intratracheal administration using the lungs immunohistochemical analysis 3 h after EV‐MSC administration.

Results : We found that the majority of EV‐MSCs were co‐localized with CD68+ lung macrophages 3 h after administration, whereas only a small amount of EV‐MSC were co‐localized with FAP+ cells, and EV‐MSC were practically not detected in αSMA+ cells. So, EV‐MSCs in vivo may have their effect by regulating lung macrophage polarization, which are considered the main effector cells of inflammation, which plays a key role in the fibrosis development. To assess the EV‐MSC effect on macrophage polarization we used an in vitro model of macrophages derived from human peripheral blood monocyte differentiation and polarization. Differentiation into macrophages was stimulated by the GM‐CSF for 6 days. Simultaneous action of polarizing factors in the M1‐proinflammatory direction (LPS + IFNg) and EV‐MSC after 4 h led to decreased expression of proinflammatory secreted protein genes of M1 macrophages (IL‐12p35, IL‐6) and increased expression of the M2 macrophage marker (CD200R1).

Summary/Conclusion : Thus, one of the possible EV‐MSC mechanisms of action in pulmonary fibrosis inhibition may be associated with their entry into alveolar macrophages after intratracheal administration and their polarization change and support of anti‐inflammatory M2 phenotype maintenance.

Funding : The study was supported by the RSF grant 23‐15‐00198, https://rscf.ru/project/23‐15‐00198/ (immunohistochemistry staining, macrophage polarization model) and State Assignment of Lomonosov MSU (animal studies).

Subtopic EV biomarker clinical translation 1

Key metabolites of serum extracellular vesicles facilitate precise identification of prostate cancer patients with PI‐RADS ≥ 3 lesions

Presenter: Zehong Peng

Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, China (People's Republic)

Introduction : The PI‐RADS scoring system is based on magnetic resonance to evaluate prostate cancer (PCa) lesions, which are divided into grades 1‐5. In clinical practice, prostate biopsy is recommended for patients with PI‐RADS ≥ 3 lesions, but the positive rate is not satisfactory, with more than half of patients undergoing unnecessary biopsy. Therefore, developing a non‐invasive method for accurately identifying PCa from patients with PI‐RADS ≥ 3 lesions is crucial. We aim to achieve accurate diagnosis of PCa patients with PI‐RADS ≥ 3 lesions by screening key metabolites from serum extracellular vesicles (EVs) metabolic fingerprints (MFs).

Methods : We used size exclusion chromatography to separate EVs and EV‐free serum from 120 patients and obtained their MFs using a ferric nanoparticle‐assisted laser desorption/ionization mass spectrometry detection platform. Subsequently, we compared the diagnostic value of EVs and EVs‐free serum MFs while screening key metabolites using machine learning and differential analysis. Furthermore, we combined key metabolites in EVs with PSA to construct a multi‐modal diagnostic model. Finally, we compared the different roles of EVs and EVs‐free serum in Gleason Score (GS ≥ 8) PCa‐related metabolic pathways through pathway enrichment analysis.

Results : We successfully constructed MFs of EVs and EVs‐free serum. Then we identified 11 key metabolites from EVs and 2 from EVs‐free serum. We found that the diagnostic ability of EV key metabolites was significantly better than that of EVs‐free serum, with area under the curve (AUC) values of 0.74 and 0.57. By further combining key metabolites in EVs with PSA, we obtained an AUC value of 0.85 for both the training and testing groups, further enhancing the diagnostic value of EVs; Finally, we found that EVs and EVs‐free serum mainly regulated five independent metabolic pathways in GS ≥ 8 PCa, suggesting that they may play different roles in related metabolic processes.

Summary/Conclusion : In this study, we achieved non‐invasive and accurate diagnosis of PCa patients with PI‐RADS ≥ 3 lesions. Among them, EVs have a better diagnostic ability, indicating their potential application in wider PCa populations in the future.

Funding : This work was financially supported by the National Natural Science Foundation of China (82103485, 82227801).

Extracellular vesicle‐associated cytokines as novel biomarkers in ER‐positive breast cancer

Meh Sameen Nawaz 1,3* , Hang Minh 1* , Shakila Jabeen 1,2 , Tonje Bjørnetrø 4 , Jürgen Geisler 1,2 , Vessela Kristensen 1 , 2, Andliena Tahiri 1,4**

1 Akershus University Hospital, Norway; 2 Oslo University Hospital, Norway; 3 Kristiania University College, Norway; 4 Vestre Viken Health Trust, Norway *Both authors contributed equally .

Introduction : Cytokines are small signalling molecules that have key roles, not only in innate and adaptive immune systems, but also in cancer development and progression. Extracellular vesicles (EVs) are small‐sized vesicles secreted by all cell types, including cancer cells, responsible for intercellular communication by transporting proteins, lipids and nucleic acids as cargo. EVs affect many cellular processes, and EV‐associated cytokines may potentially be used to investigate novel biomarkers for cancer diagnosis and for the evaluation of therapeutic responses. The aim of the project was to investigate cytokines from EVs, blood and tumour tissue, in oestrogen receptor‐positive (ER+) breast cancer (BC) patients compared to healthy individuals, and to study treatment response using two different anti‐hormonal drugs, letrozole and exemestane, in the Neoletexe trial.

Methods : EVs were isolated from plasma of BC patients, cell lines, and healthy individuals through ultracentrifugation (UC). Transmission electron microscopy, flow cytometry analysis, nanoparticle tracking analysis and western blot were used to characterize EVs. Luminex technology was used to profile EV‐associated cytokines from plasma of BC patients ( n = 46) before and during treatment with letrozole or exemestane, and from healthy individuals ( n = 13). EV‐associated cytokines were analysed in correlation to free circulating cytokines and cytokine gene expression from the tumour tissue of the same patients ( n = 22).

Results : We identified three EV‐associated cytokines, interleukin 10 (IL10), interleukin 12 (IL12) and interferon gamma inducible protein 10 (IP10) as significantly higher in BC patients at time of diagnosis compared to healthy individuals, that were also significantly reduced in levels during treatment with letrozole or exemestane ( p < 0.05).

Summary/Conclusion : EV‐associated cytokines may potentially be used as biomarkers for the detection of BC, and for monitoring the response to hormone treatment in selected breast cancer patients.

Funding : Breast cancer research network in Norway and Akershus University Hospital.

ELISA‐based assay using urinary extracellular vesicles for diagnosis of chronic kidney disease

Tatsuya Nishimura, Keiichi Takizawa, Yousuke Hirakawa, Yuko Kajiho, Shoichiro Kanda, Yutaka Harita

The University of Tokyo Hospital, Japan

Introduction : Chronic kidney disease (CKD) has emerged as a global health crisis, posing significant challenges to healthcare systems worldwide. The progressive loss of functional nephrons is a key factor in the development and progression of CKD. Early detection of CKD is crucial, as it allows for timely intervention that can significantly slow disease progression, reduce complications, and improve patient outcomes. Our previous work highlighted the potential of expression levels of mucin 1 (MUC1) on urinary EVs (uEVs) as an indicator for childhood CKD (iScience 2022), and we developed an ELISA‐based method for the quantitative assessment of uEVs (STAR Protocols, 2023). In this study, we optimized the methodology and examined its utility in a broad range of patients, including adults.

Methods : We generated monoclonal antibodies against MUC1 and evaluated their performance across two uEVs purification methods (Tim4 and CD9 antibodies) on ELISA plates using urine samples from 88 children and 160 adults with varying kidney functions who are receiving outpatient care. The MUC1‐uEVs values were standardized with urinary creatinine. This study was approved by the ethics board of the University of Tokyo. The institutional review board waived the requirement for individual informed consent. We provided comprehensive information about the study to potential participants and offered a clear opt‐out mechanism.

Results : Among the developed monoclonal antibodies, the most sensitive one, in conjunction with both Tim4 and CD9 antibodies for uEVs purification on ELISA plates, effectively captured variations in MUC1 levels on uEVs. The CD9 plate method more accurately mirrored decreased kidney function outcomes than Tim4. The area under the curve (AUC) values from receiver operating characteristic (ROC) curve analysis for predicting the presence of moderate CKD (eGFR < 60) in adults was 0.912. This predictive accuracy was consistent across gender‐specific analyses. Similar results were obtained in paediatric patients. The samples stored at 4°C or −80°C are stable for uEVs‐MUC1 quantification, allowing for future batch analysis.

Summary/Conclusion : This system demonstrates significant potential as a clinical biomarker for CKD, offering a novel, non‐invasive testing method. Its cost‐effectiveness makes it feasible for widespread implementation in clinical settings.

Cellulose nanofibre sheet‐based extracellular vesicle miRNA profiles as biomarkers for ovarian cancer

K. Yoshida, A. Yokoi, Y. Nagao, M. Kitagawa, E. Asano‐Inami, H. Kajiyama

Nagoya University, Japan

Introduction : Extracellular vesicles (EVs) are promising non‐invasive biomarkers. We developed a novel and simple EV isolation device, the cellulose nanofibre (CNF)‐based EV sheet, designed to capture EVs in micro‐volume bio‐fluids. This study aimed to investigate the utility of the EV sheet for identifying ovarian cancer biomarkers.

Methods : We collected EVs from micro‐volume ascites on multiple organ surfaces, including the liver, pelvic peritoneum, tumour, and fallopian tube, using the EV sheet during surgery. Additionally, EVs were collected from pre‐, intra‐, and postoperative body fluids (serum and urine) using the EV sheet. Small RNA sequencing was performed on 429 EV samples from ovarian cancer and benign tumour patients, followed by qRT‐PCR validation.

Results : miRNA profiles of ascites‐EVs differed based on sampling locations. Compared to normal tissues, eight miRNAs were highly expressed in ovarian cancer ( p < 0.05), with these miRNAs also showing high expression on the tumour surface. Four well‐defined miRNAs (miR‐200a‐3p, miR‐200c‐3p, miR‐425‐5p, and miR‐429) were selected as candidate targets. These four candidate miRNAs were validated by qRT‐PCR to be highly expressed on the tumour surface and in pre‐ or intraoperative body fluids in ovarian cancer. Postoperatively, urinary miR‐200a‐3p and serum miR‐429 levels were downregulated. In addition, candidate EV‐miRNAs were more highly expressed on the fallopian tubes than on tumour surfaces in some cases, facilitating location‐based differential expression comparisons.

Summary/Conclusion : EV‐miRNAs captured by CNF‐EV sheets could serve as novel biomarkers, potentially leading to new clinical strategies for ovarian cancer diagnosis, staging, and treatment efficacy.

Extracellular vesicle characterization in human faecal samples and quality control

Presenter: Kehan Hu

UKER, Medizinische Klinik 1, Germany

Introduction : Extracellular vesicles (EVs) represent a highly novel and emerging area of medical research due to their potential as novel players in inflammation in immune‐mediated inflammatory diseases (IMIDs). These nano‐sized vesicles comprise both Bacterial Extracellular Vesicles (bEV) and Host Extracellular Vesicles (hEV). Accordingly, they have emerged as promising innovative tools for the diagnosis and treatment of IMIDs. Advances in isolating and characterizing disease‐specific EV populations are currently the focus of medical research. The aim of this study is to develop and implement protocols of EVs isolation and characterization from faecal samples of healthy controls. With the goal to process samples from patients affected by IMIDs and investigate the correlation with clinical metadata.

Methods : In our research group we established protocols to isolate total EVs from human stool samples using differential ultracentrifugation, obtaining an enriched EVs fraction that we characterize through Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM). NTA provides information about the concentration of EVs and their size and purity. TEM provides information about the shape of the isolated EVs and their homogeneity. Moreover, we can quantify the ratio between bacterial Extracellular Vesicles (bEV) and host Extracellular Vesicles (hEV) through the TLR4 Cell reporter assay that measures the Lipopolysaccharide (LPS) bound to the bEV surface.

Results : We are able to isolate total EVs from stool samples in high concentration. TEM primarily reveals a round morphology, although some contaminations of short flagella or pili of bacterial origin are also observed. We also compared samples that were frozen before isolation or directly processed upon arrival, confirming that it is possible that samples can be collected and stored before processing, as demonstrated by NTA and TEM. We also obtain the first results of measurement of the bEV LPS positive fraction.

Summary/Conclusion : In conclusion, we obtained encouraging results in the isolation and characterization of faecal EVs from healthy human samples. Consequently, we plan a biobank of stool samples from patients with different IMIDs. These collected samples can be processed for further characterization and correlation with clinical metadata. EVs could serve as biomarkers, offering potential noninvasive diagnostic options for clinical application.

Funding : Interdisziplinäres Zentrum für Klinische Forschung.

Extracellular vesicles (EVs) as biomarker for chronic graft vs. host disease, and the therapeutic potential of placental cell‐derived EVs

Presenter: Mor Zavaro

Tel Aviv University, Israel

Introduction : Graft‐versus‐host disease (GVHD) is a serious complication following allogeneic haematopoietic cell transplant (HCT). Chronic GVHD (cGVHD) is a multi‐organ (e.g., lung, skin), immune‐mediated, life‐threatening disorder, occurring in 30–70% of patients. Mesenchymal stromal cells (MSCs) and their derived extracellular vesicles (EVs), originating from different tissues including the placenta, may be used for cGVHD treatment. The study explored (1) the potential use of which EVs as a biomarker of cGVHD state before and during therapy. (2) The anti‐inflammatory/fibrotic effects of placental EVs on stimulated dermal and lung cells.

Methods : EVs were obtained from 22 healthy controls (HC‐EVs) and 16 cGVHD patients before treatment (BT) and at 5 time points during placental‐expanded cells (PLX) (Pluri Inc.) therapy. EVs were characterized using nano‐tracing analysis, western blot, flow cytometry and RT‐PCR and their effects on NHDF (normal human dermal fibroblasts) were studied. Additionally, the anti‐inflammatory and anti‐fibrotic effects of placental EVs on NHDF, human epidermal keratinocytes (HACAT), lung fibroblast (WI‐38) and normal epithelial airway cells (AECs) were explored.

Results : EVs obtained from cGVHD patients display elevated αSMA protein levels ( p = 0.0002) and a fibrosis‐related miRNA‐221 expression ( p = 0.0387) compared to HC‐EVs. However, patients’ EVs do not reflect the clinical response to PLX treatment. Exposure of NHDF cells to the patients' EVs obtained from BT increased NHDF cells' TGFβ and SMAD7 expressions, indicating their pathological role. Placental EVs derived from PLX and human villous trophoblast (HVT) cells attenuated the effects of cytokine stimulation (TNFα/INFγ 0.01–0.1 ng/µL) on HACAT cells and normalized cell proliferation. The treatment of NHDF cells with a combination of TGFβ and placental EVs reduced the stimulatory effects of TGFβ on αSMA production by over 40% ( p = 0.0286). Placental EVs mitigate cytokine‐induced anti‐proliferative effects in lung epithelial cells and decrease IL‐6 gene and protein levels in TGFβ‐stimulated lung fibroblasts.

Summary/Conclusion : cGVHD‐EVs can serve as a biomarker for the disease states’ main parameters. However, EVs did not effectively reflect treatment's efficiency, potentially due to a large variation between patients (2 vs. 4 treatments, small groups). Placental EVs may be used to regulate dermal inflammation and fibrosis, warranting further investigation of their therapeutic potential.

Funding : Ministry of Health, Estates Fund, grant number 20200575.

Hyaluronan (HA)‐coated extracellular vesicles in (neuro)inflammation and improvement of long COVID symptoms by pharmaceutical HA suppression

Miriam Zentgraf 1 , Jung‐Hyun Lee 2 , Giuseppe Di Fonzo 1 , Anna Kashkanova 3 , Bettina Hohberger 1 , Oliver Drews 4 , Vahid Sandoghdar 3 , Jan Van Deun 1 , Andreas Baur 1

1 University Hospital Erlangen and Friedrich‐Alexander‐University Erlangen‐Nuremberg, Germany; 2 University of Seoul, Republic of Korea; 3 Max‐Planck‐Institute for the Science of Light Erlangen, Germany, 4 Johannes Kepler University Linz, Austria

Introduction : Systemic peripheral inflammation can lead to neuroinflammation and neurological symptoms. Long COVID is a notable example, often resulting in “brain fog” due to blood‐brain barrier disruption and type 1 interferon responses in the choroid plexus. We have previously identified the choroid plexus as an entry port for plasma extracellular vesicles (pEV) into the limbic system/hippocampus in Alzheimer's disease. Evidence pointed to a hyaluronan (HA)‐dependent mechanism. However, an HA‐coated pEV subpopulation (HA‐pEV) with clinical relevance has not been described yet. Here, we report that HA‐pEV might play important roles in inflammatory conditions, including Long COVID.

Methods : EVs from cell culture models or blood plasma samples of control subjects and patients suffering from inflammatory conditions or long COVID were purified by OptiPrep density gradient. Next, free‐flow electrophoresis (FFE) was used to selectively isolate the HA‐pEV subpopulation, making use of their distinctive surface charge. Relevant FFE fractions were pooled, concentrated by ultrafiltration and characterized by Western blotting and interferometric nanoparticle tracking analysis (iNTA). HA on EVs before and after FFE separation was quantified using ELISA. Fluorescently labelled HA‐EV uptake by target cells was imaged using confocal laser scanning microscopy (LSM).

Results : We found that HA‐pEV concentrations are significantly increased in patients with inflammatory conditions, including Long COVID. After applying FFE and iNTA on purified pEV, we were able to demonstrate and quantify a peripheral HA‐pEV subpopulation. Next, by comparing real‐time uptake of fluorescently labelled EVs with and without HA by cells from a choroid plexus in vitro model via confocal LSM, we could confirm a HA‐dependent uptake mechanism. Notably, peripheral HA‐pEV concentrations decreased upon off‐label treatment of Long COVID patients ( n = 10, informed consent) with a hyaluronan synthetase inhibitor (Hymecromone, licensed drug), correlating with an improvement of neurological symptoms.

Summary/Conclusion : Our results support a model in which inflammatory pEV reach the limbic system through a mechanism involving HA, leading to neurological sequelae.

Preoperative identification of diagnostic and functional EV biomarkers PDGFB and TIMP3 in head and neck cancer plasma using high resolution proteomics via LC‐MS/MS

Dapi Menglin Chiang 1,2,5 , Susanne I. Wudy 3 , Christina Ludwig 3 , Marlene Reithmair 2 , Laura Benecke 4,5 , Yannik da Silva 4,5 , Laurent Muller 4,5 and Michael W. Pfaffl 1

1 Division of Animal Physiology and Immunology, School of Life Sciences, Technical University of Munich, Freising Weihenstephan, Germany; 2 Institute of Human Genetics, University Hospital, LMU Munich, Munich, Germany; 3 Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich (TUM), Freising Weihenstephan, Germany; 4 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Basel, Switzerland; 5 Department of Biomedicine, University of Basel, Switzerland

Introduction : Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with more than 650,000 new cases and 330,000 deaths annually. Despite advancements in treatment, the prognosis remains poor, particularly for patients with recurrent or metastatic disease. Surgery is the primary treatment, especially for oral cavity tumours. Identifying new tumour‐related and pre‐operative biomarkers in tissues or circulation is crucial for improving diagnosis and prognosis. Liquid biopsy is a promising method for biomarker discovery, and especially extracellular vesicles (EVs) carry tumour‐derived proteins and genetic material, offering insights into cancer progression and treatment response.

Methods : This study examines the protein content of EVs before and after surgery in HNSCC patients' plasma ( n = 11). Platelet‐poor plasma was collected peripherally before operation (BOPP) and 1 h after tumour removal (AOPP), alongside local tumour venous plasma before (BOTV) and after tumour removal (AOTV). We utilized galectin‐based glycan recognition particles for EV isolation, bead‐based flow cytometry, nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo‐EM) for EV characterization, and high‐resolution liquid chromatography‐mass spectrometry (HRLC‐MS/MS) for proteomics.

Results : NTA and cryo‐EM showed comparable EV sizes, concentrations and morphology across all groups. HRLC‐MS/MS identified 14 proteins, including platelet‐derived growth factor subunit B (PDGFB) and tissue inhibitor of metalloproteinases 3 (TIMP3), which were significantly elevated in BOPP compared to AOPP. In addition, PDGFB levels were also higher in BOTV than in AOTV. Gene ontology analysis linked these proteins to cancer pathways and migration. Treatment with BOPP and BOTV plasma EVs significantly increased cell proliferation in the HNSCC cell line UPCI‐SCC‐016; hence, high expression of PDGFB and TIMP3 in solid tissue correlates with poor prognosis.

Summary/Conclusion : These results suggest that cancer surgery removes most tumour‐related EVs in peripheral and local biopsies measurable during surgery. Increased PDGFB and TIMP3 levels in BOPP plasma indicate elevated tumour growth and their decrease post‐surgery points to effective oncological treatment, suggesting their potential as powerful preoperative biomarkers during cancer ablation.

Novel peptide primes mesenchymal stem cell‐derived extracellular vesicles to suppress osteoarthritis by the induction of SOX9 and collagen type II

Hsiu‐Jung Liao 1,2 , Chih‐Hung Chang 3,4* , Ly James Lee 2* , Chi‐Ying F. Huang 2*

1 Department of Medical Research, Far Eastern Memorial Hospital, New Taipei City, Taiwan; 2 Institute of Biopharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei City, Taiwan; 3 Department of Orthopedic surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan; 4 Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan City, Taiwan

Introduction : Osteoarthritis (OA) is a progressive and irreversible degenerative joint disease characterized by the breakdown of cartilage, formation of osteophytes, remodelling of subchondral bone, and synovitis. Despite its high prevalence, there are currently no effective and safe disease‐modifying osteoarthritis drugs (DMOADs) available. In this study, we reveal an unexpected role for a specific peptide as a novel agent that stimulates chondrogenesis to prime extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) for OA treatment.

Methods : Peptide‐primed MSC‐EVs were isolated from conditioned medium via tangential flow filtration (TFF) and characterized following the minimal guidelines established by the International Society for Extracellular Vesicles (ISEV). Therapeutic evaluations included testing cartilage repair effects in an anterior cruciate ligament transection (ACLT) osteoarthritis model, analysing their impact on MSC chondrogenesis, and studying the biodistribution of peptide‐primed MSC‐EVs in an osteoarthritis mouse model.

Results : Our findings demonstrate that peptides derived from the chemokine stimulate adipose‐derived stem cell EVs (ADSC‐EVs) to activate receptor X signalling pathways, leading to increased expression of collagen type II, COMP, and SOX9 in MSCs. Intra‐articular injections of the peptide alone significantly reduced OA severity in rats following ACLT surgery. Importantly, a similarly strong therapeutic effect was observed when peptide‐primed MSC‐EVs were administered, resulting in notably thicker cartilage compared to untreated control groups. Mechanistically, peptide‐primed MSC‐EVs restore the levels of collagen type II, COMP, and SOX9 in chondrogenic lineages, thereby inhibiting MMP13 activity. Additionally, these EVs enhance chondrogenesis, significantly downregulate extracellular matrix (ECM) catabolism, and promote ECM anabolism. Our results suggest that peptide‐primed MSC‐EVs hold great potential as an effective DMOAD for OA therapy.

Summary/Conclusion : The protein enhances chondrogenesis in ADSCs by increasing SOX9, collagen type II, aggrecan, COMP, and GAG content. The protein also promotes cartilage repair in an ACLT‐induced OA model, with enhanced GAG and collagen type II staining. Using AlphaFold2‐Multimer, specific sequences within the protein were identified with stronger chondrogenesis potential. MSC‐derived EVs primed with Protein‐P significantly promoted chondrogenesis in ADSCs. These results suggest Protein‐P‐MSC‐EVs as a promising therapeutic approach for OA.

Funding : This research was funded by the Ministry of Science and Technology (MOST 111‐2314‐B‐418‐004, MOST 111‐2321‐B‐418‐002, MOST 111‐2314‐B‐418‐003, NSTC 112‐2321‐B‐418‐004, and NSTC 112‐2314‐B‐418‐010‐MY3).

Subtopic EV biomarker development 1

EPS‐urine‐derived extracellular vesicles: multi‐omics profiling for prostate cancer risk stratification

Chiara Ansermino 1,2 , Denise Drago 1 , Radmila Pavlovic 1 , Jordi Capellades Tomàs 3 , Óscar Yanes Torrado 3 , Vito Cucchiara 4 , Francesco Montorsi 4 , Alberto Briganti 4 , Annapaola Andolfo 1

1 Proteomics and Metabolomics Facility (ProMeFa), Center for OMICS Sciences (COSR), IRCCS San Raffaele Scientific Institute, 20132 Milan; 2 Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy; 3 Department of Electronic Engineering, Universitat Rovira i Virgili, Institut d'Investigació Sanitària Pere Virgili, 43007 Tarragona, Spain; 4 Department of Urology and Division of Experimental Oncology, Urological Research Institute (URI), IRCCS San Raffaele Scientific Institute, Milan 20132, Italy

Abstract unavailable

DNA associated with large extracellular vesicles outperforms circulating cell‐free DNA for the detection of oncogenic mutations in liquid biopsies of non‐small cell lung cancer patients

Katharina Maria Richter 1,2* , Smiths S. Lueong 3,4,5,6* , Marcel Kemper 1,2 , Allegra Angenendt 1,2 , Uwe Hansen 7 , Georg Evers 1,2 , Michael Mohr 1,2 , Georg Lenz 1,2 , Balazs Hegedüs 5,6,8 , Martin Metzenmacher 5,6,9 , Martin Schuler 5,6,9 , Annalen Bleckmann 1,2* , Kerstin Menck 1,2*

1 University of Münster, Dept. of Medicine A, Münster, Germany; 2 University Hospital Münster, West German Cancer Center, Münster, Germany; 3 University Hospital Essen, Bridge Institute for Experimental Tumor Therapy, Essen, Germany; 4 University Hospital Essen, DKTK Division of Solid Tumor Translational Oncology, Essen, Germany; 5 West German Cancer Center, Essen, Germany; 6 University Duisburg‐Essen, Medical Faculty, Essen, Germany; 7 University of Münster, Institute of Musculoskeletal Medicine, Münster, Germany; 8 University Medicine Essen—Ruhrlandklinik, Dept. of Thoracic Surgery, Essen, Germany; 9 University Hospital Essen, Dept. of Medical Oncology, Essen, Germany *These authors contributed equally .

Introduction : Liquid biopsies offer a minimally invasive method for detecting genetic alterations in cancer patients, with promising implications for personalized treatment. Traditionally, circulating tumour DNA (ctDNA) is analysed from cell‐free DNA (cfDNA) in plasma, but recent findings suggest that extracellular vesicles (EVs) could provide a more informative source of ctDNA. This study aimed to compare the effectiveness of EV‐DNA and cfDNA in detecting tumour mutations in plasma samples from patients with non‐small cell lung cancer (NSCLC).

Methods : Small and large EVs (sEVs/lEVs) were isolated from NSCLC cell culture supernatants or patient‐derived plasma samples (with informed consent) using differential ultracentrifugation. EV subpopulations were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting and/or mass spectrometry, highlighting differences in size and cargo. DNA was extracted with commercially available kits, isolating EV‐DNA from cell culture supernatant and, in the case of plasma, isolating both EV‐DNA and paired cfDNA from a single plasma sample. DNA quality and quantity were assessed with fluorometry and electrophoresis, and tumour mutations were detected by next‐generation sequencing (NGS).

Results : Comparative analysis showed that lEVs contained significantly more DNA than sEVs and differed in their proteomic cargo. Both EV populations carried a significant amount of DNA‐binding proteins, albeit with quantitative differences regarding the specific enrichment of histones. Treatment of lEVs with DNase I and proteinase K resulted in reduced lEV‐DNA levels, indicating that the DNA may be partially bound to protein complexes on the vesicle surface. In NSCLC plasma samples ( n = 44), total DNA yield was lower for EV‐DNA than cfDNA. However, lEV‐DNA displayed larger fragment sizes than both sEV‐DNA and cfDNA, suggesting a unique DNA profile. The larger fragment size may enhance mutation detection, increasing the diagnostic potential of EV‐DNA. Indeed, NGS analysis demonstrated that EV‐DNA yielded higher allelic frequencies and a relatively greater number of oncogenic variants compared to cfDNA, suggesting a higher sensitivity for mutation detection.

Summary/Conclusion : This study highlights lEVs as a promising source of ctDNA for mutation analysis in liquid biopsies. Further studies correlating these findings with primary tumour mutation profiles and clinical data will help define the role of EV‐DNA in molecular diagnostics, potentially improving tumour mutation detection and monitoring.

The assessment of cardiovascular risk by extracellular vesicle profiling: the importance of age‐ and sex‐related variations

Jacopo Burrello 1,2 , Jessica Goi 2 , Alessio Burrello 3 , Elena Vacchi 4,5 , Azucena Rendon 1,6 , Edoardo Lazzarini 1,6 , Vittorio Limongelli 6 , Benedetta Bussolati 7 , Giorgia Melli, 4,5 Elena Osto 8,9 , Giovanni Pedrazzini 10 , Lucio Barile 1,5,6

1 Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Labouratories for Translational Research, Ente Ospedaliero Cantonale, Bellinzona, Switzerland; 2 Division of Internal Medicine and Hypertension Unit, Department of Medical Sciences, University of Torino, Italy; 3 Interuniversity Department of Regional and Urban Studies and Planning, Politecnico di Torino, Italy; 4 Neurodegenerative Diseases Group, Labouratories for Translational Research, Ente Ospedaliero Cantonale, Bellinzona, Switzerland; 5 Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland; 6 Euler Institute, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland; 7 Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy; 8 Center for Molecular Cardiology, University of Zurich, Schlieren, Switzerland; Department of Research and Education, University Hospital Zurich, Zurich, Switzerland; 9 Division of Physiology and Pathophysiology, Medical University of Graz, Austria; 10 Division of Cardiology Cardiocentro Ticino Institute, Ente Ospedaliero Cantonale Lugano Switzerland

Introduction : Extracellular vesicles (EVs) demonstrated diagnostic and prognostic potential for pathological conditions that contribute to global disability and mortality, including cardiovascular (CV) diseases. However, the influence of systemic chronic age‐related inflammation, known as inflammaging, and sex‐specific variations of EV biomolecular fingerprint associated with CV risk indicators are still not well described.

Methods : We developed an EV‐based age index, termed ‘EVaging,’ by profiling serum‐derived EVs from 625 individuals aged 20 to 94 years. Participants were categorized into four groups according to their CV risk profile: (i) healthy controls (HC; n = 132), (ii) subjects with at least one CV risk factor ( n = 268), (iii) patients with established cardiac disease and/or organ damage ( n = 138), and (iv) patients after an acute CV event ( n = 87). The EVaging index was generated by integrating normalized mean fluorescence levels for 37 EV surface antigens, exploiting artificial intelligence and supervised learning algorithms.

Results : EVaging index was associated with chronological age in HC, whereas it mirrored the heterogeneity of EV fingerprints in patients with different CV risk profiles. Model performance indicated that variations in EV markers closely reflected the ageing process in the absence of pathological conditions. Low levels of EV‐CD31 and EV‐CD41b identified CV risk indicators in younger patients, whereas high levels of EV‐CD62P, EV‐CD41b and EV‐CD31 displayed a better discrimination performance in older patients, achieving an AUC of up to 0.946. Overall, age‐ and sex‐adjusted multivariate regression analyses identified EV‐CD11c, EV‐CD31, and EV‐HLA‐II as independent predictors of a CV risk factor, EV‐CD20, EV‐CD42a, EV‐49e, EV‐CD62P, and EV‐HLA‐II as associated with organ damage and/or established cardiac disease; and EV‐CD31, EV‐CD42a, EV‐CD44, EV‐CD49e, and EV‐CD62P as predictors of a CV event.

Summary/Conclusion : The profiling of EV surface antigens is affected by inflammaging processes in healthy individuals, and this explains why its application in CV‐compromised patients remains complex due to disease‐associated alterations in vesicle fingerprint. Nevertheless, the multiplexing profile of EVs unveils multi‐factor interactions, emphasizing their role as potential biomarkers for CV risk assessment. Our findings suggest that EV profiling can serve as a biomarker for both chronological age and ageing‐associated outcomes.

Extracellular vesicles’ lipidome as a source of biomarkers for cancer detection and monitoring

Presenter: Erika Dorado

Imperial College London, UK

Introduction : Cancer is a leading cause of death globally. Among women, breast cancer is the most common, while ovarian and colorectal cancers often lack early symptoms. Liquid biopsies based on extracellular vesicles (EVs) offer potential for early cancer detection, though their lipids remain understudied. We studied EV lipid profiles from multiple types of cancer (breast, ovarian, and colorectal cancers), as well as circulating EVs from breast and lung cancer patients.

Methods : EVs produced by breast, ovarian and colorectal cancer cell lines were isolated using size exclusion chromatography (SEC), along with EVs produced by non‐cancerous mammary and ovarian epithelial cells. Circulating EVs from breast cancer patients, collected before and after surgery, as well as from women with benign breast disease, were isolated by a combination of density gradient ultracentrifugation and SEC. Circulating EVs from lung cancer patients were also analysed. Lipids in EVs were analysed by reverse‐phase liquid chromatography‐mass spectrometry (LC‐MS), while tumour lipids and those from surrounding healthy tissue in breast cancer patients were analysed by desorption electrospray ionization‐MS imaging. The number and size of EVs were measured by nano‐flow cytometry, morphology was assessed by transmission electron microscopy, and protein markers were evaluated by immunoblotting. Informed consent was obtained from all women participating in this study.

Results : EVs were enriched with CD9/CD63/CD81 markers and circulating EVs were depleted in lipoproteins. Sphingolipids and glycerophospholipids emerged as the primary lipids distinguishing ovarian, breast, and colorectal cancer‐derived EVs. Phospholipid and ceramide species in EVs enabled accurate classification of high‐grade serous and endometrioid ovarian cancers. Similarly, cancer‐derived EVs could be reliably differentiated from non‐cancerous EVs based on phospholipid and sphingolipid species. Preliminary results show that similar phospholipid and sphingolipid classes were present in both breast tumour biopsies and circulating EVs. The lipid profile of circulating EVs in breast cancer patients enabled accurate classification of samples collected before and after tumour removal, and it distinguished cancer samples from those with fibroadenoma and from those of lung cancer patients.

Summary/Conclusion : Lipidomic analysis of EVs may help identify lipid signatures unique to different cancers, potentially aiding in non‐invasive diagnosis and monitoring.

Funding : Cancer Research UK (CRUK)

Diagnostic potential of pleural fluid‐derived extracellular vesicles in differentiating pleural‐associated diseases using machine learning

Afrodité Németh 1 , Karin Ekström 2 , Rossella Crescitelli 2 , Roger Olofsson Bagge 2 , Ádám Rák 1 , György Cserey 1 , Balázs Hegedűs 3,4 , Servet Bölükbas 4 , Tamás Visnovitz 3,5 , Tamás Garay 1,3

1 Pázmány Péter Catholic University, Hungary; 2 University of Gothenburg, Sweden; 3 Semmelweis University, Hungary; 4 University Duisburg‐Essen, Germany, 5 ELTE Eötvös Loránd University, Budapest, Hungary

Introduction : Extracellular vesicles (EVs), produced by all cells under both physiological and pathological conditions, serve as carriers of molecular information within the extracellular matrix, tumour microenvironment, and body fluids. Pleural effusion, an accessible and abundant body fluid that can accumulate in the pleura in various diseases, provides insight into disease‐related microenvironments, including those associated with cancer and mesothelioma. Thus, characterizing EVs in pleural fluid may offer a promising avenue for diagnostic applications.

Methods : EVs were characterized in 79 pleural fluid samples from 26 patients with pleura‐involved cancer, 19 patients with cancer not involving the pleura, 24 patients with mesothelioma, and 10 patients with pleuritis. EV isolation from pleural fluids was achieved using SEC and ultrafiltration, with successful isolation confirmed via TEM and nFCM. The total protein and particle concentrations of the EV fractions were measured with the Qubit protein assay and NTA, respectively. Surface marker profiling of 37 markers was conducted using the MACSPlex kit. Following EV characterization, total protein, total particle concentration, and normalized surface marker intensities were input into a machine learning algorithm employing leave‐one‐out cross‐validation (LOOCV). This AI model explored the diagnostic potential of pleural fluid‐derived EVs and aimed to identify a minimal set of markers with the highest possible diagnostic value.

Results : The machine learning algorithm successfully identified most patients with mesothelioma and pleura‐involved cancer. In contrast, patients with cancer not involving the pleura were predominantly classified as mesothelioma. Classification of pleuritis patients was not achieved efficiently with this setup.

Summary/Conclusion : This study demonstrates the potential of EV profiling from pleural fluid as a minimally invasive diagnostic approach for differentiating between pleura‐involved cancer, mesothelioma, and other pleural conditions. By leveraging machine learning, specific EV surface markers could serve as reliable indicators of disease type. This approach could reduce the need for more invasive biopsies, ultimately benefiting both patients and healthcare systems.

Funding : This research was supported by the National Research, Development and Innovation Office through the grant TKP2021‐EGA‐42.

Mitochondrial content of large and small extracellular vesicles in ageing and autoimmunity

Iris Potakowskyj 1,2 , Ee Lyn Lim 1 , Sneha Prabhakara Shastry 1,2 , Adrian Parrilla Mesas 1 , Gorjana Rackov 1

1 BioMed X Institute, Germany; 2 Heidelberg University, Germany

Introduction : Mitochondrial dysfunction and immune cell senescence are well‐established hallmarks of ageing. T cells in systemic lupus erythematosus (SLE) show signs of premature senescence and are characterized by mitochondrial dysfunction and mitophagy defects. Recent studies showed that inefficient mitophagy may lead to increased mitochondrial content in EVs.

Methods : Here, we aimed to characterize mitochondrial DNA and protein cargo in large and small EVs enriched from plasma of young vs. aged donors and SLE patients versus healthy controls. Large and small EVs were enriched from platelet‐depleted plasma using differential centrifugation coupled with size exclusion chromatography and ultrafiltration. In addition to basic EV characterization—including particle count, size distribution and protein content—mitochondrial protein content was assessed using liquid‐chromatography tandem mass spectrometry (LC‐MS/MS). MACSPlex flow cytometry array was used for assessment of EV surface markers.

Results : Enrichment of EVs was confirmed by immunoblot and nanoparticle tracking analysis, showing distinct size distribution for large and small EVs. Our results showed that mitochondrial DNA and protein cargo can be detected both in large and in small EVs, designating them as MitoEVs. Although mitochondrial proteins were more abundant in large EVs, comparison to the MitoCarta database showed that only a subset of human mitochondrial proteins is detectable in EVs. The abundance of these proteins was altered in aged compared to young donors and in SLE patients compared to healthy controls.

Summary/Conclusion : Our findings indicate that a specific subset of mitochondrial proteins is loaded in EVs. The altered levels of mitochondrial cargo in large and small EVs may serve as potential biomarkers in pathophysiological processes associated with mitochondrial dysfunction.

Funding : This study is sponsored by Merck KGaA.

Protein profile in urinary extracellular vesicles is a marker of malignancy and correlates with muscle invasiveness in urinary bladder cancer

Loïc Steiner 1 , Maria Eldh 1 , Annemarijn Offens 1 , Rosanne E. Veerman 1 , Markus Johansson 2,3 , Tammer Hemdan 4 , Hans Netterling 2 , Ylva Huge 5 , Abdul‐Sattar Aljabery Firas 6 , Farhood Alamdari 7 , Oskar Lidén 8 , Amir Sherif 2,9 , and Susanne Gabrielsson 1

1 Division of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet and Karolinska University Hospital; Center for Molecular Medicine, Stockholm, Sweden; 2 Department of Diagnostics and Intervention, Umeå University, Umeå, Sweden; 3 Department of Surgery, Urology Section, Sundsvall‐Härnösand Hospital, Sundsvall, Sweden; 4 Department of Surgical Sciences, Uppsala University, Uppsala, Sweden; 5 Department of Urology in Östergötland, and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden; 6 Department of Clinical and Experimental Medicine, Division of Urology, Linköping University, Linköping, Sweden; 7 Department of Urology, Västmanland Hospital, Västerås, Sweden; 8 Department of Surgery and Urology, Hudiksvall Hospital, Hudiksvall, Sweden; 9 Department of Biomedical and Clinical Sciences, Division of Urology, Linkoping University, 581 85 Linkoping, Sweden

Introduction : Urinary Bladder Cancer (UBC) ranks among the most prevalent cancers worldwide and has a high recurrence rate and unpredictable treatment responses. Thus, biomarkers are urgently needed. Extracellular vesicles (EVs) are released from both cancer and immune cells and provide a snapshot of the originating cell. They are abundant in urine and are therefore candidate biomarkers for UBC.

Methods : Isolated urinary EVs from 39 UBC patients were compared with EVs from healthy controls, prostate cancer patients and whole urine. Samples were from bladder urine at the time of both transurethral resection of the bladder tumour (TURB) and cystectomy, as well as urine taken from the ureter at cystectomy. EVs were isolated by a combination of tangential flow filtration (300 kDa cutoff) and differential ultracentrifugation. EVs were characterized by nanoparticle tracking analysis, electron microscopy and bead‐based flow cytometry. The protein composition of isolated and lysed vesicles was detected by proximity extension assay (PEA; Olink, immuno‐oncology panel).

Results : In UBC patients, the proteomic signature of bladder urine EVs differed from ureter urine EVs from the same individuals, and from bladder urine‐derived EVs of both healthy and prostate cancer controls. Pairwise comparison was performed with matched whole urine, revealing proteins solely detected in isolated vesicles. Additionally, a distinct signature was identified in bladder urine EVs correlating with muscle invasiveness, and a trained classifier could predict UBC with 92% accuracy. Some differentially expressed proteins, HO‐1 and MMP7, were analysed by bead‐based flow cytometry, where HO‐1 was detected on the EV surface.

Summary/Conclusion : Taken together, these results strengthen the rationale of using EVs as non‐invasive biomarkers and prognostic tools for UBC, as their protein signature was able to distinguish UBC patients from controls.

Funding : This work was funded by the Swedish Medical Research Council, the Swedish Cancer Society, the Cancer Research Foundations of Radiumhemmet, the Cancer‐ and Allergy Foundation, Karolinska Institutet's KID grant and the Karolinska Institutet Foundations.

Serum extracellular vesicles enriched for neuronal origin provide a schizophrenia‐associated miRNA signature of treatment resistance and cognitive impairment

Presenter: Michelle M. Barnett

University of Newcastle, Australia

Introduction : Mental health disorders constitute an increasingly large proportion of disease burden. Schizophrenia is a chronic and severe mental disorder with significant impact from treatment resistance and cognitive impairment. Traditional nosology fails to reconcile clinical heterogeneity within and between diagnostic categories. Non‐invasive biomarkers are needed to address these shortcomings, particularly brain relevant markers that reflect synaptic pathology. Extracellular vesicles (EVs) in the peripheral circulation provide a protected niche of nucleic acids that are derived from different tissue types. Therefore, enrichment for neuronal origin EVs may provide a quantitative readout of neuronal dysfunction and facilitate precision medicine through biologically informed characterisation of subjects.

Methods : We obtained serum samples from the Australian Schizophrenia Research Bank (ASRB) for 477 individuals. Serum was immuno‐fractionated, using anti‐microtubule associated protein 1B (MAP1B), to enrich for neuronal origin EVs. Small RNA‐seq and differential expression analysis were used to identify putative neuronal origin miRNA that differed between groups. Bioinformatics analysis was utilised to investigate the functional implications of differentially expressed miRNA.

Results : We observed miRNA profiles, from serum EVs enriched for neuronal origin, that reflect brain function and demonstrate important differences between cases and controls. Importantly, these miRNA signatures were profoundly altered in cases experiencing severe disease. Specifically, alterations in neuronal origin miRNA from schizophrenia patients with cognitive impairments function in pathways that are enriched for cognitive enhancing drug targets, such as phosphodiesterase, cholinergic and erythropoietin signalling. Additionally, treatment resistant cases harboured distinct miRNA profiles that may be leveraged to support clinical decisions for prompt initiation of clozapine, given its efficacy in treating resistant disease. Together, these results demonstrate the potential for non‐invasive analysis of neuronal origin miRNA to provide brain relevant biomarkers for schizophrenia, particularly for clinically challenging patients with unmet needs.

Summary/Conclusion : These data support the potential application of neuronal origin miRNA from serum EVs as biomarkers for schizophrenia. In stratifying patients into biologically refined subgroups, these minimally invasive, brain relevant biomarkers may guide precision medicine approaches to inform treatment selection.

Funding : NHMRC grants 1188493 and 1121474 (M.J.C.) and Australian Government Research Training Program stipend (M.M.B.).

Exploring the Role of CD9‐Positive Extracellular Vesicles in Major Depressive Disorder: Associations with Symptom Severity and Potential Physiological Implications

Presenter: Kaiping Burrows

Laureate Institute for Brain Research, USA

Introduction : Extracellular vesicles (EVs) play important roles in intercellular communication via molecular cargo transportation, thus influencing multiple physiological processes. EVs can cross the blood‐brain barrier and are detectable in blood; therefore, they may provide insight into the neurobiological basis of neuropsychiatric disorders, including major depressive disorder (MDD). CD9 is a tetraspanin, often used as an EV marker, that has regulatory functions in inflammation and immune responses. Prior research has demonstrated that EV content may contribute to the pathophysiology of major depressive disorder (MDD) by influencing neurogenesis and inflammatory processes. This pilot study explored the association between CD9 and MDD by examining the relationship between CD9 expression and symptom severity.

Methods : Blood samples were collected from 14 individuals diagnosed with MDD and 7 healthy controls (HC). Serum was isolated and stored at ‐80°C. EVs were isolated from serum using size exclusion chromatography (SEC). EV markers, size, and concentration were assessed through western blot, immunoassays, and microfluidic resistive pulse sensing. CD9 concentrations in EVs were quantified by immunoassays. Depression severity was assessed using the Montgomery–Asberg depression rating scale (MADRS) and Patient Health Questionnaire (PHQ). Differences in CD9 concentrations between MDD and HC groups were analysed using the Mann–Whitney–Wilcoxon non‐parametric test. Correlations between CD9 and depression severity (MADRS and PHQ scores) were examined using Spearman's correlation test.

Results : EVs from MDD participants exhibited significantly lower CD9 concentrations compared to HC ( p = 0.04, d = 1.15). CD9 concentration was inversely associated with higher scores on MADRS ( r = −0.45, p = 0.04) and PHQ ( r = −0.45, p = 0.04). No significant correlations were found between CD9 concentrations and age ( r = 0.23, p = 0.34) or BMI ( r = −0.18, p = 0.49). In addition, CD9 did not differ on sex ( p = 0.82).

Summary/Conclusion : Our findings demonstrate significantly lower CD9 concentrations in EVs from individuals with MDD compared to healthy controls, with an inverse relationship between CD9 levels and depression severity, as measured by MADRS and PHQ scores. Reduced CD9 may reflect impaired immune responses, disrupted vascular health, or compromised neuroplasticity, all of which could contribute to MDD pathophysiology. Future research should integrate complementary biomarkers and mechanistic studies to elucidate the precise role of CD9 in inflammation and neurobiological processes associated with depression.

Funding : R01MH123652; K99MH126950; R00MH126950; P20GM121312.

EVs as circulating biomarkers of radio‐induced cardiotoxicity in breast cancer (BC)

Presenter: Celine Loinard

IRSN, France

Introduction : BC radiotherapy (RT) can lead to irradiation of the heart, leading to an increased risk for a variety of heart diseases arising many years after radiotherapy. The release of EVs is important for biological functions, including inflammation and angiogenesis, and influences the development of cardiotoxicity. The present study tested the hypothesis that EV generation in blood after RT of BC women could predict radio‐induced cardiotoxicity.

Methods : EVs isolated from plasma of 200 women treated for BC before RT, at the end of RT, and 6 and 24 months after the end of RT were characterized as large vesicles expressing monocyte, platelet, and endothelial markers by cytometry and small EVs by Nanosight.

Results : Interestingly, large EVs generated after RT, but not small EVs, increased significantly after the end of the RT: large EVs derived from platelets and endothelial cells significantly increased at the end of the RT, 6 and 24 months after the end of the RT, by 30% and 100%, respectively compared to before RT ( p < 0.05). Large EVs derived from Monocyte increased significantly by 40% 24 months after the end of the RT compared to before the RT ( p < 0.05). Large EV isolated from patients with cardiac dysfunction (GLS < 15%) at 6 months after RT showed an increase in the wound healing process by 5%, 8%, and 10%, respectively in 2, 5, and 10 Gy irradiated HUVEC compared to EVs isolated from patients with normal GLS. VEGF, eNOS and ICAM protein levels were significantly increased by 1.8‐, 2‐ and 2‐fold change in large EVs isolated from patients with cardiac dysfunction (GLS < 15%) at 6 months after RT compared to large EVs isolated before RT. Finally, we demonstrated that Tissue Factor is significantly correlated to GLS < 15% of patients at 6 months after the RT.

Summary/Conclusion : These results demonstrate that RT increases the generation of large EVs derived from platelets, endothelial cells, and monocytes in breast cancer. Large EVs could represent an early circulating biomarker of cardiotoxicity crucial for developing effective strategies for primary and secondary prevention, which are needed to optimize the CV and cancer outcomes of BC survivors.

Characterisation of extracellular vesicles in patients with hypertrophic cardiomyopathy

Alessandra Stefania.Rizzuto 1 , Andrea Faggiano 2 , Isabella Fichtner 3 , Chiara Macchi 3 , Ilaria Giusti 4 , Vincenza Dolo 4 , Alberto Corsini 3 , Margherita Calcagnino 2 , Stefano Carugo 1,2 , Massimiliano Ruscica 2

1 Dept Clinical Sciences and Community Health, Università degli Studi di Milano; 2 Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan; 3 Dept Pharmacological and Biomolecular Sciences, Università degli Studi di Milano; 4 Dept Life, Health and Environmental Sciences, Università degli Studi dell'Aquila

Introduction : Hypertrophic cardiomyopathy (HCM), a condition characterised by chronic functional and/or structural abnormalities in the heart, is the most common genetic cardiac disease globally. Extracellular vesicles (EVs) hold promise as a liquid biopsy tool, as their circulation patterns and cargo can reflect the onset and severity of cardiac diseases. We characterised plasma‐derived EVs of 34 HCM patients and 21 volunteers (CTR).

Methods : Echocardiographic and clinical data were collected from both groups, and patients received genetic screening to identify mutations in genes most commonly associated with HCM. According to the MISEV 2023 guidelines, EVs were isolated from plasma via size‐exclusion chromatography and ultracentrifugation, and characterised via NTA, TEM, WB, and FACS analyses. Protein content of EVs was assessed by targeted proteomic analysis (Olink).

Results : The majority of subjects in both groups were male (65% and 70%), with a median age of 58 and 48 in HCM and CTR, respectively. Echocardiographic parameters (e.g., maximum wall thickness and left ventricular mass) were significantly increased in HCM. EV‐specific protein (CD63, CD9 and Alix) expression and integrity were confirmed by FACS, WB and TEM. Median EV concentration was reduced in HCM versus CTR (EV/mL/cell count: 3.3 × 10 9 vs. 4.6 × 10 9 ), while no differences were found in size. Platelet‐derived EVs (CD41a+) were doubled in HCM (EVs/µL: 36.5 vs. 17.6), likewise for EVs released from activated platelets (CD62P+ and CD40L+). In HCM, positive correlations were found between the Sudden Cardiac Death (SCD) Risk score and EVs derived from platelets ( r = 0.4577), cardiomyocytes (CD172a+; r = 0.4783) and activated endothelial cells (CD62E+; r = 0.5916). Among the analysis of 92 proteins involved in heart diseases, enrichment in proteins associated with the coagulation cascade (von Willebrand factor, tissue‐type plasminogen activator and plasminogen activator inhibitor 1) was found in plasma and plasma‐derived EVs of HCM.

Summary/Conclusion : The increase in EVs derived from platelets and activated platelets, the association between platelet‐derived EVs and SCD, and the enrichment of plasma‐derived EVs in coagulation cascade‐related proteins can pave the way to understanding the unknown aetiology of a pro‐thrombotic state observed in HCM.

Funding : PRIN 2022ZPS49L to MR.

Symptomatic menopausal women not responding to hormone replacement therapy may increase their bone and cardiovascular risk: a pilot study investigating extracellular vesicle protein cargo

Alice Hodge 1 , Emily Clarke 1 , Anders Jensen 1 , Olivia Pigden 1 , Paula Briggs 2 , Dharani Hapangama 2 , Mandy J Peffers 1

University of Liverpool, United Kingdom. 1 Liverpool Women's NHS Foundation Trust, United Kingdom. 2

Introduction : The variability in hormone replacement therapy (HRT) efficacy among menopausal women necessitates early identification of responders to optimise costs and outcomes. Extracellular vesicles (EVs), intercellular messengers released in response to triggers, including hormones, may serve as indicators of physiological response to HRT. This study is the first to explore the proteome of plasma EVs in menopausal women and investigate resistance to HRT, aiming to elucidate reasons for non‐responsiveness and to determine if EV protein cargo can predict HRT outcomes.

Methods : A cross‐sectional observational study was conducted ( n = 12) with four groups ( n = 3) stratified based on the MENQOL symptom questionnaire: ‘No HRT,’ ‘Implants,’ ‘Standard HRT responders’ (SHRT.R), and ‘Non‐responders’ (SHRT.NR). EVs were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy (TEM), and western blotting. Proteomic analysis was performed using liquid chromatography‐tandem mass spectrometry, followed by bioinformatics.

Results : Characterisation revealed a heterogeneous EV population with sizes ranging from 146.5 to 223.5 nm and concentrations of 3.63 × 10 8 to 5.95 × 10 9 particles/mL across all samples. TEM confirmed phospholipid bilayer morphology, and the western blot confirmed ALIX and CD9 surface markers in all samples. A total of 333 proteins were identified in plasma EVs; 67 were differentially abundant across groups. SHRT‐NR had a different proteome compared to all other groups. Functional enrichment analysis resulted in significantly different canonical pathways, in SHRT‐NR compared to other groups. Regarding predicted upstream analysis, TFGβ was downregulated in SHRT‐NR compared to No HRT ( p = 0.036, z = 1.114) and Implant ( p = 0.004, z = −2.172), and IL‐4 was downregulated in SHRT‐NR compared to No HRT ( p = 0.016, z = 0.447) and SHRT‐R ( p = 0.033, z = −0.343). Significant biofunctions included ‘Production ( p = 0.032, z = 0.830) and synthesis ( p = 0.048, z = 0.916) of reactive oxygen species (ROS) and necrosis’ ( p = 0.045, z = 1.525) upregulated in SHRT‐NR compared to Implant. KRT1, IGKV1‐27 and ADARB1 are potential biomarkers for SHRT‐NR.

Summary/Conclusion : SHRT‐NR plasma EVs had a different proteome from other groups. These patients may face increased harm and forgo the bone and cardiovascular protection typically provided by HRT. Identifying specific protein markers could enhance predictive capabilities regarding HRT responsiveness.

Funding : University of Liverpool funded.

Proinflammatory Phenotype of Saliva Extracellular Vesicles

Fadi Haddadin 1 , Rejina Roufegarinejad 2 , Luigi Licanti 3 , Luciano, Licanto 1 , Floyd Sarsoza 4 and Charisse Winston 4

1 University of California, San Diego, La Jolla, CA, USA; 2 University of San Diego, San Diego, CA USA; 3 San Diego State University, San Diego, CA, USA; 4 University of Southern California, Alzheimer's Disease Therapeutic Research, San Diego, CA, USA

Introduction : Saliva is becoming more recognized as a promising source for discovering biomarkers related to Alzheimer's disease (AD) because it can be collected non‐invasively and is readily accessible to diverse populations. Exciting preliminary data in blood demonstrated that brain‐derived neuronal and glial extracellular vesicles (EVs) can accurately predict the conversion of mild cognitive impairment (MCI) to AD; however, these studies have yet to be fully explored in saliva. Here, we aim to assess the feasibility of extracting EVs based on cellular origin from human saliva to predict cognitive decline amongst asymptomatic individuals at risk for AD.

Methods : EVs were extracted, precipitated, and enriched against neuronal (L1CAM, NRXN1) and glial sources (TMEM119, GLAST, PLP1) using magnetic immunocapture from patient samples enrolled in the UC San Diego Nathan Shock Healthy Aging Study and the New Ideas Study. Three methods for EV precipitation (ExoQuick (EQ)‐TC, EQ + OASIS, and Norgen) were employed per the manufacturer's recommendations. Particle size distributions were quantified using nanoparticle tracking analysis. EV concentrations and tetraspanin profiling were quantified using super‐resolution microscopy (ONI). Quantification of proinflammatory‐related EV cargos was characterized by MSD bioassay.

Results : EQ‐TC and EQ + OASIS methods successfully isolated high concentrations of saliva EVs, while the Norgen kit failed to isolate high concentrations of EVs from human saliva. Saliva EVs isolated using EQ‐TC generated subpopulations of EVs that were predominantly CD9+, while saliva EVs isolated using EQ + OASIS were predominantly CD63+ and CD9+. Concentrations of IL‐8, TNF‐α, and IL‐12p70 were significantly reduced in NRXN+ saliva EVs while IL‐6 was significantly reduced in NRXN+ and GLAST+ EVs.

Summary/Conclusion : Extracellular vesicles can be effectively isolated from human saliva, although the concentrations of tetraspanins in these EVs differ depending on the isolation method used. The levels of proinflammatory‐related cargos from EVs precipitated using EQ‐TC also varied by cellular origin, showing significantly lower concentrations in NRXN+ EVs. The positivity for tetraspanins and the specificity to cell types may influence the biomarker potential of these EVs, warranting further research. Metabolomic profiling and biomarker analyses will be conducted to assess changes in EV cargo loads concerning tetraspanin and cellular origin in asymptomatic individuals at risk for AD.

Funding : U19AG074879 4R00AG070390.

Proteomic profiling of extracellular vesicles reveals novel biomarkers for screening high‐risk patients for biliary tract cancer among those with benign biliary tract diseases

Presenter: Dong Uk Kim

CHA University Gumi Medical Center, Republic of Korea

Introduction : Benign biliary tract diseases (BBDs) can cause biliary tract cancers (BTCs) during the regeneration process of the bile duct due to repeated inflammation. In such cases, CA 19‐9 can also be elevated in BBDs. Therefore, the differentiation of BTCs from BBDs becomes very difficult and important. Novel biomarkers that can differentiate between BBDs and BTCs are necessary for early detection of BTCs. This study analysed extracellular vesicle (EV) peptides to find a novel screening biomarker for BTCs in patients with BBDs.

Methods : Plasma samples were obtained from a discovery cohort including 5 healthy people as controls, 10 patients with BBDs such as cholelithiasis, choledocholithiasis, and sclerosing cholangitis, and 29 patients with BTCs. The EV was obtained from 300 µL of plasma by using ultracentrifugation after the elimination step of plasma protein. The EV peptide samples were analysed by liquid chromatography‐data‐dependent acquisition‐tandem mass spectrometry. The acquired peptides and proteins were investigated by searching in Fragpipe‐MS Fragger. Characterization was performed using the EVs database of previous studies. differential expression protein (DEP) analysis and pathway/GO analysis are performed to know the biological process. To confirm that the differences in the excavation proteins were repeatedly observed, a validation study was performed on plasma EVs from 50 BBD patients and 101 BTC patients.

Results : A total of 25,382 peptides and 3,313 proteins were defined. Most of the gene ontology biological processes were related to vesicle‐mediated transport. On DEP analysis, compared to cancer samples, 12 proteins were up‐regulated in both healthy and benign samples, and 3 were down‐regulated. Parallel reaction monitoring analysis was performed to identify the 15 proteins in plasma EVs from 151 patients, and only three proteins were confirmed as novel screening biomarkers: PIGR, PON1, and IGHA2. The significance of three proteins and CA19‐9 was confirmed with an AUC value of 0.7790 compared to 0.5778 of CA19‐9 only.

Summary/Conclusion : This study suggested that PIGR, PON1, and IGHA2 are meaningful as screening biomarker candidates for BTCs in patients with BBDs.

Funding : Supported by Bertis Inc. Supported by a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (grant number: HA20C0009).

Proteome analysis of plasma‐isolated extracellular vesicles to understand signalling and treatment response in immune checkpoint inhibitor treated metastatic non‐small cell lung cancer patients

Petra Hååg 1 , Nupur Agarwal 1 , Lisa Liu Burström 1,2 , Bo Franzén 1 , Per Hydbring 1 , Simon Ekman 1,2 , Rolf Lewensohn 1 , Patrick Micke 3 , Klas Kärre 4 , Luigi De Petris 1,2* and Kristina Viktorsson 1*

Luigi De Petris and Kristina Viktorsson are co‐senior authors.

1 Department of Oncology Pathology, Karolinska Institutet, Sweden; 2 Karolinska University Hospital, Sweden; 3 Department of Immunology, Genetics and Pathology, Cancer Immunotherapy, Uppsala University, Sweden; 4 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Sweden

Introduction : Monitoring of immune checkpoint inhibitor (ICI) treatment effect in metastatic non‐small cell lung cancer (NSCLC) patients is hampered by the difficulty of biopsying the tumours due to the location of tumours and their heterogeneity in protein expression and signalling. Thus, liquid biopsies for non‐invasive analysis of biomarkers (BMs) are highly needed, and extracellular vesicles (EVs) constitute one way ahead. We have isolated EVs from plasma of NSCLC patients at Karolinska prior to the start and after various time points of treatment with the PD‐1 inhibitors pembrolizumab or cemiplimab with or without concomitant chemotherapy. Our aims are to identify protein BMs expression and/or signalling signatures related to ICI treatment response as well as to PD‐L1 and CD73 protein expression in EVs.

Methods : EVs were isolated from 0.5 mL EDTA‐plasma of NSCLC patients using Izon's qEVoriginal gen 2, 70 nm columns and fraction collector. Fractions 5‐10 were pooled and concentrated using Amicon concentrators with a 100k cut‐off. Nanoparticle Tracking Analysis (NTA) was applied to reveal particle size and concentration. Proteins were extracted from the EVs and profiled using proximity extension assay (PEA) on Immuno‐Oncology and Oncology II panels at the SciLifeLab Affinity Proteomics at Uppsala University. Qlucore Omics Explorer was used for data analysis and visualization. EV protein markers were validated using ELISA, FACS and WB.

Results : All samples contained particles of EV size with a median size of around 90 nm, and the EV markers CD9, TSG101 and SYND1 were found. PEA protein profiling revealed 127 proteins to be expressed in at least 75% of the samples. Among those were SYND1, PD‐L1, CD73 and other oncogenic and immune signalling proteins, with heterogeneity among the individual samples. Protein signatures that correlated with PD‐L1 or CD73 expression level in EVs included, among others, SYND1, CD40, CD244, EGF, IL18, CEACAM1, TNFRSF4, and LYN. In addition, a preliminary EVs protein signature associated with progression‐free survival is currently validated.

Summary/Conclusion : We found that multiple protein signatures rather than individual protein profiling may allow for identification of possible BM signatures in EVs of NSCLC patients when given PD‐1 inhibitors alone or combined with chemotherapy.

Funding : Stockholm Cancer Society, Swedish Cancer Society, Sjöberg Stiftelsen.

Impact of obstructive sleep apnoea‐derived extracellular vesicles on cellular ageing

Bárbara Santos 1,2,5,6 , Laetitia S. Gaspar 1,2,3 , Catarina Carvalhas‐Almeida 1,2,5,6 , Bárbara Ramalho 1,2 , Raquel Duarte 1,2,5 , Marisa Ferreira‐Marques 1,2,5 , Daniela Costa 1,2,5,6 , Gabriela Vykysala 1,2 , Elza Rocha 1,2 , Mafalda Ferreira 4 , Joaquim Moita 4 , Cláudia Cavadas 1,2,5 , Ana Rita Álvaro 1,2,3

1 CNC—UC—Centre for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2 Centre for Innovation in Biomedicine and Biotechnology (CIBB), University of Coimbra, Portugal; 3 IIIUC—Institute for Interdisciplinary Research, University of Coimbra, Portugal; 4 Sleep Medicine Centre, Coimbra Hospital and University Centre (CHUC), Coimbra, Portugal; 5 FFUC—Faculty of Pharmacy, University of Coimbra, Portugal; 6 EIT Health‐Aging PhD School

Introduction : Obstructive sleep apnoea (OSA) is a highly prevalent sleep disorder worldwide among elderly populations and may accelerate the aging process (Gaspar et al. 2017). Untreated, OSA has been associated with several chronic comorbidities such as metabolic, cardiovascular disorders, functional decline and neurodegenerative disorders. Extracellular vesicles (EVs) act as important mediators of intercellular communication, being involved in several physiological and pathological processes. The hypothesis that OSA‐related EVs may contribute to accelerated aging, through a senescence‐associated secretory phenotype, warrants further exploration. This study investigates the impact of EVs from OSA patients on aging hallmarks.

Methods : Plasma‐derived EVs were isolated from 12 men with severe OSA (54 ± 10 years) at diagnosis (T0M), after 24 months of CPAP treatment (T24M), and from control groups: age‐matched men ( n = 14; 49 ± 8 years) and young controls ( n = 12; 24 ± 2 years). EV isolation was performed using the size‐exclusion chromatography method and characterized for concentration, size, and cargo content—with transcriptomic analysis for miRNA, with samples collected at 8 AM and 10 PM. Functional assays were conducted by incubating human fibroblasts with EVs from OSA patients and controls, assessing aging hallmarks, including markers of senescence, genomic instability, and biological clock regulation. Ethical approval was obtained from FMUC and Coimbra Hospital committees.

Results : The study successfully characterized plasma‐derived EVs, verifying size and protein composition. Nanoparticle Tracking Analysis showed no differences in EV size across groups, though EV concentration was significantly elevated in OSA patients at T0M and T24M compared to controls. EV‐associated miRNA profiling was analysed in the four different groups at 8 AM and 10 PM. Regarding functional assays in human fibroblasts, EVs from OSA patients seem to cause a dysregulation in senescence genes and a disruption of biological clocks’ profile.

Summary/Conclusion : These results suggest OSA might aggravate/promotes some hallmarks of ageing, some of which are not reverted by CPAP treatment.

Funding : European Regional Development Fund (ERDF)‐Centro 2020 Programme (project ENTRO‐01‐0145‐FEDER‐000012, HealthyAging 2020), Operational Programme for Competitiveness and Internationalisation (COMPETE 2020), Fundação para a Ciência e a Tecnologia (FCT) under projects POCI‐01‐0145‐FEDER‐029002 (PTDC/MEC‐MCI/29002/2017, noOSAnoAGING), UIDB/04539/2020, UIDP/04539/2020 and LA/P/0058/2020. European Social Fund and FCT through POCH—Human Capital Operational Programme under PD/BD/135497/2018, 2020.04499.BD, and 2021.05334.BD.

Plasma EV protein profiling by proteomics identifies potential biomarkers for clinical phenotypes in systemic sclerosis

Onno J. Arntz, Sander I. van Leuven, Lenny van Bon, Irene E. van der Horst‐Bruinsma, Madelon C. Vonk, Fons A. J. van de Loo. Department of Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands

Introduction : Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, inflammation, and fibrosis affecting skin and internal organs. A frequent, life‐threatening complication is interstitial lung disease (ILD), making early diagnosis crucial for improving prognosis and treatment outcomes. Extracellular vesicles (EVs), which reflect the characteristics of their cell of origin, are promising biomarkers for assessing patient disease status. In this study, plasma EV (pEV) protein profiles were correlated with clinical parameters in SSc patients and compared to healthy controls (HCs).

Methods : EVs were isolated from platelet‐free plasma of 18 SSc patients (nine with ILD and nine without) and 10 HCs using size exclusion chromatography. EVs were characterized by nanoparticle tracking analysis, microBCA, cryoEM, and imaging flow cytometry (IFCM). Mass spectrometry (MS) was used to identify and quantify pEV proteins from each donor.

Results : No significant differences in pEV concentration, size, and protein content were observed between SSc patients (1.62 × 10 10 particles/mL, 109 nm, 3.20 fg protein/particle) and HCs (2.61 × 10 10 particles/mL, 118 nm, 2.36 fg protein/particle). IFCM confirmed the presence of CD9 and CD81 and cryoEM showed the lipid bilayer, characteristic features of EVs. MS identified 522 proteins, with 17 proteins significantly elevated compared to HC, and one protein (AMY1A) unique for SSc patients. The elevated coagulation protein von Willebrand factor, associated with vascular damage, was confirmed by IFCM and linked to vasculopathy in SSc. Skin‐specific proteins DSC3, A2ML1, and SBSN enriched in pEVs correlated, as expected, with the extent of skin involvement in SSc. The unique protein AMY1A in SSc‐pEVs may indicate salivary gland involvement in SSc, while the enriched proteins TTN and MYH1 likely reflect muscle involvement that may go undetected with standard clinical measures. In SSc patients with ILD, eight pEV proteins (PLTP, APOA2, HLA‐A, MUC5B, CD14, HGFAC, LCP1, and MUC6) were significantly reduced compared to SSc patients without ILD, with MUC5B—a key lung mucin—being particularly notable.

Summary/Conclusion : This study demonstrates, for the first time, that pEV protein profiling offers valuable insights into organ‐specific involvement in SSc patients and shows their potential as biomarkers to differentiate clinical phenotypes of SSc patients.

Discovery of brain cell‐type‐specific extracellular vesicle protein biomarkers for ischaemic stroke using integrated omics and Bayesian data analysis

Manju Babu, Deepthi Ann Thomas, Anaekshi Gogoi, Arnab Datta

Laboratory of Translational Neuroscience, Division of Neuroscience, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore 575018, Karnataka, India

Introduction : Ischaemic stroke (IS) remains a leading cause of death and disability globally, traditionally viewed as a neuronee‐focused disorder. However, the evolving concept of the neurovascular unit, which encompasses neuronees, glial cells, and endothelial components, has broadened our understanding. Recent studies highlight the role of extracellular vesicles (EVs) as key modulators of intercellular communication, carrying proteins, lipids, and nucleic acids. Notably, EVs have the ability to cross the blood‐brain barrier, positioning them as potential biomarkers for IS.

Methods : To uncover the cell‐specific role of EVs during IS, we isolated EVs from neuronees (N 2 a), astrocytes (C8‐D1A), and endothelial cells (bEnd.3) exposed to oxygen‐glucose deprivation (OGD) conditions, mimicking stroke‐induced stress. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide and lactate dehydrogenase assays were employed to determine the optimal OGD time point. EVs were isolated using polyethylene glycol‐based precipitation, adhering to MISEV 2023 guidelines. The characterization of EVs was conducted through nanoparticle tracking analysis, western blot, and transmission electron microscopy. Proteomic profiling of EVs was performed via mass spectrometry, followed by Bayesian data analysis to generate a ranked list of IS‐related EV protein biomarkers.

Results : EVs were successfully isolated following 4 h of OGD, a time point validated by metabolic assays showing no significant drop in viability and no significant increase in the cytotoxicity across all cell types. Comprehensive characterization revealed distinct size distributions, protein markers, and structural features in EVs from control and OGD conditions. Proteomic analysis identified both cell‐specific and shared EV proteins under ischaemic stress, revealing distinct differential expression patterns within each cell type and between the different cell types. Bayesian analysis integrated these findings with existing datasets, producing a prioritized list of potential EV protein‐based biomarkers for IS.

Summary/Conclusion : This study demonstrates that EVs released from neuronees, astrocytes, and endothelial cells under OGD conditions contain distinct proteomic signatures. These findings provide valuable insights into the role of EVs in IS pathology and highlight their potential as biomarkers for early diagnosis and therapeutic intervention.

Funding : DST‐SERB (SRG/2021/001357), YU/SG/169‐2024, ICMR (BMI/11(67)/2022).

Extracellular vesicles as a potential source of biomarkers in endometriosis and ovarian cancer

Barbara Bortot 1 , Alice Gualerzi 2 , Roberta Di Florio 1 , Silvia Picciolini 2 , Aurora Mangolini 2 , Francesco Valle 3 , Marco Brucale 3 , Gabriella Zito 1 , Giovanni Di Lorenzo 1 , Federico Romano 1 , Giuseppe Ricci 1,5 , Stefania Biffi 1

1 Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy; 2 IRCCS Fondazione Don Carlo Gnocchi Onlus, Milan, Italy; 3 Institute of Nanostructured Materials, Italian National Council of Research, Bologna, Italy; 4 Department of Medical and Surgical Sciences, Institute of Obstetrics and Gynecology, University of Foggia, Foggia, Italy; 5 Department of Medical, Surgical and Health Science, University of Trieste, Trieste, Italy

Introduction : Extracellular vesicles (EVs) play a crucial role in cellular communication associated with both ovarian cancer (OC) and endometriosis, involving similar immuno‐disrupting and pro‐inflammatory pathways. In OC, EVs have been shown to promote tumour growth, angiogenesis, and metastasis by influencing the behaviour of surrounding cells within the pre‐metastatic niche. In endometriosis, EVs contribute to the development and maintenance of a pro‐inflammatory environment that supports the proliferation and persistence of endometrial‐like tissue outside the uterus. Research on EVs has the potential to uncover new biomarkers for early detection and monitoring of disease progression.

Methods : This study analysed small EVs from the peritoneal fluid of women with endometriosis, women without endometriosis, follicular fluid samples, and ascites from advanced OC patients. Upon size exclusion chromatography and ultracentrifugation, atomic force microscopy (AFM) and Raman spectroscopy were utilized to characterize the EVs. Nano‐flow cytometry was employed to assess platelet activation, and a single‐particle interferometric reflectance imaging sensing method was used to determine the expression and colocalization of CD63, CD81, CD9, CD41a, CD18, CD11b, and PD‐L1. Large and small urinary EVs were isolated, and their RNA profile was examined.

Results : The study measured the activity of platelets and neutrophils, enhancing understanding of the complex interactions between these cells, which are potential therapeutic targets. Modulation of PD‐L1 and a distinct tetraspanin profile were observed, indicating the presence of specific subpopulations of EVs. Morphometric analysis through AFM revealed changes in mechanics which might correspond to different lipid content that was also detected using Raman analysis, underscoring the necessity for EVs to acquire specific features to facilitate crossing of the extracellular matrix. Urine tests represent the initial phase of a non‐invasive screening method that will be expanded to include a larger number of samples.

Summary/Conclusion : The combination of advanced techniques provided a characterization of EVs. Future studies can explore the functional implications of these findings and their potential clinical applications in the management of these diseases.

Funding : This work was supported by the Italian Ministry of Health, through the contribution given to the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy.

Investigating extracellular vesicle mitochondrial DNA levels, chronic kidney disease and mitochondrial haplogroup in the context of obesity

Presenter: Nicole Noren Hooten

National Institute on Aging, National Institutes of Health, USA

Introduction : Disparities in obesity and chronic kidney disease (CKD) burden persist in the United States between African American and White adults. Obesity is a risk factor for CKD, but little is known about the molecular mechanisms. Since extracellular vesicles (EVs) facilitate communication between cells and tissues, we investigated the association of EVs with obesity and CKD. We and others have reported that circulating cell‐free mitochondrial DNA (ccf‐mtDNA) may be EV cargo. Ccf‐mtDNA is released into the circulation in response to cellular stress or damage. Lower levels of ccf‐mtDNA in whole blood or serum have been associated with renal dysfunction. Single nucleotide polymorphisms in mtDNA can be traced to ancestry and defined as mtDNA haplogroups. mtDNA haplogroup can affect mitochondrial function. The aim of this work was to examine plasma EVs and EV mtDNA with CKD and mtDNA haplogroup.

Methods : Plasma EVs were isolated using size exclusion chromatography from a longitudinal cohort of obese African American and White adults from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. Participants were chosen who at visit 1 did not have CKD but developed CKD by visit 2 ( n = 19; CKD group) and were matched to controls who did not have CKD at either visit 1 or visit 2 ( n = 56; control group). EVs were characterized using MISEV guidelines. EV mtDNA levels were analysed by qPCR, and ancestry was determined by haplotyping participants and categorizing them into African or European mtDNA haplogroups. HANDLS is approved by the Institutional Review Board of the National Institutes of Health, and all participants provided written informed consent.

Results : EV mtDNA levels vary by mtDNA haplogroup, CKD status, age, poverty status and sex. EV mtDNA levels were lower in individuals who developed CKD overall and particularly in individuals with the African haplogroup who developed CKD.

Summary/Conclusion : Both ancestry and CKD influence EV cargo in obese individuals. These findings suggest that lower EV mtDNA levels may be a blood biomarker of CKD and may be related to mitochondrial function in CKD participants.

Funding : Intramural Research Program of the National Institute on Aging, National Institutes of Health funded this work.

Exploring potential biomarkers in malaria patient biofluids derived from extracellular vesicles’

Presenter: Shreya Bhatnagar

Birla Institute of Technology and Sciences, India

Introduction : Malaria's global impact persists, causing millions of deaths. Despite extensive efforts, shortcomings in early detection exacerbate infection severity, prompting the need for more precise diagnostic biomarkers. Recent investigations examining extracellular vesicles (EVs) released by Plasmodium ‐infected red blood cells [iRBCs] and host cells have revealed their role in disease communication, sparking our interest in their potential as diagnostic indicators in biofluids, which could lead to novel and simple detection systems. Interestingly, EVs are membrane‐protected nanovesicles that carry cell‐specific protein, nucleotide and other metabolite cargos between cells. Species‐specific biomarkers for Plasmodium infections are sparsely known. Commercially used biomarkers like HRP2 are exhibiting genomic instability in some parasite populations. Prompting the need for novel diagnostic/prognostic biomarkers.

Methods : To investigate EVs in malaria, we purified EVs from plasma samples of both healthy individuals and patients infected with either Plasmodium vivax or Plasmodium falciparum . Distinguished between severe manifestations (e.g., thrombocytopenia, hepatic dysfunction, cerebral malaria) and non‐severe samples. Isolation involved differential centrifugation and size exclusion chromatography. Biophysical characterization via particle size analysis and electron microscopy, while Western blot assessed EV markers and contamination. Proteomes of EVs were analysed by mass spectrometry.

Results : Preliminary mass spectrometry data from plasma samples included overall proteome coverage of 1339 proteins in P. vivax samples (1239 human and 100 parasite proteins) and 1409 proteins in P. falciparum (1239 human and 71 parasite proteins). Proteins unique to each species and present in over 70% of clinical isolates were filtered for further analysis. Notably, most identified parasite proteins were hypothetical or linked to basic cellular processes. To name a few, P. falciparum included PF3D7_1450400, PF3D7_0916600, PF3D7_0419900, PF3D7_1441200, and so forth, while in the case of P. vivax PVX_089580, PVX_117305, PVX_086115, PVX_002580, and so forth [patent pending]. Interestingly, changes in host proteins between severe versus non‐severe samples were also observed, which could enhance diagnostic accuracy.

Summary/Conclusion : Future work will involve expanding sample size and conducting targeted proteomics on a larger cohort of EV samples to validate diagnostic potential. Antibodies/designed peptides against selected candidate biomarkers can be assembled into a unique panel of diagnostic/prognostic markers for malaria. Devising non‐invasive or minimally invasive (finger‐prick, if possible) detection methods. Similar concepts and techniques could be applied to other diseases.

Funding : Funded by the BITS BioCyTiH Foundation.

Plasma extracellular vesicle biomarkers linked to lower muscle mass, function, and physical performance in sarcopenia

Ji Yeon Kim 1 , Tae‐Hwan Gil 1 , Ji‐Won Shin 1 , Dong‐Hyun Jang 1 , Sung Hye Kong 2 , Ok Hee Jeon 1

1 Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea; 2 Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea

Introduction : As society rapidly ages, identifying individuals at risk for sarcopenia is essential. Extracellular vesicles (EVs) have the capacity to deliver various biomolecules and regulate cellular mechanisms, making them potential biomarkers for disease. This study aimed to investigate how established sarcopenia biomarkers in plasma‐derived EVs correlate with muscle mass, function, and physical performance.

Methods : EVs were isolated from plasma using density gradient ultracentrifugation, and sarcopenia biomarkers were quantified by ELISA. In plasma‐derived EVs from a prospective osteosarcopenia cohort at Seoul National University Bundang Hospital ( n = 93), we evaluated the expression of biomarkers including CRP, TNF‐alpha, myostatin, adiponectin, and P3NP, all recognized sarcopenia biomarkers. Multivariate regression analyses were conducted, adjusting for sex, age, and body mass index. Muscle mass was assessed with DXA, functional strength by handgrip strength, and physical performance via 4m‐walking speed and the short physical performance battery (SPPB), with baseline and 2‐year follow‐up measurements.

Results : The cohort's mean age was 74.3 years, with 88.2% female participants. After adjustments, baseline muscle function significantly correlated with TNF‐alpha ( β = −0.28, p = 0.01) and CRP ( β = 0.22, p = 0.03). Additionally, baseline walking speed was significantly related to both myostatin and TNF‐alpha ( β = 0.49, p < 0.01; β = 0.36, p = 0.02; respectively). Myostatin also correlated with changes in walking speed ( β = −0.33, p = 0.01), while P3NP was significantly associated with baseline SPPB scores ( β = −0.22, p = 0.03) after adjustments.

Summary/Conclusion : Our results showed strong correlations between EV‐derived myostatin, P3NP, and TNF‐α with key sarcopenia components, particularly muscle function and performance. These findings suggest that circulating EVs could serve as reliable biomarkers for sarcopenia, offering opportunities for early detection and management through blood‐based diagnostic methods.

The role of bacterial extracellular vesicles as biomarkers for prognosis in colorectal cancer

Tian Jin, Farzaneh Malekian, Alireza Shamsian, Van Manh Hung Le, and Christoph Kahlert

Heidelberg University, Germany

Introduction : Colorectal cancer (CRC) is the third most common malignancy worldwide, with liver metastasis the major cause of death. Studies showed bacterial extracellular vesicles (BEVs) can be detected in human plasma and have an impact on immune response and gut barrier integrity. Additionally, bacterial DNA has been identified in BEVs, human CRC tissues and paired liver metastasis as well. However, the predictive role of BEVs in CRC progression and metastasis remains unclear. This study investigates the potential of BEVs prevalence or bacterial DNA load in BEVs as markers for CRC metastasis and survival.

Methods : Total circulating EVs will be isolated by size exclusion chromatography from plasma samples of 100 non‐metastatic and 100 CRC patients with liver metastases. The amount of BEVs in total plasma EVs will be detected by flow cytometry with LPS antibody, CD9 antibody and CD63 antibody. DNA will be extracted from EVs, and the bacterial DNA load, normalized to 1 × 10 10 EV particles, will be quantified using qPCR targeting the 16S rDNA gene. Clinical data, including follow‐up on tumour progression and survival, will be obtained from the database at the respective department. The data analyses will be performed by R, and the cut‐off value will be determined.

Results : Preliminary findings in indicate that in plasma samples from CRC patients, LPS + CD9 − EVs account for approximately 8% of total circulating EVs. Additionally, bacterial 16S rDNA is detectable in EV‐derived DNA. Expected Outcomes : LPS+ BEVs and bacterial DNA can be detected from plasma EVs of non‐metastatic CRC patients and those with liver metastases. LPS+ BEVs (and/or bacterial DNA) are more enriched in liver metastatic CRC patients compared to non‐metastatic CRC patients. LPS+ BEV concentration (and/or bacterial DNA level) is inversely correlated with progression free and overall survival of CRC patients.

Summary/Conclusion : Circulating LPS+ BEV concentration (and/or BEV‐derived bacterial DNA) could be used as a biomarker for CRC.

Skeletal muscle‐derived extracellular vesicles as a source of biomarkers for facioscapulohumeral muscular dystrophy

Elvira Ragozzino 1 , Lorena Di Pietro 1,2 , Elisa Orecchini 1,3 , Diego Sibilia 1 , Gurjit Kaur 1 , Flavia Giacalone 1 , Andrea Papait 1,2 , Wanda Lattanzi 1,2 , Sara Bortolani 4 , Eleonora Torchia 4 , Mauro Monforte 4 , Simona Nanni 2,5 , Luciano Giacò 3 , Giulia Mantini 3 , Manuela Papacci 6 , Andrea Sabino 6 , Ornella Parolini 1,2 , Enzo Ricci 4,6*

1 Dipartimento di Scienze della Vita e Sanità Pubblica, Università Cattolica del Sacro Cuore, Italy; 2 Fondazione Policlinico Universitario A. Gemelli IRCCS, Italy; 3 Bioinformatics Facility Core Research, Gemelli Science and Technology Park (GSTeP) Fondazione Policlinico Universitario A. Gemelli IRCCS, Italy; 4 Unità Operativa Complessa di Neurologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, Italy; 5 Dipartimento di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, Italy; 6 Dipartimento di Neuroscienze, Università Cattolica del Sacro Cuore, Italy * Contributed equally to this work .

Introduction : Facioscapulohumeral muscular dystrophy (FSHD) is a common adult‐onset muscular dystrophy characterized by selective vulnerability of muscles (some are affected while others are spared), without a predictable pattern. This variability complicates the evaluation of therapeutic treatments, underscoring the need for reliable biomarkers correlated with disease burden. Here, we aim to identify small RNA biomarkers from extracellular vesicles (EVs) isolated from skeletal muscle (Sk‐m) of FSHD patients.

Methods : Muscle biopsies were collected from seven affected and seven unaffected muscles of FSHD patients and from five healthy controls, with informed consent. Samples were cultured in EV‐depleted media overnight, and Sk‐m EVs were isolated via TEIR (Invitrogen). EVs were characterized by transmission electron microscopy and nanoparticle tracking analysis, and small RNA sequencing was performed on RNA extracted using the Trizol method.

Results : Distinct classes of small RNAs, including microRNAs and isomiRs, were identified within Sk‐m EVs. Comparative analysis revealed significant differences in Sk‐m EV microRNA and isomiR profiles among affected and unaffected FSHD muscles and healthy controls. These RNA profiles correlated with both radiological markers of muscle damage and clinical severity. Pathway analysis indicated that these RNA molecules participate in inflammatory and fibrotic pathways, crucial to skeletal muscle regeneration and degeneration.

Summary/Conclusion : Our findings identify a novel panel of Sk‐m EV‐associated microRNAs and isomiRs as promising biomarkers for FSHD‐affected muscle tissue. These insights lay the groundwork for discovering circulating biomarkers specific to muscle status, leveraging EV stability in circulation. Furthermore, the identification of altered pathways enriches our understanding of FSHD pathophysiology, offering new opportunities to elucidate and potentially counteract the mechanisms of muscle degeneration in FSHD.

Funding : This study was funded by the Solve FSHD Canada Foundation.

Investigating small extracellular vesicle microRNA as stable biomarkers for prenatal alcohol exposure

Presenter: Toby Aarons

University of Salford, UK

Introduction : Prenatal alcohol exposure (PAE) is a significant global health challenge, posing major health risks, including impacting child IQ and foetal brain development, and potentially causing foetal alcohol spectrum disorder (FASD). Developing a biomarker for PAE could enable early detection and timely interventions, supporting individuals in managing lifelong developmental challenges. Small extracellular vesicles (sEVs), particularly sEV miRNA, present a promising candidate for PAE biomarkers as a potentially minimally invasive window into brain changes that have remained stable, even after ethanol is no longer in the system. Our group aims to build on the understanding of sEV miRNA responses to ethanol, using human neural cellular models of PAE.

Methods : Human neural cells (SH‐SY5Y and neural progenitor cells) were differentiated into mature neuronees and astrocytes. Differentiated cells were chronically treated with ethanol (100 mM) and cultured after ethanol withdrawal to assess stable sEV microRNA changes. Small extracellular vesicles (sEVs) were isolated through size exclusion chromatography and characterized with TEM, fluorescence nanoparticle tracking analysis (f‐NTA), and western blotting. Cellular and sEV miRNA were analysed using real‐time qPCR. Future experiments will examine RNA epigenetic modifications in both cellular and sEV RNA from ethanol exposure using direct RNA sequencing (DRS) on the Nanopore platform.

Results : Differentiated SH‐SY5Y cells, expressing NeuN, MAP2 and β3‐tubulin, exposed to 100 mM ethanol for 7 days show miRNA transcriptional alterations, with some candidate miRNA changes persisting even after ethanol removal for an additional 7 days. sEVs were isolated from various differentiation time points in the SH‐SY5Y PAE model. Fluorescent NTA (f‐NTA) and TEM confirmed that isolated sEVs were predominantly < 200 nm, and CD81 and CD63 positive. We identified dysregulated miRNAs (miR‐9‐5p, miR‐134‐5p, and miR‐485‐5p) in sEVs 7 days after ethanol exposure. Further findings on sEV miRNA in response to ethanol in our SH‐SY5Y PAE model, alongside preliminary findings in neural progenitor cells, will be presented.

Summary/Conclusion : Identifying molecular changes specific to PAE that remain stable after ethanol withdrawal is crucial for developing new biomarkers. We show a panel of sEV miRNAs that are promising candidates for stable biomarkers of PAE that build towards this goal.

Exploring EV pathology and treatment efficacy in ALS through lymphoblastoid cell lines

Carmen Perez de la Lastra 1 , Jesus Alejandro Bueso 1 , Rocío del Carmen Bravo‐Miana 2 , Manuel Izquierdo 3 David Otaegui 2,4 and Valle Palomo 1,4

1 IMDEA Nanoscience, Spain, 2 Biogipuzkoa, Spain, 3 Instituto de Investigaciones Biomédicas Sols‐Morreale (IIBM), Spain 4 CIBERNED, Spain

Carmen Perez de la Lastra, Jesus Alejandro Bueso are co‐first authors .

Introduction : Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neuronees, leading to muscle weakness and paralysis. Lymphoblastoid cell lines from ALS patients recapitulate pathological features and are an interesting platform to study pharmacological treatments [1]. Extracellular vesicles (EVs) have emerged as potential biomarkers of ALS pathology and have been associated with TDP‐43 pathology in cell cultures and plasma from patients [2]. The specific function of EVs in ALS and their modulation with therapeutic treatments remains uncertain. This study utilizes immortalized lymphocytes derived from ALS patients as a cellular model to explore EV‐related pathology and assess potential therapeutic approaches.

Methods : Lymphoblasts were derived from ALS patients and healthy controls to analyse EV production, cargo, and molecular composition. EVs were isolated in parallel, through ultracentrifugation and with an EV precipitating reagent, and enriched EVs were characterized through nanoparticle tracking analysis (NTA), Western blotting, transmission electron microscopy and shot‐gun proteomics. Lymphoblasts were treated with therapeutic compounds, and subsequent changes in EV characteristics were assessed. Fluorescent tetraspanin labelling was performed with quantum dots (QDs) and analysed by NTA.

Results : Several characteristics significantly separate ALS lymphoblasts from healthy controls or treated samples, from concentration to molecular profile. Different isolation methodologies resulted in diverse cargo and concentration values. The proteomics study resulted in differentially expressed proteins shared between the isolation methods when comparing healthy controls and patients. When focusing on ultracentrifugation samples, a principal component analysis resulted in separate groups of controls and ALS samples. The labelling of tetraspanins with QDs resulted in values comparable to those obtained by immune‐capture techniques.

Summary/Conclusion : These findings highlight the value of ALS patient‐derived lymphoblasts as a model for studying EV pathology and therapeutic responses in ALS. The treatment‐induced changes in EV cargo indicate that EVs could serve as useful biomarkers for monitoring disease progression and treatment efficacy. This study provides a foundation for further exploration into the relevance of EVs secreted by a secondary subset of cells affected in multisystemic diseases such as ALS and their potential to be used as a monitoring biomarker in therapies to develop more effective treatment strategies for ALS.

Subtopic EV biomarker clinical translation 1

Small extracellular vesicle‐derived miRNAs detected in organ preservation solution after cold ischaemia storage impair endothelial homeostasis and influence early allograft function after liver transplantation

Daniel Vidal‐Correoso 1 , Sandra V. Mateo 1 , Fernando Lucas‐Ruiz 1 , Marta Jover‐Aguilar 1 , Sara Sánchez‐Redondo 2 , Vanesa Santos 2 , Pablo Ramírez 3 , Héctor Peinado 2 , Alberto Baroja‐Mazo 1

1 Molecular Inflammation Group, University Clinical Hospital Virgen de la Arrixaca, Biomedical Research Institute of Murcia (IMIB‐Pascual Parrilla), Murcia, Spain; 2 Microenvironment & Metastasis Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 3 General Surgery and Abdominal Solid Organ Transplantation Unit, University Clinical Hospital Virgen de la Arrixaca, Murcia, Spain

Introduction : Liver transplantation (LT) is the gold standard for patients with end‐stage liver disease. While donation after brain death (DBD) is the preferred option, donation after circulatory death (DCD) has expanded the donor pool. Despite advancements, complications such as acute rejection (AR) or hepatic artery thrombosis continue to affect post‐transplant outcomes. Biomarkers, including small extracellular vesicles (sEVs) and microRNAs (miRNAs), are emerging as valuable tools for monitoring post‐transplant events. Endothelial cells (ECs), crucial for graft survival, are the first line of defence against recipient immune cells upon reperfusion. Healthy ECs are essential for maintaining vascular homeostasis, while their activation can trigger inflammatory and immune responses. This study investigates the impact of sEV‐derived miRNAs on endothelial function and early allograft performance in LT.

Methods : sEVs were isolated from intrahepatic end‐ischaemic organ preservation solutions (eiOPS) using methods aligned with MISEV 2023 guidelines. miRNA profiling was conducted on sEVs from 79 eiOPS samples by qRT‐PCR. Functional assays were performed by transfecting miRNAs into SK‐HEP‐1 hepatic endothelial cells, and transcriptional changes were quantified via RNA sequencing. Bioinformatic analysis was conducted using R software to explore the pathways affected.

Results : No significant differences in sEV concentration were found between DBD and DCD samples. However, several miRNAs, including hsa‐let‐7d‐5p, hsa‐miR‐28‐5p, hsa‐miR‐200a‐3p, hsa‐miR‐200b‐3p, hsa‐miR‐200c‐3p, and hsa‐miR‐429, were overexpressed in DCD‐derived eiOPS. Among these, let‐7d‐5p showed a positive correlation with the Model for Early Allograft Function (MEAF), linking its expression to graft performance. A predictive model combining miR‐200b‐3p, miR‐429, and clinical variables demonstrated potential for AR detection. In vitro, transfection of SK‐HEP‐1 cells with miR‐200a‐3p and miR‐429 resulted in the dysregulation of pathways related to focal adhesion and cytokine interactions, compromising essential biological processes such as wound healing and cell adhesion.

Summary/Conclusion : These findings highlight the role of sEV‐derived miRNAs in modulating endothelial cell homeostasis and early graft function after LT. The identified miRNAs could serve as novel biomarkers for predicting post‐transplant outcomes and offer insights into the molecular mechanisms of endothelial dysfunction, providing potential therapeutic targets.

Funding : Instituto Murciano de Investigación Biosanitaria‐Pascual Parrilla (IMIB22/CI/11), Fundación Mutua Madrileña (AP171362019) and Instituto de Salud Carlos III (PI20/00185).

Role of extracellular vesicles in mediating circadian disruption effects on colorectal cancer progression and drug resistance

Presenter: Emanuele Murgo

Università degli Studi di Foggia, Italy

Introduction : Circadian rhythms play a crucial role in regulating cellular processes, including metabolism, DNA repair and immune function. Disruptions in circadian regulation are increasingly associated with tumourigenesis and drug resistance, particularly in colorectal cancer (CRC), one of the most prevalent and lethal cancers worldwide. Extracellular vesicles (EVs) serve as essential mediators of intercellular communication, contributing to the progression and adaptive resistance of tumours. However, the role of EVs in the transmission of circadian signalling disruptions within CRC has yet to be fully explored.

Methods : We generated CRC in vitro models with different levels of BMAL1‐CLOCK complex, a key circadian rhythm regulator, to induce circadian dysregulation. EVs were isolated from both circadian‐disrupted and control CRC cells and characterized by nanoparticle tracking analysis and Western blotting for canonical EV markers. The molecular contents of the EVs, including miRNA and protein profiles, were analysed via miRNA‐seq and Orbitrap QExactive HF mass spectrometer (the most advanced high‐resolution MS instruments available), respectively. Functional assays assessed the effects of circadian‐modulated EV miRNA‐protein on proliferation, invasiveness and chemotherapy sensitivity in recipient CRC cells.

Results : EVs from CRC cells with circadian rhythm disruption (specifically, altered BMAL1‐CLOCK levels) displayed distinct molecular profiles compared to EVs from control CRC cells. Proteomic analysis identified upregulation of proteins associated with cell growth and survival pathways, including those in the PI3K‐Akt and MAPK signalling pathways. miRNA‐seq analysis revealed an increase in specific miRNAs known to influence tumour proliferation and invasion. In functional assays, CRC cells treated with EVs from circadian‐disrupted cells exhibited higher rates of proliferation and invasiveness than those treated with EVs from control cells. Additionally, these cells demonstrated a significant decrease in sensitivity to standard chemotherapeutic agents, suggesting that circadian‐disrupted EVs may contribute to drug resistance mechanisms in CRC.

Summary/Conclusion : Our findings suggest that EVs play a crucial role in mediating the effects of circadian disruption in CRC by transferring altered molecular signals that enhance tumour aggressiveness and therapy resistance. Targeting EV‐mediated circadian pathways may offer a promising therapeutic approach to counteract tumour progression and improve treatment response in CRC patients.

Isolation, molecular characterization, and anti‐inflammatory potential of extracellular vesicles from Edwardsiella piscicida

Mawalle Kankanamge Hasitha Madhawa Dias, Mahanama De Zoysa

College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Republic of Korea

Introduction : Bacterial extracellular vesicles (BEVs) are nanoscale, membrane‐bound structures that play a role in intercellular communication and the transport of bioactive molecules. BEVs are gaining interest for their potential applications in drug delivery, immunomodulation, and anti‐inflammatory therapies. In this study, we present proteomic insight and evaluate the anti‐inflammatory activity of extracellular vesicles from the Gram‐negative bacterium Edwardsiella piscicida (EpEVs).

Methods : The isolation of EpEVs was performed by using the ultracentrifugation method, followed by characterization of their particle size, morphology, surface charge, and so forth. The proteome of EpEVs was analysed for peptide sequencing and protein quantification using an Orbitrap ExplorisTM 480 mass spectrometer paired with an Ultimate 3000 UHPLC liquid chromatography system. We characterized the proteomic profile of EpEVs and employed in silico methods to identify potential EpEVs proteins that may interact with host proteins. The toxicity levels of EpEVs were determined in fathead minnow (FHM) cells, murine macrophage Raw 264.7 cells, and zebrafish larvae. Additionally, the in vitro anti‐inflammatory activity of EpEVs was determined using Raw 264.7 cells.

Results : Transmission electron microscopy confirmed the globular morphology of isolated EpEVs, with an average size of 85.3 ± 1.8 nm. Proteomic analysis identified 1487 proteins, with 61.31% associated with the ‘cell’ and ‘cell part’ localizations. Key identified proteins included virulence factors, such as type III secretion system elements, flagellin, and outer membrane proteins. Protein‐protein interaction analysis with E. tarda proteins showed 673 interactions among the EpEVs proteins. In toxicity assessments, EpEVs showed no adverse effects in FHM cells up to 100 µg/mL; however, mild toxicity was observed in RAW 264.7 cells at 12.5 µg/mL. Functionally, EpEVs demonstrated potent anti‐inflammatory activity in LPS‐stimulated Raw 264.7 cells through a significant reduction of mRNAs of Tlr2, Tlr4, Il1β, Il6, and nitric oxide synthase 2 expression.

Summary/Conclusion : We have successfully isolated EpEVs using the ultracentrifugation method. These EpEVs contain distinct proteins that mediate anti‐inflammatory effects by regulating activation of TLRs and pro‐inflammatory cytokine expression, suggesting their potential as anti‐inflammatory agents.

Funding : This work was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korean government (MSIT) (2023R1A2C1006901).

Development of tumour‐specific EV‐miRNA signatures to improve lung cancer early detection (EV‐miR‐Test)

A. A. Saponaro 1,2 , V. Manuti 1 , E. Murgo 2 , G. Savino 2 , M. G. P. Marasco 2 , B. Pasculli 3 , O. Palumbo 4 , F. Mazzarelli 5 , R. Cuttano 5 , F. Bianchi 5 , T. Colangelo 1,2

1 Cancer Cell Signalling Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Italy; 2 Department of Medical and Surgical Sciences, University of Foggia, Italy; 3 Laboratory of Oncology, Fondazione IRCCS Casa Sollievo della Sofferenza, Italy; 4 Medical Genetics Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Italy. 5 Cancer Biomarkers Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Italy

Introduction : Lung cancer (LC) is the deadliest cancer worldwide, causing ∼2 million deaths annually. The two primary subtypes are non‐small‐cell lung cancer (NSCLC, ∼85%) and small‐cell lung cancer (SCLC, ∼15%). Due to late‐stage diagnoses and the lack of effective early screening, LC has a low 5‐year survival rate of less than 15%. Liquid biopsies have emerged as a promising, non‐invasive alternative to tissue biopsies, analysing circulating tumour cells (CTCs), cell‐free DNA (cfDNA), and extracellular vesicles (EVs) in bodily fluids. EVs, abundant and stable carriers of molecular cargos like microRNAs (miRNAs), are critical for understanding cancer biology as they regulate cancer development, progression, and resistance to therapies. Additionally, cell‐free miRNAs (cf‐miRNAs) are highly stable and tissue‐specific, making them promising candidates for LC diagnosis and prognosis.

Methods : To identify a tumour‐specific EV‐miRNA signature, a global miRNA expression profiling was performed on early‐stage NSCLC patients' plasma samples compared to healthy controls. EVs were isolated using ultracentrifugation and immunoaffinity beads, followed by characterization via nanoparticle tracking analysis and immunoblotting. Epithelial cell‐specific EVs (EP‐EVs), enriched for EpCAM+ markers, were captured using an EpCAM antibody. This profiling method was extended to compare miRNA signatures in tumour and normal lung cells, further distinguishing epithelial‐origin (EP‐EV‐miRNAs) from inflammatory‐origin (TME‐EV‐miRNAs). Tumour‐specific EV‐miR signature was validated through calibration, validation, and clinical groups, while functional assays investigated EV‐miRNAs roles in NSCLC cell proliferation, migration, and invasion.

Results : A new EV‐miRNA signature was identified, distinguishing LC patients from healthy individuals. The origin of LC‐derived EV‐miRs (epithelial vs. inflammatory) was elucidated. The analysis of the functional role of EP‐EV‐miRs and TME‐EV‐miRs in NSCLC provided insights into their involvement in cancer progression, revealing that dysregulated EV‐miRNAs enhanced cell proliferation and invasion in NSCLC cell lines compared to controls.

Summary/Conclusion : EV‐miRNA could serve as non‐invasive biomarkers for monitoring LC progression over time. Developing an accurate, tumour‐specific EV‐miR test may reduce false positives in NSCLC screening and refine risk assessment, especially for individuals selected for LDCT screening. Clarifying EV‐miRNA origins and functions could lead to an effective early detection test for NSCLC, improving diagnosis, survival rates, and treatment outcomes while potentially lowering healthcare costs.

Decoding EV‐miRNA‐regulated neurodegenerative pathways in the aftermath of COVID‐19

Presenter: Gunjan A. Bawne

Biomedical Primate Research Center, The Netherlands

Introduction : Post‐Acute Sequelae of COVID‐19 (PASC) is a growing concern in public health, with the majority of PASC patients suffering from lingering neurological symptoms like extreme fatigue and cognitive impairment. This suggests a potential link between COVID‐19 and neurodegenerative diseases like Alzheimer's (AD) and Parkinson's (PD). Viral infections, including SARS‐CoV‐2, may accelerate neurodegeneration, but the mechanisms driving PASC remain unclear. Extracellular vesicles (EVs) in biofluids mediate intercellular communication, transporting molecules (e.g., miRNAs) that can potentially impact disease progression and have already emerged as biomarkers for HIV‐induced cognitive problems and as markers for AD and PD. In this study, we investigate EV‐miRNA‐regulated pathways driving SARS‐CoV‐2‐induced neurodegeneration to gain insights into mechanisms underlying the pathophysiology of PASC.

Methods : Differentially expressed EV‐miRNAs from published datasets of patients suffering from COVID‐19, AD and PD were annotated to target genes, followed by pathway enrichment analyses. Pathways shared between SARS‐CoV‐2, AD, and PD were further compared to free miRNA data from other viral infections and neurodegenerative diseases. The IsoSeek pipeline for effective EV isolation, quantification and subsequent transcriptomic analysis was utilised to analyse the EV‐miRNAs from plasma of 6 control and 6 SARS‐CoV‐2‐infected macaques (3 months post‐infection). Subsequently, pathways regulated by human orthologs of differentially expressed miRNAs were identified using a similar bioinformatics approach as described above.

Results : Pathway enrichment analysis of human plasma EV‐miRNA targets revealed significant overlap in inflammation, immune response, and oxidative stress between SARS‐CoV‐2 infection, AD, and PD. This signature remained consistent across several viral infections known to influence neurodegeneration in humans. Moreover, dysregulated EV‐miRNAs in macaques' plasma at 3 months following SARS‐CoV‐2 infection were linked to neurodegeneration pathways, similar to EV‐miRNAs from human studies on acute infection.

Summary/Conclusion : These findings highlight the potential of EV‐miRNAs as biomarkers for detection and monitoring of SARS‐CoV‐2‐induced neurodegeneration. While analysed human data reflects acute cases, the macaque model reveals EV‐miRNA‐regulated neurodegenerative pathways months post‐infection, suggesting potential long‐term effects. Further validation of these miRNAs in plasma from PASC patients will show their relevance to the human disease and will form a framework for future research into diagnostics and targeted therapies.

Subtopic Cancer biomarkers 1

Chemotherapy resistance in triple‐negative breast cancer mediated by extracellular vesicles and vesicular RNA

Presenter: Efrain A. Perez‐Moreno

National Autonomous University of Mexico, Mexico

Introduction : Triple‐negative breast cancer (TNBC) is an aggressive and heterogeneous disease mainly treated with chemotherapy. However, resistance to therapy significantly impacts patient survival. Extracellular vesicles (EVs) play a role in this resistance by transferring RNA between tumour cell subpopulations, influencing gene expression and host cell traits, which contribute to tumour aggressiveness and drug response. This study investigates the role of vesicular RNA in TNBC chemoresistance.

Methods : EVs were isolated from the culture media of TNBC cells: MDA‐MB‐468, MDA‐MB‐231, and BT‐549, which vary in drug response, using PEG precipitation and sequential differential ultracentrifugation (dUC). Small EVs were characterized both physically and biochemically. Sensitive cell lines were exposed to 10,000 sEVs per cell from resistant cells. Transcriptomic analysis of the EVs and conditioned cells was conducted with microarrays, and the findings were correlated with public expression data from TNBC patients resistant to chemotherapy. Four RNA transcripts were identified as key players in EV‐mediated chemoresistance. Their functional roles were validated in vitro through siRNA and miRNA mimic transfections, assessing their impact on chemotherapeutic responses and tumour aggressiveness.

Results : Conditioning with dUC‐enriched EVs, which express inclusion markers like CD9, CD63, and Hsp70 without high exclusion marker levels, from docetaxel‐ or doxorubicin‐resistant cells reduced sensitivity to chemotherapy in recipient cells. The biodistribution of EVs in these cells is primarily perinuclear and cytoplasmic, with docetaxel treatment decreasing EV quantity. EVs also contain ABCG2, a protein associated with chemotherapy resistance, which is upregulated in conditioned cells, while miRNA biogenesis proteins are downregulated. Transcriptomics of EVs, conditioned cells, and public clinical data indicated that CDH6, TMPRSS4, miR‐574‐5p, and miR‐7977 are linked to chemoresistance. Modulating these genes affected cell proliferation, viability, migration, invasion, and apoptosis. Additionally, transcriptional analysis showed enrichment in DNA repair and cell growth pathways, highlighting their role in cell plasticity and survival in EV‐induced chemoresistance.

Summary/Conclusion : We have characterized an RNA signature, first described in EVs, that may mediate the transfer of chemoresistance in TNBC, contributing to the understanding of resistance mechanisms in this disease.

Funding : PAPIIT‐UNAM‐ IA206422.

Exosomal and cfDNA in diagnosis and treatment of colorectal cancer

Presenter: Alisa Petkevich

Peoples' Friendship University of Russia (RUDN), Russia

Introduction : Exosomes are one of the promising sources of biomarkers, indicating the processes that occur but do not yet have clinical manifestation, which is partly explained by the intercellular messenger role of exosomes. The aim of this study was to show the potential of exosomes in the prognosis and shaping of treatment of colorectal and prostate cancer.

Methods : Exosomes were isolated from plasma samples of patients with colorectal cancer of I‐II ( n = 21) and III‐IV stages ( n = 21) by differential centrifugation. NTA was performed, and CD9 and CD63 expression was assessed by WB. Circulating cell‐free DNA was isolated with QIAamp MinElute ccfDNA (Qiagen, Germany). Exosomal DNA was isolated with the same kit from previously isolated exosomes. Exome sequencing was performed for every plasma sample in triplicate: sequencing of circulating DNA, of circulating cell‐free DNA and of exosomal DNA (Illumina, USA). Afterwards, in an attempt to increase the specificity of sequencing, DNA regions susceptible to mutations (for 21 samples) were amplified with customized primers and then sequenced (Illumina, USA).

Results : Mutation signatures of cell‐free DNA and exosomal DNA were compared to the circulating DNA sequencing. Cell‐free DNA and exosomal DNA showed similar mutational signatures for patients with stages III–IV, differing from circulating DNA by 17%–23% and not showing significant differences between each other. In patients with stages I–II stages mutational signatures of cell‐free DNA and exosomal DNA showed a more significant difference of up to 15%. When sequencing DNA from exosomes, the proportion of mutant DNA to normal DNA is on average 14% higher than in circulating DNA. Obviously, sequencing DNA regions provides increased sensitivity in detecting mutations and may allow us to identify mutations in biomaterial represented by less than 1% compared to WT alleles.

Summary/Conclusion : We supposed to find that exosomal DNA should reflect the mutational landscape of the tumour to a greater, or at least not lesser, degree than circulating cell‐free DNA, and thus its sequencing data should differentiate from circulating DNA sequencing. During this study, we partially received some confirmation of this hypothesis and plan to continue working in this direction.

Bacille Calmette‐Guérin immunotherapy failure in bladder cancer patients: unlocking the world of miRNAs derived from urinary extracellular vesicles

Ana Teixeira‐Marques 1,2 , José Pedro Sequeira 1,2 , Sara Monteiro‐Reis 1 , Rui Freitas 1,3 , Marta Dueñas 4,5,6 , Rui Henrique 1,7,8 , Carmen Jerónimo 1,8

1 Cancer Biology and Epigenetics Group, Research Center of IPO Porto Research Center/Porto Comprehensive Cancer Center (Porto.CCC); 2 Doctoral Programme in Biomedical Sciences, ICBAS School Medicine and Biomedical Sciences, University of Porto, R. Jorge de Viterbo Ferreira 228, 4050 313 Porto, Portugal; 3 Urology Clinic & Department of Urology, Portuguese Oncology Institute of Porto (IPO Porto); 4 Research Institute Hospital 12 de Octubre (Imas12), University Hospital 12 de Octubre, Av Cordoba s/n, 28041 Madrid, Spain; 5 Molecular Oncology Unit, CIEMAT (Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas), Avenida Complutense 40, 28040 Madrid, Spain; 6 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III, SPAIN; 7 Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto); 8 Department of Pathology and Molecular Immunology, ICBAS‐School of Medicine & Biomedical Sciences, University of Porto; Porto, Portugal

Introduction : According to the EAU guidelines, bladder cancer patients classified into intermediate‐ or high‐risk groups are typically treated with BCG immunotherapy. However, despite this treatment, 40%–50% of patients experience tumour recurrence/progression. There are no FDA/CE‐approved biomarkers to predict BCG failure, contributing to poor outcomes and overtreatment. Small non‐coding RNAs (sncRNAs) derived from extracellular vesicles (EVs) have gained attention as potential biomarkers, as they play a key role in cell‐to‐cell communication in cancer. This study aimed to profile the EV‐sncRNAs in BCG‐treated patients to uncover candidate biomarkers that could predict BCG failure, as well as to explore the role that EV‐sncRNA targets may play in disease relapse.

Methods : Urinary EVs were separated from BCG non‐failure ( n = 8) and BCG failure ( n = 7) patient samples (informed consent was obtained; IPO Porto ethical approval—CES_62/023) using differential ultracentrifugation and characterized according to the MISEV2023 guidelines. RNA was isolated, and sncRNA‐sequencing was conducted. SncRNAs were considered statistically significant when |log2(FoldChange)| ≥ 0 and p value < 0.05. KEGG and GO analyses were performed on the respective miRNAs’ targets.

Results : SncRNA‐seq identified 63 miRNAs differentially expressed between the BCG non‐failure and BCG failure groups, with 5 miRNAs being significantly downregulated and 12 significantly upregulated in the BCG non‐failure group. Overall, patients who experienced BCG failure showed fewer miRNAs than the BCG non‐failure group (647 vs. 458 miRNAs, respectively), adding to the fact that these numbers were lower in progressions than in recurrences (377 and 403 miRNAs, respectively). The MAPK signalling pathway showed the highest gene ratio and count by KEGG analysis, and differences in biological processes related to small GTPase‐mediated signal transduction were found by GO analysis.

Summary/Conclusion : Urinary EV‐miRNAs revealed potential as promising biomarkers for predicting BCG failure. Additionally, the lower EV‐miRNAs number in BCG failure patients, particularly in those with tumour progression, suggests that these miRNAs may have a tumour‐suppressive role and may be involved in driving disease progression. Furthermore, the regulation of MAPK pathway targets by EV‐miRNAs may be associated with BCG failure, underlying the need for a deeper understanding of these sncRNAs in BCG failure.

Funding : UCIPredict‐2023/TRANSCAN3/0001/2021; FCT PhD Fellowship 2023.01232.BD.

Subtopic EV biomarker clinical translation 1

Circulating extracellular vesicles participate in platelet aggregation in patients with burn injury complicated by sepsis

Martina Schiavello 1 , Barbara Vizio 1 , Ornella Bosco 1 , Filippo Mariano 1 , Stefania Bruno 1 , Maria Chiara Deregibus 1 , Anna Pensa 2 , Loris Delmastro 1,3 , Emanuele Pivetta 1 , Alberto Sciarrillo 2 , Simon F De Meyer 4 , Fulvio Morello 1 , Giovanni Camussi 1 , Giuseppe Montrucchio 1 and Enrico Lupia 1

1 Department of Medical Science, University of Turin, Turin, Italy; 2 Burn Centre and Plastic Surgery, University Hospital City of Science and Health, CTO Hospital, Turin, Italy; 3 School of Specialization in Emergency Medicine, University of Turin, Turin, Italy; 4 Laboratory for Thrombosis Research, KU Leuven Campus Kulak, Kortrijk, Belgium

Introduction : Sepsis is the leading cause of death in patients with burn injury and represents a real diagnostic challenge. Circulating extracellular vesicles (EVs) are novel intercellular mediators involved in the downstream inflammatory response during sepsis that have promising potential as biomarkers and mediators of sepsis. Here, we characterized EVs circulating in burn patients during sepsis by multiplexed phenotyping and explored their role in platelet aggregation.

Methods : EVs were isolated from 33 patients with burn injury, 23 with (burn septic patients—BSP) 10 without sepsis (burn non‐septic patients—BnSP), and 10 healthy subjects (HS) used as controls, by ultracentrifugation and precipitation‐based methods. EVs were analysed by NTA, TEM and flow cytometry. EV surface antigens were studied by bead‐based multiplex cytofluorimetric analysis. In vitro functional assays on platelet aggregation were studied in platelet‐rich plasma (PRP) using light‐transmission aggregometry.

Results : BSP expressed a more specific pattern of EV epitopes than BnSP and HS. Of note, we found an increase of CD42a expression in BSP‐ compared to BnSP‐ ( p < 0.05) and HS‐ ( p < 0.0001) derived EVs. Moreover, the CD42a‐EVs expression level was gradually increased during the severity of sepsis in BSP. In vitro experiments showed that EVs from BSP, but not from HS, primed platelet aggregation, and this effect was reduced by a CD42 neutralizing antibody.

Summary/Conclusion : Our results suggest the potential of CD42a‐EVs as diagnostic and prognostic markers of sepsis in a heterogeneous population of burn patients. Moreover, we proposed CD42a‐EVs as pathogenic mediators of increased platelet activation in burn patients complicated by sepsis.

A multiomic investigation of human milk extracellular vesicles in perinatal HIV and antiretroviral therapy exposure

Presenter: Allison Kupsco

Columbia Mailman School of Public Health, USA

Introduction : Human milk (HM) is beneficial for early infant development, and breastfeeding is recommended globally for women with HIV (WHIV) on suppressive antiretroviral therapy (ART). HM has a high extracellular vesicle (EV) content, containing small metabolites and microRNAs, which are responsive to the maternal environment. Our goal was to investigate the impact of maternal HIV/ART exposure on the HM EV‐encapsulated metabolome and miRNAs and to compare metabolites between whole HM, skim milk, and EVs.

Methods : HM was collected at 1 month postpartum from WHIV receiving ART and HIV‐seronegative women living in South Africa (N ≈ 20 per group). Skim milk was separated with differential centrifugation, from which EVs were isolated using size‐exclusion chromatography. Successful EV isolation was confirmed with nanoparticle tracking analysis, transmission electron microscopy, and tetraspanin analysis. Panels of 700 metabolites and 1500 lipids were measured with Ultra Performance Liquid Chromatography‐Mass Spectrometry on whole HM, EVs, and skim milk. EV‐miRNAs were sequenced on an Illumina NovaSeq. We used linear models to compare levels of lipids, metabolites, and miRNAs in HM EVs of HIV/ART versus seronegative controls, adjusting for relevant covariates. Ethical approval was obtained from participating institutions, and participants provided informed consent.

Results : There were 293 lipids and 62 metabolites detected across all HM fractions, with 4 lipids and 15 metabolites detected solely in EVs. Of 89 small metabolites detected in HM EVs, 17 were significantly associated with HIV/ART status (false discovery rate [FDR] < 0.05), including methylmalonic acid, octanoyl‐carnitine, and ergocalciferol. Concentrations of these metabolites in skim and whole HM were not associated with HIV/ART status. Of 187 detected EV‐miRNAs, six were significantly lower in milk from WHIV, and hsa‐miR‐186‐5p was significantly higher in milk from WHIV (FDR < 0.05). The top 3 miRNAs were members of the miR‐30 family. KEGG pathway analysis revealed pathways related to insulin signalling, endocrine resistance, prolactin, and mitochondria.

Summary/Conclusion : These preliminary results suggest that HIV/ART exposure may be associated with HM EV pathways relevant for vitamin metabolism (i.e., ergocalciferol and methylmalonic acid) and metabolic health. Future research will examine the relationship between HM EV contents and child health.

Proteomic profiling of extracellular vesicles from iPSC‐derived brain organoids in Alzheimer's disease

Presenter: Vasiliki Machairaki

Johns Hopkins School of Medicine, USA

Introduction : Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia. AD patients frequently suffer from neuropsychiatric symptoms (NPS, agitation, depression, psychosis) from early disease stages, correlating to dysfunctional serotonergic systems. We have been generating iPSC‐derived hindbrain organoids (HT‐organoids) enriched in serotonergic neuronees from healthy individuals and AD patients with and without NPS. Proteomic analysis has been performed in both the organoids and derived EVs to detect differentially expressed proteins. Hindbrain organoids and EVs can be used to evaluate patient‐specific differences in disease progression and accelerate EV‐based biomarker discovery.

Methods : EVs have been isolated from the organoid‐cell culture supernatants by size exclusion chromatography and characterized by nanoflow cytometry. Proteomic profiling has been done on healthy and AD HT‐organoids and secreted EVs.

Results : The comparison between the identified proteins that showed significant differences in expression when distinguishing healthy and AD samples revealed 503 proteins positively identified for the organoids and 322 for the EVs. Interestingly, among those, only 32 (4%) were significant in both sets of comparisons. Even more surprisingly, among the 18 out of 32 proteins that were more highly expressed in healthy organoids, only 7 (∼39%) were also more highly expressed in their respective EVs. Conversely, among the 14 of the 32 shared significant proteins that were downregulated in healthy organoids compared to AD, 10 (71%) were also downregulated in their respective EVs. The overall concordance of down‐ or upregulation for the 32 proteins that were significant both when looking at organoids and EVs was thus 17/32, or ∼53%, when discriminating between healthy and AD.

Summary/Conclusion : Proteomic analysis of organoids and their derived EVs has important implications for the application of EVs as biomarkers of disease progression.

Funding : NIH R01 grant; NIH R21 grant.

Extracellular vesicles as carriers of pathogenic α‐synuclein in neurodegenerative diseases

Suman Dutta, Stelios Chatzimichail, Achillefs N. Kapanidis, George K. Tofaris

University of Oxford, UK

Introduction : Pathogenic alpha‐synuclein (α‐syn) protein aggregation is a hallmark of neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Given the potential of extracellular vesicles (EVs) as transport vehicles for misfolded proteins within the central nervous system (CNS), we investigated α‐syn in brain‐derived EVs to assess their role in disease propagation and as biomarkers for distinguishing PD/DLB from MSA.

Methods : EVs were isolated from post‐mortem brain tissues of healthy controls (HC) and patients with PD, DLB, and MSA. A combination of ultracentrifugation and size‐exclusion chromatography was used for EV enrichment, followed by nanoparticle tracking analysis (NTA) and western blotting for characterization. Single‐molecule localization studies with direct stochastic optical reconstruction microscopy (dSTORM) were employed to visualize EV‐associated α‐syn and the expression of various CNS cell‐type‐specific markers.

Results : The composition of neuronal and glial marker‐positive EVs in the brain was determined and found to be similar in healthy brains and those with α‐synucleinopathies. Neuronee‐originating EVs (L1CAM+) in PD and DLB samples exhibited increased association with α‐syn compared to MSA or healthy controls. In MSA samples, α‐syn showed a slightly higher association with oligodendrocyte‐originating EVs (MOG+) than with neuronee‐originating EVs, suggesting a potential disease‐specific EV signature.

Summary/Conclusion : There is a distinct pattern of α‐syn trafficking via EVs across neurodegenerative diseases, highlighting the potential of EVs as biomarkers for early diagnosis and differentiation between PD/DLB and MSA.

Funding : This project is supported by the Kavli Application for Research Development (K.A.R.D.) Fund, the Kavli Institute for Nanoscience Discovery, University of Oxford, and the Alzheimer's Research UK (ARUK) Thames Valley Network Pilot Award 2024.

High blood concentration of CD45‐EPCAM+ extracellular vesicles is associated with metastatic disease and poor survival in patients with pancreatic cancer

Presenter: Davide Brocco

University of Chieti, Italy

Introduction : Circulating tumour‐derived extracellular vesicles (EVs) represent a dynamic source of tumour biological information. In our study, we explore the clinical relevance of blood circulating EVs with a tumour‐like phenotype (CD45 − EPCAM + ) in patients with pancreatic cancer (PC). Furthermore, we established an immunocompetent, syngeneic murine PC model to correlate blood concentration of CD45‐EPCAM+ EVs with tumour growth.

Methods : Blood samples were collected from 60 patients with a histologically confirmed diagnosis of PC and sixty healthy controls. To recapitulate clinical findings in an in vivo model, we inoculated FC1199P3 and B6KPC3 murine cell lines in mice, and we longitudinally collected blood samples before and weekly after tumour inoculations. EVs were identified, enumerated, and phenotypically characterized from fresh whole blood samples by applying a polychromatic flow cytometry (FC) protocol based on a combined EV staining with a lipophilic cationic dye (LCD) and phalloidin. Surface subtyping of the total EV population was performed by combined selection of EPCAM positivity and CD45 negativity.

Results : Median blood concentrations of CD45 − EPCAM + EVs were doubled in the cancer population compared to HCs ( p = 0.003). Blood levels of EVs with tumour‐like phenotype were significantly associated with metastasis ( p = 0.03). Survival analysis revealed a significant correlation between low CD45 − EPCAM + EV concentration and improved overall survival ( p = 0.005). Notably, blood concentration of CD45 − EPCAM + EVs significantly increased following tumour inoculation in B6KPC3 and FC1199P3 mice.

Summary/Conclusion : Overall, these results provide interesting perspectives into the application of FC to enrich blood‐circulating tumour‐associated EVs and identify blood‐based EV biomarkers for personalized clinical management in patients with pancreatic cancer.

Funding : ‘National Center for Gene Therapy and Drugs based on RNA Technology. CN00000041 ‐ CUP: D73C22000810006,’ funded by Ministero dell'Università e della Ricerca—Piano Nazionale. Ripresa e Resilienza Missione 4, Componente 2, Investimento 1.5.

Hairy cell leukaemia as a paradigm of rare tumours: multiparametric profiling of plasma small and large extracellular vesicles unveils a biology‐related and disease‐specific signature

Filippo Maltoni 1,2 , Alessandro Broccoli 1,2 , Ghazal Narimanfar 1 , Simone Tiberi 1 , Roberto Maria Pellegrino 3 , Martina Barone 2 , Dorian Forte 1 , Giulia Gabrielli 1,2 , Lisa Argnani 1 , Carla Emiliani 3 , Pier Luigi Zinzani 1,2 , Lucia Catani 1,2

1 University of Bologna, Italy; 2 IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Italy; 3 University of Perugia, Italy

Introduction : Hairy cell leukaemia (HCL) is a rare and incurable lymphoproliferative B‐cell tumour characterized by infiltration of malignant hairy cells (HCs) within bone marrow. No disease‐specific biomarkers have yet penetrated clinical practice. Small (S‐) and large (L‐) extracellular vesicles (EVs) are particles promoting intercellular communication with a role in the ‘education’ of tumour niche. However, their function in HCL is unknown. Moreover, studies integrating protein, lipid and mRNA cargo of different plasma EV populations from (rare) cancer patients are completely lacking.

Methods : L‐ and S‐EVs were isolated from plasma of 8 HCL patients and 10 healthy donors (HD) by ultracentrifugation and characterized for surface protein expression (flow cytometry), lipid (mass spectrometry) and mRNA cargo (sequencing).

Results : Comparing HCL S‐ and L‐EVs, the former were enriched in immune (CD8, CD25), cancer/stem cell (CD326, CD133‐1), and EV (CD9, CD63, CD81) markers, while the latter resulted enriched in CD42a (platelet marker). Notably, CD25 is an HC marker. L‐EVs were also significantly enriched in phosphatidylethanolamine (PE), plasmenyl phosphatidylethanolamine (PE‐P), phosphatidylserine (PS), and phosphatidylinositol (PI) lipid classes. Compared to HD EVs, HCL S‐ and L‐EVs overexpress B (CD19, CD20) and T (CD8) cell markers and the CD44 adhesion molecule. HCL S‐EVs are enriched in HC (CD25), monocyte (CD14), and endothelial (CD105) markers. Lipidomic analysis revealed a significant increase in PE, PE‐P, and PS in both HCL EV subpopulations, with HCL L‐EVs also enriched in sphingomyelin. Exploring mRNA content, we found clear separation between S‐ and L‐EVs and between HCL and HD samples and revealed that S‐EVs carry transcripts of various lengths, while L‐EVs rarely express the shortest ones. Gene Ontology analysis showed that, comparing HCL S‐ and L‐EVs, the latter are enriched in transcripts encoding membrane structures. Comparing patients and HD, we found that HCL L‐EVs were enriched in transcripts related to HCL B‐cell origin/function and vesicle biogenesis, while HCL S‐EVs showed higher expression of transcripts related to cytoplasmic components, RNA splicing and B‐cell origin/function.

Summary/Conclusion : We identified a tumour‐associated signature of circulating HCL EVs that provides pathogenesis‐related information and highlights EVs for future liquid biopsy approaches in rare tumours.

Funding : The work was funded by the Italian Ministry of Health, RC‐2025‐2797531.

Proteomic and lipidomic profiling of plasma extracellular vesicles in multiple sclerosis

Presenter: Drishya Mainali

The University of Sydney, Australia

Introduction : Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) driven by immune‐inflammatory and neurodegenerative mechanisms. Identifying reliable biomarkers for MS remains crucial for understanding disease pathophysiology and monitoring clinical progression. EVs cross the blood‐brain barrier and mediate cellular crosstalk, making them promising candidates for non‐invasive biomarkers. This study investigates the proteomic and lipidomic profiles of plasma‐derived EVs from individuals with relapsing‐remitting MS (RRMS) and progressive MS (PMS) compared to healthy controls (HCs).

Methods : Large and small EVs were isolated from platelet‐reduced plasma of untreated RRMS ( n = 5), PMS ( n = 5), and healthy controls (HCs, n = 5) via ultracentrifugation. Additional EV isolation using size exclusion chromatography was performed on HCs ( n = 10), RRMS ( n = 10), and PMS ( n = 8). EVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and liquid chromatography/tandem mass spectrometry for proteomic and lipidomic profiling.

Results : Lipidomic analysis of sEVs revealed significantly lower levels of sphingomyelin (SM) lipid species (SM 34:1, SM 34:2, SM 36:1, SM 40:1, SM 40:2, and SM 42:2) in RRMS, and SM 34:1, SM 34:2, SM 36:1 in PMS compared to HCs. Proteomic analysis identified unique protein profiles in RRMS and PMS cohorts, with HCN1 and HCN2 (potassium/sodium hyperpolarization‐activated cyclic nucleotide‐gated channel 1 and 2) enriched in plasma EVs of 7 out of 8 individuals with progressive MS. EV‐associated VIM (Vimentin) and SAA4 (Serum Amyloid A‐4) were enriched in RRMS, while proteins such as C4BP4 and C4BPA (C4b‐binding protein), PROS (Vitamin K‐dependent protein S), and PRG4 (Proteoglycan‐4) were significantly decreased. In PMS, CO9 (Complement C9) was significantly increased, alongside significantly decreased levels of IGHM (Immunoglobulin Heavy Constant Mu) and A2MG (Alpha‐2‐Macroglobulin).

Summary/Conclusion : This study reveals distinct EV lipid and protein profiles in MS subtypes, identifying decreased sphingomyelin levels in RRMS. The detection of HCN1 and HCN2, uniquely expressed in PMS within peripheral blood plasma, is notably underreported. Ongoing cohort expansion aims to validate these findings for biomarker development and therapeutic targeting.

Funding : NHMRC.

Clinical development of multi‐omic protein and miRNA biomarker signatures for multiple myeloma EV liquid biopsy: identification, validation, and AI‐assisted analysis

Angelique Cheryl 1 , Rebecca Sheridan 1 , Kieran Brennan 1 , Despina Bazou 1,4 , Peter O'Gorman 2 , David Matallanas 3 , Luis F. Iglesias‐Martinez 3 , Margaret Mc Gee 1

1 School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin; 2 Mater Misericordiae University Hospital Dublin; 3 Systems Biology Ireland, School of Medicine, University College Dublin; 4 Department of Biology, Maynooth University, Ireland

Introduction : Multiple myeloma (MM) is an incurable haematologic malignancy characterised by malignant plasma cell expansion in the bone marrow (BM), often progressing to treatment‐resistant stages. Currently, MM diagnosis and monitoring rely on invasive BM biopsies and M‐protein quantification, lacking the sensitivity needed for early detection and disease staging. Extracellular vesicles (EVs) offer a promising alternative, carrying proteins and miRNAs that reflect disease status, representing a minimally invasive biomarker source for MM monitoring and staging.

Methods : Peripheral blood samples were collected from 36 individuals at Mater Hospital: healthy controls ( n = 10), newly diagnosed MM ( n = 12), relapse ( n = 8), remission ( n = 3), and MGUS ( n = 3). EVs were isolated per MISEV2023 guidelines using density gradient ultracentrifugation (DG‐UC) and characterised for identity and purity via nanoparticle tracking analysis (NTA) and Western blotting of markers (HSP90B1, HSP70, CD147, TSG101, CD9). Proteomic content was analysed by mass spectrometry, with RNA sequencing for transcriptomic profiling. Differential expression analyses (FDR < 0.05) were applied to proteomic and miRNA data, identifying potential biomarkers for disease staging. Additionally, supervised machine learning (ML) models, including Random Forest, Naive Bayes, and LASSO regression, optimise biomarker selection for MM staging.

Results : Our analysis identified a robust 13‐protein and 9‐miRNA EV biomarker panel capable of distinguishing MM disease stages. Notably, protein LFQ intensities were elevated in a stage‐specific manner, with distinct protein markers identified for initial diagnosis, relapse, remission, and MGUS. Furthermore, six miRNAs were upregulated and three significantly downregulated in MM compared to controls. Biomarker signatures are refined using ML integration to enhance diagnostic accuracy, identifying proteins and miRNAs with the highest predictive power for disease classification.

Summary/Conclusion : This study highlights a blood‐based EV biomarker panel as a promising non‐invasive approach for MM diagnosis and staging, supporting clinical decision‐making. Integrating ML models strengthens this panel's diagnostic potential, paving the way for standardised EV‐based assays for clinical validation in independent cohorts. This advancement could complement or replace BM biopsies, facilitating routine monitoring of MGUS and MM progression and improving patient outcomes. Disclosure: Certain biomarker details are withheld due to a pending patent application.

Funding : Supported by an Irish Cancer Society postgraduate scholarship and University College Dublin School of Biomolecular and Biomedical Science.

Platelet‐derived exosomes may function as a prospective diagnostic marker and therapeutic target in sepsis

Presenter: Hong‐Yue Lai

China Medical University, Taiwan (Republic of China)

Introduction : Coagulopathy is a major factor linked to mortality in septic patients, ranging from mild thrombocytopenia to life‐threatening disseminated intravascular coagulation (DIC). Generally, DIC is characterized by both thrombosis (exposed to tissue factor) and uncontrolled bleeding (secondary to platelet consumption), leading to organ failure. Accordingly, the effects of anticoagulant treatments for sepsis‐associated coagulopathy remain uncertain, and therapies beyond antimicrobials are rarely recommended globally. While the mortality rate for sepsis‐associated coagulopathy is high, early detection and prompt intervention are crucial for improving patient outcomes. Recent studies have highlighted the substantial role of extracellular vesicles, such as exosomes, in sepsis, but their clinical utility remains uncharacterized. In this study, we focused on enhancing diagnostic accuracy via exosome phenotypes to better stratify patients with specific coagulation features who might benefit from the corresponding therapies.

Methods : We prospectively recruited healthy controls ( n = 3), sepsis patients ( n = 3), and septic shock patients ( n = 7), and their blood specimens were collected. Clean exosomes were isolated from platelet‐poor plasma utilizing the ExoQuick precipitation solution (System Biosciences). Subsequently, exosome size and concentration were determined by ZetaView analysis, and exosome phenotypes were defined through flow cytometry.

Results : We observed that exosome size and concentration were not significantly changed among healthy controls, sepsis patients, and septic shock patients. Notably, using flow cytometry to detect exosome phenotypes, we found that the expression of the platelet marker (P‐selectin, CD62P) on exosomes was significantly reduced in sepsis and septic shock patients compared with that in healthy controls. Nevertheless, there was no significant alteration in the expressions of endothelium marker (CD31) and tissue factor (CD142) on exosomes.

Summary/Conclusion : Platelet‐derived exosomes, but not endothelium‐derived exosomes, may serve as a promising early diagnostic marker and a potential therapeutic target without increased risk of bleeding for septic patients with coagulopathy.

FCN3 and ITIH2 expression in the extracellular vesicles from the plasma of breast cancer women are novel candidates to be used as predictive biomarkers

Presenter: Rocio Castillo Sánchez

National Polytechnic Institute (Cinvestav), Mexico

Introduction : Breast cancer is the second most common cancer among women worldwide. The development of predictive biomarkers for breast cancer would allow the identification of patients that will benefit from early treatment. The liquid biopsy emerges as a proposal for a non‐invasive method in the diagnosis and prognosis of breast cancer. There are several cell components that can be analysed in a liquid biopsy, such as circulating tumour DNA (ctDNA), circulating tumour cells (CCTs) and extracellular vesicles (EVs). EVs refers to particles that are released from cells, are delimited by a lipid bilayer, and cannot replicate on their own. EVs cargo is an indicator of its cell of origin, containing biomolecules such as lipids, genetic material and proteins. EVs secretion in breast cancer has been well‐described, making it an effective tool for diagnosis.

Methods : Protein expression in EVs from plasma of breast cancer women and healthy donors was analysed using flow cytometry, western blot and HPLC mass spectrometry. All studied participants signed informed consent, and the protocol was approved by the ethics and research committee of INCan, Mexico (CI/600/15; CEI/710/15).

Results : Proteomic analysis revealed that EVs contain proteins associated with different cellular processes, including ficolin (FCN3), which is a protein of the complement activation lectin pathway, and the inter‐α trypsin inhibitor heavy chains (ITIH2), which are proteins that play an important role in inflammation and carcinogenesis. Our result showed that FCN3 and ITIH2 were differentially expressed in the EVs from plasma of healthy donors and breast cancer patients. Moreover, expression of proteins was analysed by using Cox proportional hazard regression and the Kaplan–Meier model, and results demonstrated that FCN3 and ITIH2 are both independent prognostic biomarkers for overall survival in breast cancer patients.

Summary/Conclusion : FCN3 and ITIH2 in the EVs are protective factors, because a decrease in their expression is an adverse prognosis in breast cancer.

Glycan‐ and protein‐sensing nanotech for extracellular vesicle‐based lung cancer screening and immunotherapy response prediction

Presenter: Quan Zhou

The University of Queensland, Australia

Introduction : Lung cancer is the leading cause of cancer‐related deaths worldwide, largely due to late‐stage diagnosis that limits treatment options. Current screening methods like low‐dose CT lack accuracy in distinguishing early‐stage malignant from benign nodules, highlighting the need for more precise, cost‐effective tools. Additionally, identifying patients likely to benefit from immunotherapy is crucial due to variable responses and possible side effects. Reliable biomarkers could guide immunotherapy decisions, improving outcomes and personalizing care. Extracellular vesicles (EVs), as valuable components in liquid biopsy, show promise for lung cancer screening and assessing immunotherapy response.

Methods : We developed a multiplex microfluidic platform that integrates alternating current electrohydrodynamics and surface‐enhanced Raman scattering (SERS) nanoparticle barcodes to capture and analyse EVs for lung cancer screening and immunotherapy prediction. For screening, a specific protein and glycan panel was identified through an isogeneic cancer cell line model and subsequently applied to the SERS microfluidic platform in a clinical cohort, distinguishing between benign and malignant nodules. For immunotherapy prediction, bioinformatic analyses identified glycosylation‐associated genes enriched in lung cancer, correlating with immune infiltration. Knockdown experiments in lung cancer cell lines allowed for lectin microarray analysis of glycan alterations in EVs, with functional assays elucidating their immunomodulatory roles. The glycan signature was validated on the SERS microfluidic platform in lung cancer patients with variable immunotherapy outcomes.

Results : The SERS microfluidic platform demonstrated high specificity and sensitivity in distinguishing early‐stage malignant lung nodules from benign nodules using both protein and glycan EV panels, with AUC values above 0.85. Distinct EV glycan profiles between late‐stage and early‐stage lung cancer patients indicated progressive alterations in glycosylation associated with tumour progression and immune evasion. Furthermore, alternations in EV glycan profiles were identified in knockdown experiments, which were found to correlate with outcomes in lung cancer patients receiving immunotherapy by the SERS microfluidic platform.

Summary/Conclusion : This SERS microfluidic platform offers a robust, minimally invasive approach for lung cancer screening and immunotherapy response prediction. By leveraging EV‐based protein and glycan panels, this platform provides an accurate and efficient alternative to traditional methods, addressing limitations of current screening techniques and advancing personalized immunotherapy strategies in lung cancer.

Subtopic Biomarker technologies 1

Circulating extracellular vesicles with immunomodulatory properties and biomarker potential in obesity‐related complications

Maiken Mellergaard 1,2 , Rikke Bæk 3 , Javier Alejandro Donoso Quezada 1 , Katja Lund Cliff 1 , Malwina Ulanowska 1 , Rikke Wehner Rasmussen 1 , Malene Møller Jørgensen, 2,3 Aase Handberg 1,2

1 Department of Clinical Biochemistry, Aalborg University Hospital, Denmark; 2 Department of Clinical Medicine, Aalborg University, Denmark; 3 Department of Clinical Immunology, Aalborg University Hospital, Denmark

Introduction : Obesity will affect 24% of the world's population in 2035, and > 50% of obese individuals develop complications related to non‐alcoholic fatty liver disease (NAFLD) and the metabolic syndrome (MetS). These individuals are at high risk of developing type 2 diabetes and cardiovascular disease via imbalanced immunity and chronic low‐grade inflammation. Biomarkers for stratification of obesity phenotype according to risk of developing complications are lacking. Extracellular vesicles (EVs) are known to regulate immunity via various context‐dependent mechanisms, including delivery of mediators like lipids and cytokines, or they can directly activate or suppress T cell function via surface expressed molecules. However, little is known about adaptive immunomodulatory EVs in obesity‐complications.

Methods : We did a longitudinal intervention study focusing on obese individuals with NAFLD and MetS (NAFLD‐MetS; high‐risk obesity) undergoing weight‐loss intervention for 20 weeks compared with obese individuals with either NAFLD or MetS (obese controls; low‐risk obesity). We used plasma samples to screen for candidate EV‐biomarkers to distinguish obesity phenotype by EV array analysis. Promising candidate biomarkers were validated by high‐resolution flow cytometry and related to clinical metabolic parameters.

Results : Among the top EV‐candidates distinguishing the high‐ and low‐risk group, several had immunomodulatory properties (patent pending). Especially, two classes of EVs with adaptive immunomodulatory properties were significantly increased in the NAFLD‐MetS group compared with obese controls. Moreover, these were significantly decreased during intervention, in line with levels of ectopic liver fat (57% reduction) and homeostatic model assessment for insulin resistance (42% reduction). Interestingly, clinical parameters were significantly decreased after 4 weeks, whereas immunomodulatory EVs were reduced after 20 weeks of weight loss.

Summary/Conclusion : We show that levels of EVs with T‐cell modulatory properties follow central features of developing metabolic conditions, possibly suggesting involvement in disease development. We are currently characterizing the cellular origin and intra‐vesicular content (cytokines and metabolites) of these EVs and how this relates to circulating levels of cytokines and metabolites to understand their potential functional properties and role in high‐risk obesity. Understanding the impact of EVs on disease development is essential for their future use as diagnostic biomarkers for stratification of obesity phenotype.

Funding : Independent Research Fund Denmark: 3101‐00394B and Research Cube AAUH 2022.

Unveiling the role of extracellular vesicle miRNAs in NAFLD pathogenesis and therapy

Manuti Virginia 1 , Saponaro Anna Alessia 1,2 , Murgo Emanuele 1 , Savino Giulia 2 , Marasco Maria Greta Pia 2 , Villani Rosanna 3 , Serviddio Gaetano 3 , Colangelo Tommaso 1,2

1 Cancer Cell Signalling, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy; 2 Department of Medical and Surgical Sciences, University of Foggia, Italy; 3 C.U.R.E. (University Center for Liver Disease Research and Treatment), Liver Unit, Department of Medical and Surgical Sciences, University of Foggia, Italy

Introduction : Non‐alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease, characterized by excessive hepatic fat accumulation. The disease can progress from simple hepatic steatosis to non‐alcoholic steatohepatitis (NASH), fibrosis, and, in severe cases, liver cirrhosis and cancer. Understanding the molecular mechanisms underlying this progression is crucial for developing targeted therapies. Extracellular vesicles (EVs), carrying miRNAs, play critical roles in intercellular communication, impacting metabolic and inflammatory pathways. This study seeks to uncover the role of EV‐derived miRNAs in NAFLD progression.

Methods : EVs were isolated from hepatocyte cell lines with induced steatosis via fatty acid exposure and characterized using nanoparticle tracking analysis and Western blotting. miRNA sequencing was performed to identify differentially expressed miRNAs in EVs derived from steatotic versus control hepatocytes. Select miRNAs of interest were validated using digital PCR. Functional assays were conducted by treating healthy hepatocyte cultures with miRNA‐enriched EVs to evaluate their impact on lipid accumulation, insulin signalling pathways and inflammatory markers (ELISA).

Results : Key miRNAs were identified in NAFLD‐derived EVs, notably those influencing lipid accumulation and inflammatory response. Functional studies demonstrated that these EV miRNAs regulate pathways critical to NAFLD pathogenesis, highlighting their potential as therapeutic targets. miRNA sequencing revealed a distinct miRNA expression profile in EVs derived from steatotic hepatocytes compared to controls such as miR‐122 and miR‐34a. These miRNAs are known to influence lipid storage and insulin resistance. Furthermore, when hepatocytes were treated with EVs containing steatosis‐associated miRNAs, there was an increase in intracellular lipid accumulation. In addition, Western blot analysis of insulin signalling pathway markers showed a decrease in phosphorylation of key proteins, such as Akt, in hepatocytes treated with miRNA‐enriched EVs. Finally, ELISA results revealed elevated levels of inflammatory cytokines (e.g., IL‐6 and TNF‐alpha) in hepatocytes exposed to steatosis‐associated EVs.

Summary/Conclusion : In the context of NAFLD, EV‐derived miRNAs may play a significant role in modulating pathways involved in lipid metabolism, insulin resistance and inflammation—three key aspects of the disease pathology. Our findings reveal EV‐miRNAs as modulators of metabolic dysregulation and inflammation in NAFLD, underscoring their promise as biomarkers and therapeutic targets.

Mirnomic analysis of urine extracellular vesicles from type 2 diabetic patients under SGLT2 inhibitor treatment: identification of biomarkers for therapeutic response

Beatrice Spokeviciute 1 , Fabrizio Buffolo 1 , Maddalena Arigoni 2 , Raffaele Calogero 2 , Ilaria Barchetta 3 ,Salvatore Cocola 3 , Gisella Cavallo 3 , Maria Felice Brizzi 1

1 Department of Medical Sciences, University of Turin, Italy; 2 Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy; 3 Department of Experimental Medicine, University of Sapienza, Rome, Italy

Introduction : SGLT2 inhibitors (SGLT2i) are oral medications that lower blood glucose in type 2 diabetes mellitus (T2DM). In addition to their glucose‐lowering effects, SGLT2i improve cardiovascular and renal outcomes by reducing HbA1c and blood pressure, decreasing inflammation, and improving kidney function, although the underlying mechanisms remain unclear. Extracellular vesicles (EVs) offer a non‐invasive way to monitor cellular responses to SGLT2i, as they carry bioactive molecules like miRNAs and reflect the physiological state of their origin cells. This study investigates renoprotective mechanisms of SGLT2i by analysing EVs from urine in T2DM patients treated with dapagliflozin.

Methods : EVs were isolated from patient urine samples via ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and western blotting for CD63 and TSG101 markers. miRNA expression profiles were assessed via next‐generation sequencing. Differentially expressed miRNAs were annotated using miRBase and linked to target genes through miRTarBase, followed by pathway analysis with Enrichr and network clustering with Gephi. The MACSPlex Assay was performed to identify changes of EV surface proteins following treatment.

Results : NTA confirmed a homogeneous EV population (100–150 nm), while Western blot verified EV identity. Sequencing identified 3827 miRNAs, with 16 miRNAs differentially expressed between baseline and 7‐month follow‐up samples. Pathway analysis linked these miRNAs to NLRP3 inflammasome regulation, platelet calcium homeostasis, and ion transport, suggesting reduced inflammation and stress responses. EV protein analysis revealed increased CD326 (EpCAM), indicating renal epithelial regeneration, and decreased CD146, associated with reduced kidney injury.

Summary/Conclusion : Urine EVs from SGLT2i‐treated patients showed shifts in miRNA expression and protein markers, suggesting reduced inflammation, cellular stress, and kidney injury alongside enhanced epithelial repair. Our findings suggest that EVs may serve as potential biomarkers for monitoring therapeutic response in T2DM and provide the basis to uncover the biological process associated with SGLT2i‐induced renal protection.

Funding : Supported by the University of Turin, BRIM_RILO‐19_01.

Extracellular vesicles in hepatic metabolism: insights into vesicle‐mediated signalling pathways in liver disease

Presenter: Giulia Savino

University of Foggia, Italy

Introduction : Extracellular vesicles (EVs) are nano‐sized particles released by cells that play a critical role in intercellular communication by transporting proteins, lipids, and RNAs. In hepatic metabolism, EVs have emerged as potential regulators of metabolic processes and liver pathology. This study explores the role of EVs in modulating liver metabolism, with a focus on their influence on lipid and glucose homeostasis and their contribution to the progression of liver diseases, including non‐alcoholic fatty liver disease (NAFLD).

Methods : EVs were isolated from plasma samples of patients with diagnosed liver disease and matched healthy controls following MISEV2023 guidelines. The EVs were characterized by nanoparticle tracking analysis (NTA), electron microscopy and immunoblotting. Proteomic and RNA‐seq/miRNA‐seq analyses were conducted to identify metabolic modulators enriched in EVs. Functional studies involved treating hepatocyte cultures with isolated EVs from different metabolic conditions to observe effects on lipid accumulation and inflammatory responses.

Results : EVs from liver disease patients (NASH_MASH) display a distinct metabolic signature compared to healthy controls, showing significant enrichment in molecules (protein‐miRNA) associated with lipid metabolism, particularly those influencing fatty acid oxidation and lipogenesis. These EVs affect hepatocytes by promoting lipid accumulation and inflammation, underscoring their role in metabolic disruption in liver disease. Furthermore, proteomic data suggest that EV cargo may serve as early biomarkers for liver metabolic disease progression.

Summary/Conclusion : This study supports the hypothesis that EVs act as critical mediators in hepatic metabolism and liver disease progression. The enrichment of lipid‐related molecules within EVs and their effects on hepatocytes suggest that targeting EV release or uptake may offer a novel therapeutic approach for metabolic liver diseases. Further research is warranted to fully understand EV‐mediated signalling pathways in liver metabolism and their potential for clinical applications.

Immuno‐qPCR as a highly sensitive and scalable diagnostic tool for profiling surface proteins on blood‐derived EVs

Presenter: Lucia Ciglar

Austrian Institute of Technology GmbH, Austria

Introduction : The molecular cargo of EVs is reflective of their cellular origin, making them a promising source of biomarkers for various diseases. Traditionally, biomarker research has focused on nucleic acids rather than proteins, largely due to the limitations of existing methods for protein detection. Current methods often lack the sensitivity to quantify low‐abundance proteins in complex biological fluids, they are difficult to scale up, and they may require specialized equipment. To address these challenges, we developed an immuno‐qPCR method, which significantly enhances sensitivity while offering a cost‐effective and high‐throughput approach for detecting surface proteins on EVs.

Methods : To adapt immuno‐qPCR for EV surface protein detection, we evaluated different protein immobilization strategies. The final optimized protocol employed magnetic beads to capture EVs, followed by surface protein detection using commercially available antibodies conjugated to DNA tags. These DNA tags were then amplified and quantified through qPCR. In a proof‐of‐concept study, we compared the performance of our immuno‐qPCR method to conventional flow cytometry‐based detection in clinical serum samples from patients with atopic dermatitis.

Results : The use of magnetic beads, both general protein‐binding and exosome‐specific, allowed for efficient capture of EVs, with the exosome‐specific beads providing an additional advantage of increased specificity and faster workflow. When tested on a cohort of 16 atopic dermatitis patients and 16 healthy volunteers, our optimized immuno‐qPCR method simultaneously detected 10 different surface proteins and demonstrated superior sensitivity compared to conventional flow cytometry‐based detection. Notably, for one of the tested proteins, CD25, we detected a significant difference in levels between patients and healthy volunteers, which was not uncovered with flow cytometry. As CD25‐positive immune cells have been previously shown to be enriched in the blood of atopic dermatitis patients, this finding further validates our approach.

Summary/Conclusion : The immuno‐qPCR method offers a highly sensitive, scalable, and versatile platform for the detection of EV surface proteins. This approach holds great promise for biomarker discovery and non‐invasive diagnostics, with potential applications extending beyond blood and atopic dermatitis.

Funding : This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No. 853995 with EU Horizon 2020 and EFPIA support.

Alteration of the circulating EV miR profiles in hypertensive conditions

Shuzhen Chen 1 , Smara Sigdel 1 , Gideon Udoh 1 , Annie Chen 1 , Brandon Yu 1 , Rakan Albalawy 2 , Jinju Wang 1

1 Department of Biomedical Sciences; 2 Department of Internal Medicine, Marshall University, Huntington, WV 25755, USA

Introduction : Vascular dementia (VD) is a chronic cerebrovascular syndrome caused by various factors, with cerebral vascular damage being one of the key pathological features. Circulating extracellular vesicles (cEVs) are emerging as novel players in neurodegenerative diseases, but the roles and implications of cEVs in hypertension‐related VD are largely unknown. In this study, we studied the cEV miR profiles/signatures in hypertensive transgenic mice.

Methods : The cognitive functions of mice were assessed. CEVs were isolated from hypertensive transgenic and wild‐type mice (denoted as HPN‐cEVs and WT‐cEVs). The total RNAs, including small RNAs, were extracted from each cEV sample. RNA quality and integrity were determined using the Agilent 2100 Bioanalyzer. The libraries were prepared with the QIAseq miRNA Library Kit (Qiagen) and sequenced using an Illumina NGS system. Data analysis was performed using the RNA‐seq Analysis Portal.

Results : Our data showed that the middle‐aged hypertensive transgenic mice showed impaired learning capability and short‐term memory. They had remarkably reduced cerebral microvascular density. We also found that the cells reside along the brain's blood vessels, exhibiting senescence‐like characteristics, in the hypertensive mouse brain, suggesting cerebrovascular dysfunction under hypertensive conditions, which could precede VD. According to our preliminary study of RNA‐seq, 74 miRs had a fold change less than −2 or larger than 2. Among those 74, 23 miRs had a fold change less than −4 or larger than 4 combined with an FDR p value less than 0.05. Some of those altered miRs, such as miR‐133a‐3p, miR‐196a‐5p, and miR‐296‐5p, are highly related to vascular repair and neuronee functions in the brain.

Summary/Conclusion : Taken together, our data suggests the possible involvement of cEVs in hypertension‐related VD. It will shed light on establishing new strategies that interfere with the onset and progression of hypertension‐related VD and provide a new perspective for alleviating and delaying VD in individuals with hypertension.

Funding : American Heart Association (AHA)‐Career Development Award (935826), AHA‐Transformational Project Award (24TPA1291189), and the National Institute of General Medical Sciences (U54GM104942).

Subtopic EV therapeutics development 1

Delivery of HIF‐1α via extracellular vesicles with collagen‐binding activity for enhanced wound healing

Presenter: Sungmi Jeon

Seoul National University, Republic of Korea

Introduction : This study investigated the potential of engineered extracellular vesicles (EVs) to deliver a stable, constitutively active form of HIF‐1α (scHIF‐1α) to promote wound healing. A collagen‐binding activity was integrated into EVs to enhance their retention at wound sites, and collagen sponges were employed as scaffolds to ensure sustained, localized release of scHIF‐1α EVs. These findings suggest that the delivery of scHIF‐1α via EVs, particularly when combined with collagen‐based sustained‐release systems, offers a promising and patient‐friendly therapeutic strategy for treating chronic wounds.

Methods : EVs were isolated by sequential centrifugation, purified using a tangential flow filtration system and a 0.45‐µm PVDF syringe filter, and finally ultracentrifuged at 150,000 × g for 90 min. EVs were characterized through various methods, including Western blot analysis for marker identification, zeta potential measurement using a ZetaView system, and cryo‐EM to observe EV morphology and structure. In vitro functional tests included cell proliferation assays, transwell migration assays, tube formation assays, and collagen‐binding assays. The EV retention and wound healing abilities were evaluated using C57BL/6 (B6) mice and the db/db mouse model. The methods used in this study were based on the MISEV2023 guidelines, which serve as the standard for EV research.

Results : In vitro studies have demonstrated that scHIF‐1α EVs significantly improved cell proliferation, migration, and angiogenesis in dermal fibroblasts, endothelial cells, and keratinocytes—key cells involved in the wound healing process. In vivo, scHIF‐1α EVs accelerated wound closure, enhanced tissue regeneration, and promoted angiogenesis in various wound healing models, including excisional wounds, surgical skin flaps, and diabetic wounds. The integration of collagen‐binding activity further enhanced EV retention, amplifying therapeutic outcomes.

Summary/Conclusion : The use of engineered scHIF‐1α EVs offers a breakthrough in wound healing therapy, providing a stable and targeted means of HIF‐1α delivery to the wound site. Collagen‐binding activity integration further enhances the clinical applicability of this approach, offering a sustained, localized, and non‐invasive therapeutic option. This study lays the groundwork for the future clinical applications of EV‐based therapies in wound healing, with potential benefits that extend beyond traditional treatment approaches.

Mesenchymal stem cell apoptotic bodies: warriors against inflammation

Presenter: Fiona Buckley

University of Galway, Ireland

Introduction : Mesenchymal stem cells (MSCs) are multipotent stem cells with regenerative potential. Despite the low survival rate post‐transplantation, MSCs retain their potent immunomodulatory and tissue repair capabilities. These observations gave rise to the ‘dying stem cell hypothesis,’ whereby the beneficial effects of transplanted MSCs are attributed to the immunomodulatory effects of apoptosis. The process of apoptosis results in the formation of apoptotic bodies (ABs), which can be cleared by phagocytosing immune cells. The current gold standard for inducing apoptosis for AB isolation involves the use of staurosporine. However, staurosporine is a known carcinogen and therefore limits the translational applications of these studies. It is hypothesized here that UV irradiation of MSCs produces ABs with the potential to reduce the inflammatory profiles of efferocytosing immune cells.

Methods : Apoptosis was induced in the D1 ORL UVA MSC cell line using UV irradiation in serum‐free conditions to avoid contaminating the sample with serum EVs. To isolate the ABs from the conditioned media of the apoptotic MSCs, differential centrifugation was used. First, the conditioned media was centrifuged at 500 × g for 10 min to pellet dead cells. The supernatant was collected and further centrifuged at 3000 × g for 30 min. ABs were washed once. ABs were characterised by TEM, DLS, BCA protein quantification, and flow cytometry for EV markers CD9, CD63 and CD81, and MSC markers CD73, CD90 and CD105. The immunomodulatory potential of MSC‐ABs was tested using allogenic murine phagocytes in vitro.

Results : UV irradiation induces apoptosis in the D1 ORL UVA MSC cell line. MSC‐ABs isolated at 3,000 x g are approximately 1 µm in diameter and express EV markers CD9, CD63 and CD81, and MSC markers CD73, CD90 and CD105. MSC‐ABs are efferocytosed by phagocytes and show potential to reduce the inflammatory profiles in vitro, including surface markers, migratory properties and inflammatory cytokine secretion.

Summary/Conclusion : This work demonstrates a rapid and chemical‐free method of generating MSC‐ABs with immunomodulatory potential, providing a promising insight into novel therapies against inflammation.

Funding : This project is funded by the European Research Council under an ERC Starting Grant, #852152.

Biomimetic nanostructural materials composed of placental amniotic membrane‐derived extracellular vesicles for self‐healing and anti‐adhesion during caesarean section

Presenter: Xiao Yi

Southern Medical University, China (People's Republic)

Introduction : Post‐caesarean section (CS) adhesions and impaired uterine healing severely impact female reproductive health. It is urgent to promote wound healing and tissue regeneration and prevent adhesion in minimizing CS‐induced trauma. Cell‐free therapies represented by extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs) are promising in tissue repair. And extracellular matrix (ECM)‐derived materials show the potential of preventing adhesion and promoting tissue regeneration. Herein, we designed a fully amniotic membrane (AM)‐derived biomimetic nanostructural materials (AM‐BNMs) composed of AM‐MSCs‐EVs and AM‐ECM nanofibres, validating the synergistic effect of anti‐adhesion and promoting tissue regeneration of AM‐BMNs in a rat CS model, which offered an innovative strategy for EVs in the management of postoperative wounds.

Methods : AM‐ECM and AM‐MSCs were isolated from human AM. Simultaneously, the ECM was fabricated into nanofibres, with which we constructed a three‐dimensional (3D) culture system. Characterization of the MSCs‐EVs was performed through nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting (WB), followed by an in‐depth proteomic analysis. Subsequently, we integrated MSCs‐EVs with the ECM nanofibres to develop AM‐BMNs. Finally, the efficacy in enhancing uterine wound healing and mitigating adhesion formation of AM‐BMNs was then assessed in a rat (CS) model.

Results : We constructed an AM‐ECM nanofibre bionic culture system. Through the culture system, high‐concentration EVs could be harvested. These EVs contained miR‐302a‐3p involved in TGF‐β and YAP pathways, which exerted multifaceted effects on wound healing by regulating fibroblast and migration and reducing the excessive deposition and misalignment of ECM. We designed AM‐BNMs composed of AM‐MSCs‐EVs and AM‐ECM nanofibres and regulated ingredient concentrations to construct AM‐BNMs patches and injectable AM‐ECMs. The CS rat model proved that AM‐BNMs could promote wound healing, prevent postoperative adhesion significantly and enhance tissue regeneration.

Summary/Conclusion : EVs had a great effect in preventing adhesion after the CS, facilitating various wound healing and promoting the repair of postoperative damaged tissues. AM‐BNMs provided a new scheme for the application of EVs in the treatment of postoperative adhesion.

Funding : This work was supported by the financial support of the National Natural Science Foundation of China (82371725 and 32301204) and the Guangdong Basic and Applied Basic Research Foundation for Distinguished Young Scholars (2024B1515020024).

Arg1 protein delivered by exosome restores arginase 1 expression, reduced arginine concentration in plasma and elongates Arg1 − / − mice survival

Presenter: Minghao Sun

Capricor Therapeutics, Inc., USA

Introduction : Arginase 1 deficiency (ARG1‐D) is a rare genetic disorder characterized by the loss of arginase 1 (Arg1), the final enzyme in the urea cycle. In patients with ARG1‐D, hepatocytes are unable to convert ammonia into urea, leading to elevated levels of arginine and ammonia in the blood and cerebrospinal fluid, which in turn results in progressive neurological symptoms. Current therapeutic strategies primarily focus on managing plasma arginine and ammonia levels, but long‐term outcomes remain poor. As of now, there is no approved treatment specifically for ARG1‐D available in the United States.

Methods : Utilizing Capricor's StealthX platform, researchers engineered the human Arg1 enzyme for expression on the exosome membrane in either intra‐ or extra‐exosomal orientations. The resulting Arg1‐exosomes were evaluated for their in vitro functionality as a disease‐specific therapeutic approach for ARG1‐D. Remarkably, regardless of their localization on the exosome membrane, Arg1 exhibited biological activity and effectively converted arginine into urea. Notably, only nanograms of Arg1 delivered via exosomes proved as potent as micrograms of human recombinant Arg1 protein. Additionally, engineered exosomes containing intra‐exosomal Arg1 were capable of delivering the Arg1 protein into 293F and HepG2 cells in a time‐ and dose‐dependent manner.

Results : These findings suggest that Arg1 exosomes not only catalyse arginine conversion efficiently at lower doses but also protect the Arg1 protein during delivery to target cells, maintaining its enzymatic activity in vitro. In further investigations in vivo, a single dose of Arg1‐exosomes was able to restore Arg1 activity in the liver and reduce plasma arginine levels to those comparable to wild‐type mice within 2 h. Repeated dosing extended the lifespan of Arg1 knockout mice by 50% in preliminary tests.

Summary/Conclusion : Together, these results indicate that StealthX platform‐engineered Arg1 exosomes have the potential to serve as an enzyme replacement therapy, delivering Arg1 to target hepatocytes and possibly providing broader clinical benefits for patients with ARG1‐D.

Delivering immunomodulatory cytokine licensed human bone marrow‐derived MSC‐EVs to the ocular surface using an innovative fluid gel system

Seyedmohammad Moosavizadeh 1,2 , Jiemin Wang 1,2 , Manon Jammes 1,2 , Anastasia Walsh 4,5 , Ellen Donohoe 1,2 , Aoife Canning 1,2 , Abbas Tabasi 1,2 , Trung Bach 1,2 , Thomas Robinson 3 , Richard Moakes 3 , Aideen E Ryan 1,2,4,5 , Liam Grover* 3 , Thomas Ritter* 1,2

1 Regenerative Medicine Institute (REMEDI), School of Medicine, College of Medicine, Nursing and Health Sciences, University of Galway, Galway, Ireland; 2 CÚRAM, SFI Research Centre for Medical Devices, University of Galway, Galway, Ireland; 3 Healthcare Technologies Institute (HTI), School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom; 4 Discipline of Pharmacology and Therapeutics, School of Medicine, College of Medicine, Nursing and Health Sciences, University of Galway, Galway, Ireland; 5 Lambe Institute for Translational Research, School of Medicine, College of Medicine, Nursing and Health Sciences, University of Galway, Galway, Ireland

Introduction : MSC‐EVs demonstrate immunomodulation and tissue regeneration properties while addressing the challenges associated with cell therapy. Licensing MSCs with pro‐ and/or anti‐inflammatory cytokines enhances their therapeutic effects. Delivering MSC‐EVs to the target site in a controlled manner may enhance MSC‐EVs therapeutic efficacy. Gellan gum fluid gel (GGFG) is a biocompatible delivery system which can extend the ocular retention time and overcome the obstacles of commercial eye drops due to its shear‐dependent viscoelasticity and phase transition properties. The aim of this study was to investigate the MSC‐EVs delivery to the ocular surface and evaluate their therapeutic effects.

Methods : Naïve‐ and cytokine‐licensed‐MSC‐EVs were isolated by size exclusion chromatography and characterized extensively following MISEV guidelines. MSC‐EVs were investigated for their immunomodulation and tissue regeneration effects on macrophage polarization and in human corneal epithelial (HCEpi) and keratocyte (HCK) wound healing assays, respectively. GGFG was synthesized by the shear field technique and characterized for its viscosity, viscoelasticity, transparency, and biocompatibility. MSC‐EVs cumulative release from GGFG was examined over 6 hr incubation.

Results : MSC‐EVs had spherical bilayer morphology with a mode size of 80 nm. The MSC‐EVs were positive for CD9, CD63, and CD81, and TSG101 markers were analysed by flow cytometry and western blotting. Both MSC‐EVs can polarize pro‐inflammatory macrophages to the anti‐inflammatory phenotype by upregulating CD206 and CD163. Furthermore, licensed MSC‐EVs were more effective at upregulating CD206 across various concentrations. The licensed MSC‐EVs better‐promoted cell migration and wound healing in HCEpi and HCK scratch models analysed by an Olympus microscope. Transparent GGFG acts as solids at rest but as fluids under force. GGFG didn't show any toxicity on the human corneal epithelial and keratocyte cells or on the bovine eye ex‐vivo model. The GGFG released 20% of the MSC‐EVs after 6 h and demonstrated a controlled profile.

Summary/Conclusion : Together, these results evidenced that cytokine licensed MSC‐EVs have enhanced immunomodulation and tissue regeneration activities in 2D in vitro corneal models. In addition, the GGFG demonstrates a promising system for the MSC‐EVs ocular delivery in a biocompatible and controlled manner in 2D in vitro and ex vivo models, necessitating further investigation of the therapeutic efficacy on in vitro and in vivo corneal injury models.

Encapsulation within bacterial vesicles increases the effectiveness of antibiotics against gram‐negative bacteria

Presenter: Meishan Wu

Lehigh University, USA

Introduction : Gram‐negative bacteria are intrinsically resistant to many antibiotics because of their dual‐membrane cell wall. Small, hydrophilic antibiotics are able to pass through protein channels, called porins, to access the bacterial cytosol; however, bacteria can downregulate the expression of these porins, thereby decreasing penetration of these drugs. One means by which bacteria deliver functional molecules to other bacteria is through the use of extracellular vesicles. We therefore hypothesized that incorporating antibiotics within the vesicle lumen would improve drug uptake and, as a result, increase the effectiveness of these drugs in Gram‐negative bacteria.

Methods : To test this hypothesis, we first investigated the optimal method to load small molecule antibiotics of varying molecular weight and hydrophobicity into vesicles produced by a hypervesiculating strain of Escherichia coli (JC8031). We then compared the antibacterial activity of vesicle‐encapsulated antibiotic and unencapsulated antibiotic against several gram‐negative bacteria, including multidrug‐resistant (MDR) clinical isolates.

Results : We found that small molecule antibiotics, including fluoroquinolones and β‐lactams, could be readily loaded within the vesicles. We next demonstrated that vesicle‐encapsulated imipenem was as effective as unencapsulated imipenem in killing Pseudomonas aeruginosa strain PAO1, a common Laboratory strain. However, in MDR isolates, vesicle encapsulation improved the activity of the drugs. Vesicles alone had no effect on cell viability, indicating that it was not the mere presence of the vesicles that increased bacterial death but more likely an improved delivery of the drugs. We hypothesized that by fusing with the bacterial membrane, vesicles enable porin‐independent delivery of antibiotics. To test this hypothesis, we compared the delivery of unencapsulated and vesicle‐encapsulated drugs in a panel of porin knockouts from the E. coli Keio collection. We observed that in the ΔompF strain (JW0912), imipenem was less effective than in the parent strain (BW25113), demonstrating that this antibiotic is transported through the OmpF porin. When we delivered vesicle‐encapsulated imipenem to the ΔompF knockout, the effectiveness of the antibiotic increased, supporting our model that vesicles deliver their cargo in a porin‐independent manner.

Summary/Conclusion : Together, these results show that vesicle‐mediated delivery increases the activity of existing antibiotics simply by improving their transport to the bacterial cytosol.

EVs from glycolytic MSCs: a promising therapy for osteoarthritis

Presenter: Maria J. Araya

Universidad de Los Andes, Chile

Introduction : Osteoarthritis (OA) is a degenerative joint disease in which there is cartilage loss and chronic inflammation. Currently, only palliative treatments are available. We have shown that glycolytic mesenchymal stem cells (MSCglyco) release extracellular vesicles (EV) with properties that could be appealing for developing new therapies for OA.

Methods : MSCs were treated with oligomycin to induce a glycolytic metabolism. EVs were isolated through ultracentrifugation, quantified by nanoparticle tracking analysis (NTA) and characterized through flow cytometry and transmission electron microscopy (TEM). EVs from MSCglyco were added to the culture media of either chondrocytes or synovial macrophages from OA patients. After 24 h we evaluated EV internalization and OA‐associated markers in both target cells. MSCglyco‐derived EV‐treated synovial macrophages were further analysed with single‐cell RNA sequencing. Finally, we injected EVs from MScglyco intraarticularly into a collagenase‐induced osteoarthritis (CIOA) murine model to assess their regenerative, antiapoptotic, and anti‐inflammatory effects.

Results : EVs from MSCglyco showed no significant differences in identity markers compared to those from naive MSC and were internalized by both target cells. Indeed, EVs from MSCglyco promoted a recovery of the healthy chondrocyte phenotype, while decreasing inflammatory markers in synovial macrophages. Moreover, single‐cell data revealed an increase in pro‐regenerative macrophage populations. Indeed, our in vivo data reveals cartilage regeneration, a decrease in chondrocyte apoptosis and a reduction in joint inflammation upon intraarticular injection of MSCglyco‐derived EVs.

Summary/Conclusion : Our results show significant evidence of EVs from MSCglyco acting as a multifunctional treatment for OA, targeting both main symptoms, cartilage loss and synovial inflammation.

Funding : Research supported by ANID‐Chile through Beca Doctorado Nacional (folio 21220015); FONDECYT Regular (N°1211353); FONDECYT Iniciación (N°11220549); FONDEF‐ID (21I10194); IMPACT‐FB (210024); and Tecnologías Avanzadas (TA24I10054).

Delivery of HIF1α via Extracellular Vesicles Induces Macrophage Reprogramming into a Resolutive Phenotype in Sepsis‐Induced Liver Damage

Presenter: Yeji Lee

Korea Institute of Science and Technology, Republic of Korea

Introduction : In sepsis, the liver plays a key role as a central filter, where hepatic macrophages serve as the primary antimicrobial defence by removing bacteria and regulating immune responses. Modulating these hepatic macrophages is essential to trigger crucial defence mechanisms. In our approach, we aim to modulate this immune system by delivering the transcription factor HIF1α, a key player in reprogramming macrophages during sepsis. Transcription factors are promising proteins due to their ability to dynamically adjust gene expression across various conditions, although their delivery remains challenging. In this study, we aimed to load HIF1α into EVs and hypothesized that delivering HIF1α through EVs could reprogram monocytes/macrophages to induce a resolutive environment.

Methods : EVs were isolated following the MISEV2023 guidelines. Sepsis was induced in C57BL/6 mice through LPS administration. Thirty micrograms of scHIF1α‐EVs were administered intravenously, and immune profiling was analysed using FACS, scRNA‐seq, and Visium.

Results : To encapsulate HIF1α into EVs, we fused HIF1α to the C‐terminus of ΔI‐CM and attached CD81 to its N‐terminus. The functionality of encapsulated scHIF1α in EVs was validated using a reporter system. We evaluated the therapeutic potential of scHIF1α‐EVs and found that only the scHIF1α‐EV treatment group demonstrated an increased survival rate and reduced liver toxicity. scRNA‐seq analyses confirmed that scHIF1α‐EVs induced the differentiation of MoMFs, with elevated expression of Nr1h3 and C/EBPb. We showed that the differentiation of Nr1h3‐dependent pro‐efferocytic MoMFs, which clear damaged hepatocytes and immune cells, and Cebpb‐dependent pro‐survival MoMFs, which activate innate memory responses, occurs in an scHIF1α‐dependent manner. Additionally, Visium analysis revealed that scHIF1α‐EVs promoted the formation of resolutive macrophages localized near the portal vein.

Summary/Conclusion : Our study highlights the role of scHIF1α‐EVs in promoting resolutive macrophage activity during sepsis‐induced liver damage. Additionally, this research opens avenues for refining transcription factor delivery methods, with the potential to enhance recovery outcomes in sepsis patients.

Neuroprotective role of colostrum extracellular vesicles in Parkinson's disease

Presenter: Dawson Hollingsworth

UNMC, USA

Introduction : Parkinson's disease (PD) is the second most common multi‐system neurodegenerative disorder, linked to immune dysfunction that impacts the clinical symptoms. Current PD treatments for motor and non‐motor symptoms are palliative, with a failure to affect the cause of nigrostriatal degeneration. In past decades, our labouratories have focused on innate and adaptive immune pathways that control neuroinflammation and provide neuroprotection in animal disease models and the clinic. Recently, bovine colostrum‐derived EVs (CEVs) have garnered interest as immune modulators, with high safety profiles and yields, and have been shown to traffic to the brain. Considering their favourable biodistribution pattern, safety, scalability, and anti‐inflammatory properties, we evaluated their potential as neuroprotective agents in the methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of PD.

Methods : CEVs were isolated by differential ultracentrifugation from bovine colostrum, collected 24 h after birth. They were characterized for size, distribution, concentration, and morphology by biochemical and molecular tests and by positron emission tomography for tissue distribution. Parallel studies evaluated the expression of neuroinflammatory and neurodegeneration biomarkers after intravenous injection of the CEVs into the MPTP‐intoxicated mice.

Results : CEVs had a size range of 50‐150 nm, were positive for exosome markers, and had a cup‐shaped double membrane morphology when imaged by transmission electron microscopy. RNA sequencing and transcriptomic analysis from the substantia nigra and striatum of MPTP‐intoxicated mice treated with CEVs demonstrated changes in biomarkers associated with reductions in immunocyte recruitment and activation, controlled microglial activation, and reduced pro‐inflammatory gene expression. This was in addition to diminished canonical inflammasome activation, and cytokine and chemokine responses in CEV‐treated MPTP mice. These changes corresponded with decreased expression of upstream regulatory molecules linked to inflammation. Concomitantly, the regulatory T‐cells increased in the blood and spleen of CEV‐administered MPTP mice. Anti‐inflammatory responses led to neuroprotection, as evidenced by a significant increase in the survival of tyrosine hydroxylase nigral dopaminergic neuronees following CEV treatment in the MPTP mice.

Summary/Conclusion : CEVs control neuroinflammatory activities and demonstrate dopaminergic neuronal protection in a PD model. CEVs could be developed as part of a comprehensive therapeutic strategy, offering a unique disease‐modifying PD treatment.

Funding : This research was supported by the University of Nebraska Foundation.

Müller glia‐secreted small extracellular vesicles as potential therapy for early‐stage diabetic retinopathy

Presenter: Sun Young Lee

University of Southern California, USA

Introduction : Müller glia (MG) are the primary glial cells unique to the retina and serve as a major source of neuroprotective factors and regulators of vascular permeability. Increasing evidence indicates that diabetes adversely affects the retinal neurovascular unit (NVU), impacting its interdependent vascular, neuronal, glial, and immune cells as diabetic retinopathy (DR) progresses. MG have been shown to release small extracellular vesicles (MG‐sEVs), which play an important role in cell‐to‐cell communication. The current study aims to investigate the therapeutic potential of intraocular treatment with MG‐sEVs to protect the NVU in DR.

Methods : sEVs were isolated from the conditioned cell culture media (CCM) of the primary mouse MG cells using the tangential flow filtration (TFF) method and comprehensively characterized as established in our lab. Two DR mouse models, the STZ‐induced model for type I and the db/db model for type II DR, received three weekly intravitreal injections of MG‐sEVs post‐DR induction. We assessed neuroretina thinning, retinal vascular leakage, macrophage infiltration, and leukocyte recruitment through in vivo fluorescein angiography (FA) and optical coherence tomography (OCT) and immunohistochemistry (IHC) of retinal sections using anti‐F4/80 ab and FITC‐ConA perfusion of retinal flat mounts. In addition, electroretinograms (ERGs) were conducted.

Results : We confirmed DR induction in both models ( n = 10) through inner retinal thinning, increased vascular leakage (in db/db mice only), and heightened retinal macrophage and vascular leukocyte infiltration. In contrast, intraocular MG‐sEVs treatment significantly preserved retinal thickness, particularly in the inner neuronal layers, and reduced vascular leakage, macrophage infiltration, and leukocyte recruitment. Although there was a mild reduction in a‐ and b‐wave amplitudes in scotopic ERG for DR, MG‐sEV treatment did not significantly improve these ERG findings.

Summary/Conclusion : MG‐secreted sEVs may have the potential to mitigate early‐stage DR by protecting NVU, which addresses the gap in early‐stage DR treatment. Despite advances in understanding DR pathophysiology, no treatments currently exist for early‐stage DR beyond screening and managing systemic risk factors. Additionally, treatments for advanced DR, such as anti‐VEGF injections and laser photocoagulation, often fail to restore vision. While these initial results are encouraging, additional studies in biological cargos of MG‐sEVs are underway.

Funding : National Institutes of Health (NIH)/National Eye Institute (NEI) R21EY035425.

3D bioprinting of osteoblast‐derived small extracellular vesicle scaffolds for enhanced osteogenic differentiation and bone regeneration

Presenter: Chun Liu

The University of Queensland, Australia

Introduction : Bone regeneration remains a critical focus in regenerative medicine, with an increasing demand for innovative approaches to enhance bone healing and repair. Among additive manufacturing techniques, 3D bioprinting stands out as a cutting‐edge technology for fabricating 3D biological constructs, enabling precise control over hierarchical architecture and shapes through the layer‐by‐layer addition of bioinks and biological materials. Recent advancements have highlighted the potential of combining 3D bioprinting and small extracellular vesicles (sEVs) as a promising ‘cell‐free’ strategy for regenerative medicine applications. Our approach combines gelatin methacryloyl (GelMA) with small extracellular vesicles (sEVs) derived from primary human osteoblasts (hOBs) and gingival fibroblasts (hGFs) to create a 3D bioprinted scaffold model aimed at improving osteogenic differentiation and bone regeneration in vitro and in vivo.

Methods : sEVs were isolated from primary hOBs and hGFs using size exclusion chromatography (SEC) and characterized by cyto‐TEM, NTA, BCA, FTIR, and multiple cytokine FACS. The sEVs were encapsulated in 6% GelMA and 3D bioprinted via microextrusion. sEV release from the constructs was validated using DiO‐labelling, confocal imaging, NTA, and CD9 ELISA. hOB attachment and osteogenic differentiation were assessed on GelMA scaffolds, with and without hOBs‐sEVs and hGFs‐sEVs, using qRT‐PCR, ALP staining, and Alizarin Red staining. In vivo analysis utilized a 26‐rat skull defect model, where bone regeneration was evaluated through micro‐CT, H&E staining, and immunohistochemical markers: Bone Sialoprotein (BSP), Collagen Alpha‐1(I) Amino‐Propeptide (CAAP), Osteopontin, Periostin, and von Willebrand Factor (vWF).

Results : hOBs cultured on GelMA/hOBs‐sEV constructs demonstrated significantly improved cell attachment and focal adhesion, with upregulation of genes associated with mechanotransduction (YAP, Rac1, TAZ, FAK, and RhoA) and osteogenic differentiation (BSP, OSX, OPN, ALP, and OCN) compared to GelMA/hGFs‐sEV and GelMA‐only groups. After 2 weeks in vitro, ALP activity and Alizarin Red staining were markedly increased in hOBs on GelMA/hOBs‐sEV constructs. In vivo, GelMA/hOBs‐sEV constructs showed enhanced bone regeneration, with H&E and immunohistochemical analyses (BSP, CAAP, vWF, osteopontin, and periostin) confirming increased osteogenic marker expression and bone matrix formation compared to controls.

Summary/Conclusion : Our findings indicate that 3D bioprinted GelMA scaffolds embedded with hOB‐derived sEVs significantly promote osteogenic differentiation and bone regeneration in vitro and in vivo.

Targeting extracellular vesicle secretion in combination with venetoclax synergistically induces apoptosis in FLT3‐ITD+ AML

Mariagiovanna Barresi 1,2 , Jonas Lang 1,2 , Katharina Enghofer 1,2 , Christoph Nuernbergk 1,2 , Grace Wagner 1 , Jennifer Rivière 1 , Michèle Buck 1 , Rupert Oellinger 2,8 , Ali Bashiri Dezfouli 2,6,7 , Gabriele Multhoff 2,7 , Robert Oostendorp 1 , Bernd Giebel 3 , Roland Rad 2,5,8 , Florian Bassermann 1,2,4,5 , Katharina S. Götze 1,5 , Judith S. Hecker 1,2,4,5

1 Technical University of Munich (TUM), School of Medicine, Department of Internal Medicine III, Munich, Germany; 2 TranslaTUM, Center of Translational Cancer Research, Technical University of Munich (TUM), Munich, Germany; 3 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany; 4 Bavarian Center for Cancer Research (BZKF), Partner Site Munich, Munich, Germany; 5 German Cancer Consortium (DKTK), partner site Munich and German Cancer Research Center (DKFZ), Heidelberg, Germany; 6 Department of Otolaryngology, Head and Neck Surgery, TUM School of Medicine and Health, Technical University of Munich (TUM), Munich, Germany; 7 Department of Radiation Oncology and Central Institute for Translational Cancer Research (TranslaTUM), Technical University of Munich (TUM), School of Medicine and Health, TUM University Hospital, Munich, Germany; 8 TUM School of Medicine and Health, Institute of Molecular Oncology and Functional Genomics, Technical University of Munich, Munich, Germany

Introduction : Apoptosis resistance is a hallmark of human cancers, including acute myeloid leukaemia (AML). Disposal of pro‐apoptotic factors via extracellular vesicle (EV) release has been observed in several cancer models as a way to achieve resistance. This study focuses on characterizing apoptotic factors within primary AML‐derived EVs and examines the impact of EV release inhibition with FTY720, an FDA‐approved multiple sclerosis drug, combined with venetoclax (VEN), a Bcl‐2 inhibitor used in AML therapy, to assess its effect on apoptosis.

Methods : BM‐derived EVs were purified from healthy donors and AML patients (FLT3‐WT and ‐ITD, n = 25) with informed consent and ethical approval (2022‐559‐S‐KK). EVs were isolated using ultracentrifugation or the ExoEasy Kit (Qiagen) and characterized by transmission electron microscopy (TEM), western blotting, and nanoparticle tracking analysis (NTA). Proteomic and RNA analyses assessed EV sample content. AML samples were tested for FTY720‐induced apoptosis via flow cytometry. Immunostaining analysed EV release inhibition and intracellular EV accumulation induced by FTY720. RT‐qPCR quantified miRNA expression, while western blotting measured EV and apoptosis markers. Lastly, co‐treatment with FTY720 and VEN was evaluated.

Results : TEM and immunoblotting verified pure EV fractions (125–136 nm) with expression of characteristic markers (e.g., CD63, CD81, HSP70). NTA assessed particle size and concentration, revealing fewer but larger particles in AML‐derived samples compared to healthy controls. Content significantly differed between healthy and AML‐derived EVs, with apoptosis‐regulating pathways enriched specifically in FLT3‐ITD AML EVs. Notably, several FLT3‐ITD AML‐enriched miRNAs (e.g., miR‐155‐5p, miR‐1‐3p, miR‐10a‐5p, miR‐196b‐5p, and miR‐30c‐5p) target key apoptosis components, including Bcl‐2 and MCL‐1. Concordantly, FLT3‐ITD AML samples showed higher sensitivity to FTY720 than FLT3‐WT or healthy cells (e.g., EC50 = 2.1 and 12.2 µM, respectively, for FLT3‐ITD AML patients and healthy cells). Interestingly, FTY720 co‐treatment increased VEN‐induced apoptosis in FLT3‐ITD AML, while FLT3‐WT cells remained VEN‐resistant, with no impact on healthy cells.

Summary/Conclusion : Altogether, these results suggest that FLT3‐ITD AML cells establish a protective mechanism against apoptosis by disposing of pro‐apoptotic factors via EV release. Blocking this disposal, combined with BCL‐2 inhibition, sensitizes FLT3‐ITD AML cells to apoptosis, highlighting a promising therapeutic strategy to overcome resistance in FLT3‐ITD AML. Ongoing studies aim to identify the specific factors responsible for this sensitization.

Funding : DJCLS13‐R/2023.

Extracellular vesicle therapy with HIF‐1α for hindlimb ischaemia

Presenter: Seongeon Cho

Korea Institute of Science and Technology, Republic of Korea

Introduction : Peripheral arterial disease (PAD) is a vascular condition marked by reduced blood flow to the limbs, causing ischaemia, pain, and tissue damage. Hindlimb ischaemia, a common PAD model, helps evaluate therapies for vascular recovery and tissue repair. Despite existing treatments, their effectiveness remains limited, requiring novel approaches. This study explores the use of extracellular vesicles (EVs) loaded with hypoxia‐inducible factor 1‐alpha (HIF‐1α), a key regulator of angiogenesis and arteriogenesis. Delivering HIF‐1α via EVs aims to enhance stability, promote vascular remodelling, and improve tissue repair.

Methods : Male Balb/c mice (10–12 weeks old) were anaesthetized, and the right hindlimb was shaved and disinfected. A small incision was made to expose the femoral artery, and a ligation was performed between the femoral and saphenous arteries to induce ischaemia. Phosphate‐buffered saline (PBS) or extracellular vesicles (EVs) were injected intramuscularly into the ischaemic limb at multiple sites. Blood flow recovery was assessed using laser Doppler imaging at various time points. The muscles from both ischaemic and non‐ischaemic limbs were harvested for histological and molecular analyses to evaluate treatment efficacy.

Results : Blood perfusion was significantly restored in mice treated with HIF‐1α‐loaded EVs compared to those injected with PBS or control EVs. Tissue damage following ischaemic injury exhibited a similar trend, with reduced damage observed in the HIF‐1α EV group. While angiogenesis plays a role in recovery from hindlimb ischemia, arteriogenesis is more critical for restoring higher volumes of blood flow. In the mouse hindlimb ischemia model, arteriogenesis in the adductor muscle was assessed by measuring the diameter and circumference of the gracilis collateral artery. Both parameters showed the greatest increase in mice treated with HIF‐1α EVs, indicating enhanced vascular remodelling and improved blood flow recovery.

Summary/Conclusion : HIF‐1α‐loaded EVs enhanced blood flow recovery, reduced tissue damage, and promoted vascular remodelling by stimulating angiogenesis and arteriogenesis in a mouse hindlimb ischaemia model. These results suggest that HIF‐1α EVs offer a promising new approach for cell‐free therapy in ischaemic diseases.

VivaZome proprietary human cell‐derived extracellular vesicles improve functional outcomes after moderate traumatic brain injury in a preclinical model

Min Chen 1 , Baani Bagga 1 , Smriti Krishna 2,3 , Ella Johnston 2 , Johannes Weber 2 , Xenia Sango 2 , David Reutens 1 , David Haylock 2,3

1 Centre for Advanced Imaging, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia; 2 VivaZome Therapeutics Pty Ltd, Jenny Graves Building, La Trobe University, Bundoora, Victoria 3083, Australia; 3 La Trobe University, School of Molecular Science, Kingsbury Drive, Bundoora, Victoria, 3083, Australia

Introduction : Inflammatory mechanisms exert a profound influence on the outcome of traumatic brain injury (TBI). Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have been shown to modulate the inflammatory response and improve functional recovery in preclinical TBI models (J Mol Neurosci 2020; 70(5): 677–688). VivaZome's proprietary cells (VZT‐PC) share many of the characteristics of MSCs including cell surface markers, immunomodulatory properties and differentiation potential (Eur J Cell Biol 2020; 99(7): 151123). VZT‐PC‐conditioned culture medium has been shown to accelerate wound healing, reduce inflammation and increase angiogenesis (NPJ Regen Med 2020;5(1):24.). EVs derived from VZT‐PCs contain several microRNAs known to modulate inflammation and have anti‐inflammatory activity in a cell‐based assay. Based on these properties, this study evaluated the treatment effect of VZT‐PC‐derived EVs in a preclinical TBI model.

Methods : Adult C57 mice were subjected to a cortical contusion by compressing the cortex to a depth of 1.0 mm at a velocity of 3.5 m/s and 400 ms duration with a bevelled tip 3 mm in diameter (TBI‐0310). Mice were randomLy allocated to treatment groups: 1. sham injury + vehicle, 2. TBI + vehicle, or 3. TBI + EVs (2.19 × 1010 particles, iv, post‐injury). Rotarod trials, open field tests, elevated plus maze, and Barnes maze were performed by investigators blinded to the group to assess functional recovery.

Results : Fall latency of rotarod trials was significantly increased in EV‐treated TBI mice compared to vehicle‐treated TBI mice, indicating that EV treatment effectively improved the recovery of grip strength, balance, and motor coordination after TBI. Velocity and distance travelled during the open field test were comparable across the three treatment groups, suggesting that locomotor function was not affected by TBI and EV treatment. In the elevated plus maze test, exosome treatment enhanced exploratory behaviours in TBI mice compared to vehicle, as evidenced by the increased duration in the open arms. Barnes maze results showed that TBI caused impairment in spatial learning, which was not reversed by EV treatment.

Summary/Conclusion : VZT‐PC‐derived EVs improved motor function and increased exploratory behaviour. The accessibility and high proliferative potential of GFs make them an attractive source of EVs for treating TBI.

Funding : Cooperative Research Centres Projects (CRC‐P) Grants.

Magnetic ion channel activation (MICA) enabled extracellular vesicles (EVs) synthesis: a new therapeutic pathway to promote osteogenic differentiation

Presenter: Afeesh Rajan Unnithan

University of Bradford, UK

Introduction : Extracellular vesicles (EVs) hold significant promise for therapeutic applications due to their inherent ability to facilitate intercellular communication and deliver bioactive molecules. However, existing production techniques yield low quantities of EVs, which critically limits their clinical applicability. This work introduces a novel strategy to enhance EV production by harnessing Magnetic Ion Channel Activation (MICA) technology. By integrating these advanced stimuli into the EV production process, we aim to elicit cellular responses that significantly increase EVs' yield and functionality. The therapeutic efficiency of MICA‐derived EVs was evaluated by assessing their ability to promote osteogenic differentiation.

Methods : A nanoplatform of magnetic nanoparticles combined with graphene oxide (GOMNPs) was developed to enhance EV synthesis via MICA‐based non‐invasive activation. TREK1‐targeted GOMNPs were incubated with MC3T3 cells to harvest EVs under MICA stimulation. After activation, EV concentration was measured using BCA analysis, and size was evaluated by NTA and TEM. The osteogenic differentiation of MICA‐derived EVs was assessed in hBMSCs through Alizarin Red staining and proteomics analysis for osteogenesis‐related proteins.

Results : MICA activation led to a significant increase in EV production in cells compared to controls, as confirmed by BCA analysis and FACS. When these MICA‐derived EVs were applied to hBMSCs, they exhibited a notably higher osteogenic potential. Alizarin‐Red staining indicated increased mineralization in hBMSCs treated with MICA‐derived EVs, suggesting enhanced osteogenic differentiation. Proteomics analysis further confirmed the presence of osteogenesis‐related proteins, reinforcing the efficacy of MICA‐EVs in promoting bone formation pathways. These results highlight the potential of MICA‐derived EVs as a powerful, non‐invasive strategy for enhancing osteogenesis in regenerative medicine applications.

Summary/Conclusion : In conclusion, this study demonstrates the significant impact of Magnetic Ion Channel Activation (MICA) on enhancing the yield of extracellular vesicle (EV) production. MICA effectively increased the quantity of bioactive EVs from cells, addressing limitations in traditional methods that often yield low‐quality EVs. The developed nanoplatform using magnetic nanoparticles and graphene oxide provided a non‐invasive activation technique that improved EV synthesis while preserving their functional integrity. Our findings show that MICA‐derived EVs promote osteogenic differentiation, offering a promising strategy for regenerative medicine and bone tissue engineering and overcoming current challenges in EV production for therapeutic applications.

First‐in‐human application of human umbilical cord‐derived extracellular vesicles for neuroprotection in spina bifida surgery

Presenter: Matthias Krause

University Hospital Salzburg, Austria

Introduction : Spina bifida is a congenital neural tube defect that has a high risk of secondary neurological deterioration due to tethering of the spinal cord. We present the case of a child with spina bifida who underwent spinal cord release surgery accompanied by human umbilical cord‐derived mesenchymal stromal cell‐derived extracellular vesicle (UC‐MSC‐EV) therapy for the first time in a human. Here, we discuss the surgical procedure and post‐operative outcome and highlight the potential benefits of extracellular vesicle therapy in the management of spina bifida. The administration of extracellular vesicles containing potentially therapeutically active agents has emerged as a potential new treatment modality for neurological disorders. By direct intrathecal application during surgery, UC‐MSC‐EVs can deliver therapeutic payloads to target cells and the extracellular environment, offering a novel approach to neuroprotection and tissue repair.

Methods : Case presentation—a 2‐year‐old female child with a diagnosis of spina bifida presented with progressive syringomyelia as a sign of secondary tethered cord syndrome with an intramedullary dermoid inclusion tumour after postnatal spina bifida repair. After thorough pre‐operative assessment and multidisciplinary consultation, it was decided to proceed with spinal cord release surgery combined with extracellular vesicle application. During the surgical procedure, the tethered spinal cord was released, and the dermoid as well as lipoma tissue were completely resected using intraoperative neurophysiologic monitoring to minimize trauma to neural structures. Concurrently, UC‐MSC‐EVs were administered directly onto the released and neurulated placode and the spinal cord.

Results : The postoperative MRI demonstrated a good de‐tethering effect, no postoperative medullary oedema, and diminished syringomyelia. No adverse events were reported. The neurological deficit remained stable at the pre‐existing level.

Summary/Conclusion : Intraoperative application of UC‐MSC‐EVs might be a feasible option to ameliorate intrathecal scarring and neurodegeneration following spina bifida surgery. Whether EVs will result in significant effects for the long‐term neurological outcome needs to be studied thoroughly in randomized controlled clinical trials. EV therapy might open new horizons to prenatal and postnatal spina bifida therapy if UC‐MSC‐EVs can efficiently diminish spinal cord tethering and neurodegeneration for the many severely handicapped children and thus reduce the burden of disease for the patients and their families.

Exosome platform: ExoPTEN as a breakthrough therapy for acute spinal cord injury, nerve regeneration, and beyond

Presenter: Noa Avni

NurExone, Israel

Introduction : NurExone is a platform company pioneering exosome‐based therapies to address nerve damage and support nerve regeneration across multiple clinical indications. Our first product, ExoPTEN, targets acute spinal cord injury (SCI), utilizing exosomes derived from bone marrow mesenchymal stem cells (MSCs) to deliver a proprietary siRNA targeting PTEN, an inhibitory regulator in the mTOR pathway. Exosomes, as natural carriers, provide a highly effective and targeted delivery system for therapeutic molecules, while their ability to cross biological barriers makes them ideal for regenerative therapies.

Methods : our exosome platform has been tested in two well‐established preclinical rat models of SCI: full spinal cord transection and spinal cord compression. The siRNA‐loaded exosomes were administered via intranasal and intrathecal routes. Efficacy was measured through MRI, BBB motor scoring, von Frey sensory tests, and immunohistochemistry. Homing ability was confirmed through fluorescent labelling, up to 7 days post‐injury.

Results : ExoPTEN demonstrated significant efficacy in both tested preclinical rat models. Improvements were observed in motor, sensory, and structural functions, as confirmed by MRI and histological analyses. ExoPTEN exhibited strong and effective homing capacity to the injury site, up to 7 days post‐injury. The exosome production process was successfully optimized from small‐scale to large‐scale 3D cultures while maintaining product potency and integrity.

Summary/Conclusion : These results, combined with orphan drug designation for acute SCI and strong FDA support from the pre‐IND submission, highlight the robustness and versatility of our exosome technology. This manufacturing platform allows for the loading and delivery of diverse therapeutic payloads, extending its application to other clinical indications marked by nerve damage, such as glaucoma. NurExone is advancing this platform to serve unmet medical needs in regenerative medicine, particularly in conditions requiring nerve regeneration.

Funding : NurExone Biologic.

Therapeutic potential of umbilical cord mesenchymal stromal cell‐derived extracellular vesicles on atopic dermatitis: animal study

Presenter: Maimonah E. Al‐Masawa

DTERM, The National University of Malaysia, Malaysia

Abstract unavailable

Mesenchymal‐derived bone cells regulate osteoclastogenesis via an extracellular vesicle‐mediated mechanism

Mimma Maggio 1 , Carolina Martins 1 , Rawan Almasri 1 , Stéphane Petrousek 1,2 , Mathieu Y. Brunet 1 , Tara Ní Néill 1,2 , Conor T. Buckley 1,2,3 , Lorraine O'Driscoll 1 , David A. Hoey 1,2,3

1 Trinity College Dublin, Ireland; 2 Advanced Materials and Bioengineering Centre (AMBER), Ireland; 3 Royal College of Surgeons in Ireland, Ireland

Introduction : Osteoporosis is a skeletal disease arising from an imbalance in bone remodelling leading to increased fracture risk. As key regulators, osteocytes mediate osteoclastogenesis via secreted paracrine factors in a mechanical loading‐dependent manner. Recently, extracellular vesicles (EVs) have emerged as major physiological mediators of cell‐cell communication in bone. However, their involvement in osteoclastogenesis remains unestablished. Hence, this study aimed to investigate the role of mesenchymal‐derived bone cells and secreted EVs in osteoclastogenesis upon the lineage commitment stage and mechanical environment of parental cells.

Methods : Human marrow stromal/stem cells (MSC), human osteoblasts (OB) and MLO‐Y4 osteocyte‐like cells (OCY) were mechanically stimulated by oscillatory fluid shear (OFS). EVs from static (EV‐S) and mechanically activated (EV‐F) bone cells were obtained via conditioned media (CM) differential ultracentrifugation (70Ti fixed‐angle rotor, 110,000 × g , 75 min, 4°C) and characterized via NTA, TEM and flow cytometry. The osteoclast differentiation of human CD14+ monocytes was assessed upon CM and EV treatments. EV‐microRNA sequencing was performed, and selected miRNA was transfected into monocytes.

Results : CM from MSC cultured in static (CM‐S) and exposed to OFS (CM‐F) significantly inhibited osteoclastogenesis. Inhibition was observed to a greater extent upon treatment with CM‐S. Static‐cultured OB secretome didn't affect osteoclast differentiation; however, OB CM‐F significantly inhibited osteoclastogenesis compared to CM‐S. Osteocyte CM‐S and CM‐F both inhibited osteoclastogenesis, with stronger inhibition following mechanical stimulation. Following EVs collection, MSC‐EVs inhibited osteoclastogenesis independent of the mechanical environment. Similarly, OB‐EVs elicited osteoclast inhibition, with stronger inhibition upon treatment with EV‐F. OCY‐EVs strongly inhibited osteoclastogenesis, evidenced to a greater extent following mechanical stimulation of the parent cell, on par with what was observed in the whole secretome. Finally, miR‐150‐5p was identified within OCY‐EVs, and its transfection suggested a dose‐dependent decrease in osteoclastogenesis.

Summary/Conclusion : These results showcased osteocytes as regulators of osteoclastogenesis via a novel EV‐mediated mechanism. MSC and OB secretomes inhibited osteoclast differentiation to differing degrees dependent on their mechanical environment, found to be EV‐mediated. These findings offer an important stepping stone towards the understanding of EV‐mediated osteoclastogenesis, paving the way for the development of cell‐free osteoporosis therapies.

Funding : SFI FFP (19/FFP/6533) (DH), EU Horizon 2020 IND, EVPRO [814495] and IRC Advanced Laureate Award EVIC (LOD) [IRCLA/2019/49]

Engineered extracellular vesicles with anti‐senescence mRNA attenuate cellular senescence‐associated secretory phenotype in human primary cells

Presenter: Victor Pham

Invitrx Therapeutics, USA

Introduction : Ageing and associated cellular decline present significant challenges for health and longevity. Exosomes, nanoscale vesicles capable of delivering molecular payloads, have emerged as promising vectors for targeting ageing‐related pathways. This study introduces novel engineered extracellular vesicles loaded with a proprietary anti‐senescence mRNA, which exert anti‐senescence, anti‐inflammatory, and regenerative properties in multiple human primary cells.

Methods : We engineered a stable cell line overexpressing a proprietary anti‐senescence mRNA and cultured them in a 3D bioreactor (iCellis Nano) for 14 days, collecting conditioned media every 48 h to enable scalable exosome production. Extracellular vesicles were isolated via tangential flow filtration (Spectrum Labs) with a 300 kDa cutoff, diafiltrated with plasma‐lyte, and characterized by nanoparticle flow cytometry (Nano FCM) for count, size, and surface markers, with qPCR confirming the mRNA presence. Biological activities of the engineered exosomes were evaluated and compared with non‐engineered exosomes and rapamycin (a well‐known anti‐senescence agent) as controls. To assess senescence‐associated secretory phenotype (SASP) effects, we induced senescence in human primary dermal fibroblasts (NHDFs), human mesenchymal stem cells (MSCs), and monocytes (THP‐1) using etoposide. We measured β‐galactosidase and SASP markers (p21, IL‐1β, IL‐6, IL‐8, and TNF‐α) via CellEvent/CellReporter and ELISA, respectively. Muscle scratch assays assessed regenerative potential in exosome‐treated muscle cells.

Results : Our engineered MSC line overexpressed anti‐senescence mRNA without affecting cell morphology or markers (CD90+, CD73+, CD34‐). Serum/xeno‐free manufactured in the 3D bioreactor yielded an exosome count at 10 1 2 particles/mL, averaging 80 nm in size, and displayed CD9, CD63, and CD81. RT‐qPCR confirmed high mRNA levels. Our senescence induction protocols in NHDFs, MSCs, and THP‐1 cells upregulated β‐galactosidase and SASP markers significantly. We observed up to a 50% reduction in SASP markers and an 80% decrease in β‐galactosidase expression by engineered exosomes. Muscle scratch assay confirmed enhanced regenerative potential compared to controls.

Summary/Conclusion : These findings highlight the potential of RNA‐loaded exosomes to reduce senescence markers in human primary cells, suggesting a promising strategy against reducing ageing hallmarks in age‐associated diseases, particularly those driven by accumulating senescent cells.

WNT3A‐loaded bi‐layered microwell scaffolds enhance stemness and extracellular vesicle production in salivary gland stem cells for radiation‐induced dysfunction recovery via the YWHAZ‐PI3K‐AKT pathway

Presenter: Yeo‐Jun Yoon

Yonsei University College of Medicine, Republic of Korea

Introduction : Salivary gland (SG) dysfunction, often induced by irradiation treatment for cancer therapy and Sjögren's Disease, can lead to fibrosis characterized by excess extracellular matrix accumulation. Enhancing stemness and promoting extracellular vesicle (EV) production from SG tissue stem cells may offer therapeutic strategies for restoring SG function.

Methods : We developed a double‐layered microwell scaffold incorporating a biochemical niche to enhance stemness and EV production in SG tissue stem cells. The scaffold comprised a WNT3A protein‐loaded poly(D,L‐lactide‐co‐glycolide) (PLGA) electrospun nanofibre combined with a polycaprolactone (PCL) microwell array to ensure sustained WNT3A release and support three‐dimensional (3D) cell culture. Human SG‐derived epithelial stem cells (sgEpSCs) were cultured on various substrates: 2D plastic dishes, 3D PCL microwells, 3D bare PLGA + PCL microwells, and WNT3A‐loaded PLGA + PCL microwells. The regenerative effects of sgEpSC‐derived EVs were evaluated using irradiated murine SG models and a human SG organoid system. All animal experiments were approved by the institutional animal care committee, and human cells were obtained with informed consent under approved protocols.

Results : The PCL microwells integrated with the WNT3A‐loaded PLGA nanofibre scaffold promoted the transformation of sgEpSCs into 3D spheroids, ensured sustained WNT3A release, and significantly increased EV production. EVs derived from sgEpSCs cultured in WNT3A‐releasing microwells (3DWNT‐EVs) were injected into the SG ducts of irradiated mice, resulting in reduced epithelial and progenitor cell death and preserved SG function. In vitro organoid assays demonstrated that 14‐3‐3 protein zeta/delta, highly expressed in 3DWNT‐EVs, significantly enhanced the proliferation of SG progenitor cells and increased the expression of phosphorylated phosphoinositide 3‐kinases/protein kinase B (p‐PI3K/AKT).

Summary/Conclusion : The use of WNT3A‐releasing microwell scaffolds to grow 3D spheroids enhances EV production from sgEpSCs, which in turn can mitigate SG dysfunction post‐radiation exposure. These findings suggest that the engineered microwell scaffold supports stem cell maintenance and EV‐mediated regenerative processes, offering a promising approach for restoring SG function after radiation‐induced injury.

Funding : This work was supported by the National Research Foundation (NRF) of Korea and funded by the Ministry of Science and ICT (2020M3A9I4039045); and the NRF grant funded by the Korea government (RS‐2024‐00432946).

Extracellular vesicles as immunomodulatory tools: From mechanisms to therapies for inflammation and regeneration

Ritu Raj, Sam Hulme, Ivana Milic, Andrew Devitt

Aston University, UK

Introduction : Extracellular vesicles (EVs) are important mediators in various biological processes, including inflammation, playing a crucial role in cellular communication and regulation of immune responses. This interaction between EVs and inflammation has been studied extensively, highlighting their dual role in the initiation and resolution of inflammation. The ability of EV from apoptotic cells to modulate inflammation offers potential therapeutic applications. This study builds on over a decade of EV research from the Devitt lab to design EVs with targeted immunomodulatory properties. By examining the interaction between engineered EVs and recipient cells, this work seeks to develop novel therapeutic strategies to resolve inflammation and promote tissue regeneration.

Methods : EVs were isolated from genetically modified human embryonic kidney (HEK‐293‐F) cells, engineered to express specific immunomodulatory enzymes, in accordance with MISEV2023 guidelines. Post‐isolation, EVs were characterized using an array of bioanalytical and biophysical techniques, including flow cytometry, mass spectrometry, and protein/enzyme biochemistry assays. The immunomodulatory efficacy and therapeutic potential of engineered EVs were validated through in vitro functional assays.

Results : Genetically modified 293‐F cells showed upregulated expression and activity of immunomodulating enzymes. EVs secreted from engineered ‘active’ cells show immunomodulatory enzymatic activity and upregulate the presence of specialised pro‐resolving mediators of inflammation and their precursors. ‘Active’ EVs induce pro‐repair macrophage surface phenotypes and cytokine profiles and improved wound closures in in vitro models. Overall, EVs secreted from genetically modified HEK 293 cells transfer active biomolecular components to recipient cells, thereby modifying recipient metabolic activity towards pro‐repair.

Summary/Conclusion : Extracellular vesicles are essential mediators of inflammation, capable of both promoting and resolving immune responses. This study illustrates the role of EVs as an active metabolic compartment, capable of modulating immune responses to support tissue repair via regulating inflammation. As research progresses, EVs may become critical tools for treating a wide range of inflammatory diseases and chronic wounds.

Funding : BBSRC Super Follow‐on Fund (APP3887): EVolution sFOF: Engineering active EV for control of chronic inflammatory responses.

Subtopic Biologic activity and therapeutic potency of EVs 1

Donor cell age and type influence extracellular vesicles therapeutic potential for osteoarthritis treatment

Presenter: Karyna Tarasova

Veterinary Medicine University of Vienna, Austria

Introduction : Mesenchymal stem cells (MSCs) demonstrate promising therapeutic effects for osteoarthritis, predominantly mediated by paracrine signals. Notably, foetal and perinatal cells exhibit greater efficacy than adult cells. This study evaluates the therapeutic efficacy of extracellular vesicles (EVs) derived from foetal and perinatal MSCs on inflamed adult ovine chondrocytes and synoviocytes in vitro.

Methods : EVs were isolated from the supernatant of ovine foetal umbilical cord‐derived MSCs (fMSCs) and chondrocytes (fCCs) ( n = 4 biological replicates) and immortalized human perinatal MSCs cell lines (Wharton's jelly (WJ‐MSC/TERT273) and amniotic membrane (P‐MSC/TERT308)), which had been cultured in serum‐free medium in hollow fibre bioreactors, using tangential flow filtration (300 kDa cut‐off). EVs were characterized for particle size distribution and number and marker proteins. The therapeutic effects of foetal and perinatal‐derived EVs (1 × 10 9 particles/mL) were assessed on inflamed (1 ng/mL TNF‐α and IL‐1β) chondrocytes in 2D and 3D culture. Healthy, inflamed untreated and inflamed dexamethasone‐treated chondrocytes served as controls. Multi‐omics analyses (RNA‐Seq, miRNA‐Seq, mass spectrometry proteomics) were employed as readouts.

Results : All 4 EV types were effectively internalized by inflamed and healthy chondrocytes, with no difference between allogeneic and xenogeneic EVs. Inflammation significantly increased uptake of all 4 EV types. Notably, fCC‐derived EVs exhibited the highest uptake. All treatments significantly reduced the inflammatory response at 24 h post‐treatment, comparable to dexamethasone. Ingenuity pathway analysis revealed that while all 4 EVs inhibited ‘HIF1A signaling,’ foetal and perinatal EVs showed distinct pathway modulation. Foetal EVs uniquely downregulated the ‘neutrophil extracellular trap signalling,’ ‘HOTAIR regulatory,’ and ‘leukocyte extravasation signalling’ pathways, with fCC‐EVs leading to the highest activation of ‘sirtuin signaling.’ Foetal MSCs and fCCs derived EVs showed a superior pro‐regenerative effect compared to perinatal‐derived EVs with significant upregulation of several mRNAs (aggrecan (ACAN) and collagen type II alpha 1 (COL2A1)).

Summary/Conclusion : Treatment outcomes demonstrated clear distinctions based on donor age (foetal vs. perinatal) and cell differentiation (MSC vs. articular chondrocytes). Selection of EVs sources tailored to specific clinical needs may thus optimize therapeutic efficacy in osteoarthritis treatment.

Non‐canonical supramolecular protein assembly within extracellular vesicles

Diego Baranda Martínez Abascal 1,2* , Thomas Glynn 1* , Julieta Sánchez 5,4,2,7 , Marc Moltó‐Abad 1,2 , Giovanni Lerussi 1 , Verónica, Villagrasa 1,2 , José Luis Corchero 2,5,4 , María Fidel‐Lledó 1 , Roser Ferrer 1,3 , Simó Schwartz 1,2,3 , Ibane Abasolo 1,2,3,6 , Joaquin Seras Franzoso 1,2,4

1 Vall d'Hebron Research Institute (VHIR), Spain; 2 Centro de Investigación Biomédica en Red, Bioingeniería; 3 Biomateriales y Nanomedicina (CIBER‐BBN), Spain; 4 Universitat Autònoma de Barcelona (UAB), Spain; 5 Institut de Biotecnologia i de Biomedicina (IBB), Spain; 6 Instituto de Química Avanzada de Cataluña (IQAC), Spain; 7 Instituto de Investigaciones Biológicas y Tecnológicas, Argentina *Diego Baranda Martínez Abascal and Thomas Glynn equally contributed to this work .

Introduction : Interest in protein therapeutics is driven by their broader functional range and better biocompatibility than small drugs. Recombinant proteins are particularly suited for restoring missing biological activities via replacement therapies. However, they usually exhibit a short half‐life, may be difficult to produce and often display a poor ability to cross biological barriers. In this context, evolutionarily conserved delivery systems such as extracellular vesicles (EVs) have been shown to naturally surpass these barriers to deliver proteins and other biomolecules with unexpected efficiency. Here, we have recombinantly produced three structurally and functionally distinct proteins with potential for replacement therapies in lysosomal storage disorders (LSDs): alpha‐galactosidase, cathepsin K and NPC intracellular cholesterol transporter 1. We have loaded them in vivo into EVs and characterized the protein intermolecular interactions and the subsequent supramolecular protein architecture to unravel structural cues supporting EV's outstanding performance.

Methods : EVs were isolated by a combination of TFF and SEC procedures from transiently transfected high‐density HEK293F cell suspension cultures. EV characterization included NTA, TEM and WB. Protein conformation was assessed by FTIR and TEM. The biological activity of the protein was determined in vitro via specific enzymatic and/or cell‐based assays.

Results : The protein of interest was found in EVs in all cases after transient overexpression, and it was able to retain its biological activity. Looking at the protein nanoarchitecture within the EVs, significant amounts of intermolecular antiparallel beta sheets were detected. Additionally, TEM direct observation of proteinase K (PK) digested EVs revealed a fibrillar core of PK‐resistant protein. Such findings were complemented with a remarkably higher stability of the EV‐loaded protein in comparison to its soluble counterpart. Note that only EV‐loaded protein exhibited measurable amounts of enzymatic activity after 14 days of incubation at RT and a 5‐fold higher cumulative signal, even after EV membrane disruption by sonication.

Summary/Conclusion : Such protein supramolecular organization, characteristic of amyloid structures, may endow the EV‐loaded protein with higher mechanical and enzymatic stability, providing also a sustained and slow‐release profile capable of outperforming conventional recombinant proteins.

Funding : Instituto de Salud Carlos III and co‐funded by the European Union: DipEV (PI22/1301), NEX‐TRY (PI21/0936); European Commission: Mimic‐KEY ( GA964386 ).

Oral delivery of MSC‐EVs as a novel approach to treat inflammatory bowel disease

Mona Belaid 1,2 , Wei Heng Chng 2 , Ram Pravin Kumar Muthuramalingam 2 , Yun Wei Lim 3 , Bertrand Czarny 3 , Giorgia Pastorin 2 , Driton Vllasaliu 1

1 King's College London, United Kingdom; 2 National University of Singapore, Singapore; 3 Nanyang Technological University, Singapore

Introduction : Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. IBD affects over 10 million people worldwide, and current treatments are associated with poor clinical outcomes and severe systemic side effects. Our previous work in vitro showed that mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) are promising therapeutics for IBD due to their regenerative potential and immunomodulatory properties. The aim of this study is to explore the therapeutic potential of MSC‐EVs to treat IBD in vivo. MSC‐EVs are most commonly administered by injection; however, the oral route offers advantages in treating IBD, as it can directly target the site of action and is a more convenient route of administration. Here, we report a novel formulation of MSC‐EVs for oral delivery, and we investigate the therapeutic effect in an in vivo model of IBD.

Methods : MSC‐EVs were isolated by ultracentrifugation on a sucrose cushion and characterized for particle size, concentration, protein content, marker analysis and morphology. The stability of MSC‐EVs was evaluated in gastric and intestinal fluids using NTA, nanoflow cytometry, and cryo‐EM. A double‐coating formulation consisting of chitosan and Eudragit S100 was developed to improve the stability of MSC‐EVs for oral administration and target colon delivery in a dextran sodium sulphate (DSS)‐induced colitis mouse model. C57BL/6J mice received 3% DSS in drinking water to induce colitis, and MSC‐EVs were administered daily by oral gavage (1E9 particles/dose) for 5 days. Mice were scored daily for disease activity, and at necropsy, colon tissue was collected for histological examination and quantification of inflammatory markers.

Results : The double coating protected MSC‐EVs from enzymatic degradation and facilitated the release of intact EVs in the colonic fluid. Coated MSC‐EVs significantly reduced the disease activity index and inflammation in the colon of DSS mice, while non‐formulated MSC‐EVs showed similar results to the negative control group.

Summary/Conclusion : A novel formulation of MSC‐EVs was developed for oral delivery and reduced inflammation in mice with DSS‐induced colitis. This study highlights the potential of MSC‐EVs as novel therapeutics for IBD and suggests that oral delivery could be an effective route of administration.

Funding : The in vivo work was supported by NAMIC grant A‐8001601‐00‐00.

EVs from reprogrammed WNT‐enriched human bone marrow‐derived MSCs can mitigate intestinal injury

Subhrajit Saha 1 , Rishi Man Chugh 1 , Payel Bhanja 1 , Dongsheng Gu 2 , Brian H. Johnstone 2

1 University of Kansas Medical Center, Kansas City, KS, USA; 2 Ossium Health, Indianapolis, IN, USA

Introduction : Currently, there is a need for strategies that mitigate gastrointestinal acute radiation syndrome (GI‐ARS). Although a few drugs are available as prophylactic agents, there is nothing available as a mitigator. We have developed a human bone marrow mesenchymal stem cell (MSC)‐derived extracellular vesicle (EV) therapy which can mitigate GI‐ARS and improve survival when applied 24 hrs after an otherwise lethal dose of radiation exposure. Repair and regeneration of ISCs depends on the activation of canonical WNT/β‐catenin signalling. MSCs are one of the major sources of WNT ligands, which bind to the WNT receptor present on the cell surface of ISCs and intestinal epithelial cells. In the present study we have discovered that activation of canonical β‐catenin signalling suppresses WNT expression in BM MSCs. EVs from β‐catenin inhibitor treated BM MSCs promoted intestinal stem cell regeneration and mitigated intestinal injury.

Methods : The regulatory role of β‐catenin in WNT expression in BM MSCs was examined by treating these cells with a tankyrase inhibitor. Pharmacologically modulated BM MSCs were examined for WNT expression by qPCR. EVs were isolated from pre‐conditioned WNT‐enriched BM MSCs and infused in irradiated C57BL/6 mice to determine the regenerative response of intestinal stem cells (ISCs) and epithelial repair.

Results : Tankyrase inhibitor treated BM MSCs induce multiple WNT ligands (WNT 3a/4/5a/5b/6/7a/8b/9a/9b/11) expression several fold. Transfusion of these pre‐conditioned WNT‐enriched human MSCs into immune‐competent mice at 24 h post irradiation effectively mitigated GI‐ARS with significant survival by 100% ( p < 0.0005) beyond 30 days without any symptom of graft‐versus‐host disease. Mice treated with/without cell therapy are all exposed to partial body irradiation, having 2.5% bone marrow shielding. Cell‐derived extracellular WNTs are primarily carried via extracellular vesicles (EV). EVs from pre‐conditioned MSCs exhibited significant WNT activity by TOPflash assay ( p < 0.0005) compared to untreated MSCs EVs. Preconditioned‐MSC‐EV‐treated mice show significantly better restitution of crypt‐villous structure ( p < 0.0001). Irradiated organoids developed from Lgr5‐eGFP‐IRES‐CreERT2 mice demonstrated a significant presence of Lgr5 + GFP + cells and normal budding‐crypt formation in response to EVs treatment ( p < 0.05).

Summary/Conclusion : In summary, our studies demonstrated that WNT‐enriched pre‐conditioned BM MSC‐derived EVs are a potential countermeasure for GI‐ARS.

Adipose stem cell‐derived nanovesicles attenuate myocardial ischaemia–reperfusion injury via miR‐24‐3p

Presenter: Wei Heng Chng

National University of Singapore, Singapore

Introduction : Extracellular vesicles (EVs) and EV‐mimetics derived from stem cells have shown therapeutic benefits in myocardial injury; however, the molecular mechanisms underlying their therapeutic effect remain unclear.

Methods : The Mayo Clinic Biospecimens Review Group (ID: 17‐010290) approved the use of de‐identified residual lipoaspirate material as a starting material for the isolation of adipose‐derived stem cells (ADSC)‐EVs from a human subject. Cell‐derived nanovesicles (CDNs) are EV‐mimetics produced by forcing cells through an extruder or a spin cup fitted with membrane filters. CDNs from U937 monocytes, ADSCs or HEK293 kidney cells require less time while achieving higher (100–200‐fold) yields compared to conventional approaches for EVs. Mouse myocardial ischaemic/reperfusion (I/R) studies were approved by the National University of Singapore Institutional Animal Care and Use Committee (IACUC, Protocol Number: R18‐1452). The model was established by subjecting male C57BL/6 mice to left anterior descending coronary artery ligation, which was removed after 30 min to allow reperfusion. Five min prior to reperfusion, 40 µg of protein of CDNs or EVs or an equivalent volume of saline was injected via tail vein. Animals were sacrificed at 24 h.

Results : In this study we demonstrate that EVs derived from human ADSC, and CDNs derived from ADSCs or U937 monocytes, but not HEK293 cells, showed similar cardioprotective effects. We also evaluated the inflammatory response, oxidative stress response and apoptotic levels within the infarct myocardium upon treatment with ADSC‐CDNs in I/R injury. The expression of catalase and superoxide dismutase 2 was upregulated in the infarct area of the treatment group, suggesting an enhanced anti‐oxidation capability induced by ADSC‐CDNs. Moreover, TUNEL staining revealed fewer apoptotic cells in the treatment group, with a 66% decrease in apoptosis ( p value = 0.029). Collectively, these data demonstrate that ADSC‐CDN treatment mitigates inflammation, oxidant stress and cell apoptosis in the I/R‐injured hearts. Finally, by analysing the microRNA content of EVs and CDNs, we identified miR‐24‐3p as the main therapeutic component suppressing cardiomyocyte apoptosis via upregulation of Nrf2.

Summary/Conclusion : Taken together, these data indicate that miR‐24‐3p mediates the beneficial effects of ADSC‐CDNs against myocardial injury.

Funding : National University of Singapore NanoNASH Program (A‐0004336‐00‐00), Ministry of Education Singapore (A‐0008496‐00‐00), NAMIC grant (A‐8001601‐00‐00), and NRF (A‐0000065‐08‐00).

Human macrophage derived EVs for tissue repair: the role of phenotype and how this can be modulated via metabolic reprogramming

Cansu Gorgun 1,2 , Paula Klavina 1 , Carolina Martins 2 , Brenton L. Cavanagh 3 , Annie M. Curtis 1,3,5* , David A. Hoey 2,3,5*

1 School of Pharmacy and Biomolecular Sciences, RCSI, Dublin, Ireland; 2 Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College, Dublin, Ireland; 3 Advanced Materials and Bioengineering Research Centre, Trinity College Dublin & RCSI, Dublin, Ireland; 4 Cellular and Molecular Imaging Core, RCSI. Dublin, Ireland; 5 Tissue Engineering Research Group, RCSI, Dublin, Ireland *Authors contributed equally .

Introduction : Macrophages, both pro‐inflammatory (M1) and anti‐inflammatory (M2) phenotypes, along with their extracellular vesicles (EVs), are essential for tissue repair. The biological effects of M1‐EVs and M2‐EVs are well‐studied in mice, but data translating to humans are limited. However, macrophage phenotypes are shaped by metabolic pathways that vary across species, and inflammatory activation is closely linked to shifts in metabolic state. Therefore, this study investigates (i) the biological function of human M1‐EVs and M2‐EVs and (ii) utilizing Pyruvate Kinase M2 (PKM2) to alter the pro‐inflammatory profile of M1‐macrophages and produce metabolically modulated macrophage‐derived EVs for enhanced tissue regeneration.

Methods : Primary human monocytes were isolated from buffy coats and polarized to macrophages: M0 (untreated), M1 (LPS and IFN‐gamma), and M2a (IL‐4 and IL‐13). Macrophages were treated with the PKM2 activator DASA‐58 for 2 h prior to M1 polarization. Metabolic profiling was performed using a Seahorse XF‐analyser, and EVs were separated by ultracentrifugation at 110,000 × g with TH660 rotor. EV characterization included NTA, WB, and TEM, while surface markers were analysed via flow cytometry using a bead‐based multiplex assay. The therapeutic potency of EVs was assessed in vitro on HUVECs for angiogenesis, as well as on human MSCs for osteogenesis.

Results : Our findings show that human macrophage polarization doesn't change EV core characteristics but significantly affects EV yield based on the donor. Importantly, surface marker expression on EVs varies according to the polarization state of the originating macrophages, reflecting differences in their functional properties. M1‐derived EVs inhibited HUVECs migration and angiogenesis, while M2‐derived EVs enhanced these processes, demonstrating the role of M2‐EVs in vascular processes. Conversely, while M1‐derived EVs enhanced the hMSCs migration, no differences were observed with M2‐EVs. Additionally, metabolic modulation of human macrophages via PKM‐2 enhanced the angiogenic potential of these macrophage‐derived EVs without affecting their osteogenic potential, highlighting a selective influence on regenerative pathways.

Summary/Conclusion : We demonstrated that altering the M1‐phenotype in human macrophages through metabolic modulation with PKM‐2 activation with DASA‐58 generates EVs with M2 properties, linking metabolism to EV generation and suggesting strategies for promoting tissue repair.

Funding : This project is funded by the European Union under the Marie Skłodowska‐Curie Postdoctoral Fellowship No. 101106209, ‘METABOLATE.’

GMP‐grade human mesenchymal stromal cell‐derived extracellular vesicles promote anti‐inflammatory and pro‐regenerative effects in a murine model of inflammatory bowel disease

Alice Zaramella 1,2 , Paola Bisaccia 1,2 , Agner Henrique Dorigo Hochuli 1,2 , Raquel Moll‐Diaz 1,2 ,Miriam Duci 1,2 , Rudra Kashyap 3 , Marcin Jurga 3 , Maurizio Muraca 2 , Andrea Porzionato 4 , Michela Pozzobon 1,2

1 Department of Women's and Children's Health, University of Padova, Italy; 2 Institute of Pediatric Research Città della Speranza, Padova; 3 EXO Biologics, Liège, Belgium; 4 Department of Neurosciences, University of Padova, Padua, Italy

Introduction : Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that has a multifactorial nature, including the environment, microbiome, genetics and immune suppression/stimulation balance. The inflammation of colon tissue resulting from impaired immune system regulation affects the integrity of the intestinal barrier. The loss of intestinal barrier integrity leads to increased intestinal permeability and, in turn, mucosal barrier dysfunction. Since a definitive cure for IBD is still not available, the need for new therapeutic targets remains fundamental. Our group already demonstrated the therapeutic effect of murine mesenchymal stromal cell‐derived Extracellular Vesicles (EVs) in IBD. Here, good manufacturing practice‐grade (GMP‐grade) produced EVs from human mesenchymal stromal cells were used to evaluate their anti‐inflammatory and pro‐regenerative effects in an in vivo model of IBD.

Methods : For six days (from day one to day six), 3% dextran sulphate sodium (DSS) was administered in drinking water to induce colitis, and on day ten, the animals were sacrificed. Human GMP‐grade EVs were administered by five intraperitoneal (ip) injections (daily from Day 3 until Day 7) in mice with acute colitis induced by DSS. The experiments included a control group (CTRL, no DSS and EVs administration, n = 15 mice), a DSS+placebo (PBS) group ( n = 25), and a DSS + EVs group ( n = 30). EVs were characterized by Nanosight, CryoTEM, and cytofluorimetric analysis of antigen surface. The clinical efficacy, the histological score and protein expression by immunofluorescence were evaluated.

Results : Mice's weight in experimental groups was reduced compared to the CTRL without significative differences. The percentage of damaged mucosa was significant decreased in the EV‐treated group compared to the DSS group. The colon length of the EV‐treated mice significantly improved. The secretion of MUC2, MUC5, iNOS and CD163 was evaluated by immunofluorescence. The EV administration increased the secretion of regenerative proteins such as mucins. Moreover, a reduction of iNOS and a parallel increase of CD163 were also observed.

Summary/Conclusion : We proved for the first time that human GMP‐grade EVs have significant pro‐regenerative and anti‐inflammatory activity in a murine model of IBD.

Telomerized MSCs of various tissues and donors: designer cell factories for standardizable and scalable production of clinical‐grade EVs

Regina Grillari 1,2 , Marieke Roefs 1 , Matthias Wieser 1 , Alessia Brancolini 1 , Johanna Gamauf 1 , Matthias Postl 1 , Marianne Pultar 3 , Giulia Corso 1 , Matthias Hackl 3 , Madhusudhan Reddy Bobbili 2,4,5 , Johannes Grillari 2,4,5

1 Evercyte GmbH, Austria; 2 Austrian Cluster for Tissue Regeneration, Austria; 3 TAmiRNA GmbH, Austria; 4 BOKU University, Austria; 5 Ludwig Boltzmann Institute for Traumatology, Austria

Introduction : Human MSC‐EVs are widely recognized for their potential therapeutic applications in age‐related diseases and tissue regeneration due to their immunomodulatory and pro‐regenerative properties. However, their clinical use is constrained by the lack of standardized and scalable EV production. Primary MSCs have a limited replicative lifespan and progressively lose differentiation capacity at early population doubling levels (PDLs), casting doubt about the resulting bioactivity of later PDL‐derived MSC‐EVs. Telomerized cell lines can circumvent this limitation; they offer indefinite proliferation potential while maintaining cell‐type specific characteristics. Here, we established different telomerized MSC lines and investigated their potential for therapeutic EV production.

Methods : MSCs were isolated from different tissues, including umbilical cord, bone marrow, adipose tissue, placenta, endometrium and dental pulp under xeno‐free conditions with full documentation. Thereafter, the replicative life span was extended by non‐viral overexpression of the catalytic subunit of human telomerase (hTERT). Cell‐type‐specific characteristics were analysed and mRNA profiles were evaluated by NGS. EVs were produced under xeno‐free 2D or 3D culture conditions and isolated using tangential flow filtration. EVs were characterized for their in vitro anti‐inflammatory and anti‐fibrotic properties. Moreover, EVs were characterized for safety aspects, especially in terms of the presence of hTERT.

Results : Telomerized MSCs maintained the typical markers and function of primary MSCs while exhibiting an unlimited replicative lifespan. mRNA profiling of the top 10% of genes revealed tissue‐specific differences in MSCs isolated from various human organs, with MSCs derived from different parts of the placenta clustering together. EVs isolated from all telomerized MSCs showed particle sizes in the typical EV range (100–200 nm) and expression of tetraspanin markers. Moreover, they displayed anti‐inflammatory and anti‐fibrotic activity, showing slight differences depending on their MSC origin. Importantly, EVs from primary and telomerized cells do not carry hTERT DNA, full length hTERT mRNA or hTERT protein.

Summary/Conclusion : This study demonstrates that telomerized MSCs retain the essential properties of primary MSCs and produce EVs with biological activity without any risk of transferring hTERT to the recipient cells. These findings suggest that telomerized MSCs are a reliable and safe cell factory for producing clinical‐grade EVs, circumventing the limitations of primary MSCs for EV production.

Funding : EU‐Horizon‐2020 (#101072766; #721975). Evercyte GmbH; TAmiRNA GmbH.

Targeted malignant wound healing with miRNA‐enhanced MSC‐derived EVs in a sustained‐release atelocollagen hydrogel

Presenter: Marina R. Hadjicharalambous

Theramir Ltd, Cyprus

Introduction : Malignant wounds in advanced cancer patients present a complex therapeutic challenge, with existing treatments mainly offering palliative care. This study aims to develop a novel approach by evaluating the therapeutic efficacy and safety of mesenchymal stem cell‐derived extracellular vesicles (EVs) formulated in an atelocollagen‐based hydrogel for application in open wounds. To optimize this formulation, we investigated the effects of native versus lyophilized EVs, as well as unmodified EVs compared to EVs engineered to carry single or multiple miRNAs with well‐characterized anti‐cancer and wound‐healing properties.

Methods : EV characterization was performed following ISEV guidelines. TRPS technology quantified EV concentration and size distribution, while western blot analysis confirmed the presence of EV‐specific markers. Both native and lyophilized EVs, either unmodified or miRNA‐modified, were incorporated into a custom atelocollagen hydrogel matrix enriched with anti‐inflammatory and anti‐microbial plant extracts to create a sustained‐release delivery system. Molecular and physical characterizations confirmed the stability and functionality of the formulation. The therapeutic efficacy was assessed in vitro using scratch assays on keratinocyte monolayers to monitor wound closure and tube formation assays on HUVEC cells to evaluate angiogenesis. Expression of key wound‐healing markers and pathways, including inflammatory and redox‐related pathways, was quantified through RT‐PCR and western blotting. Anti‐cancer properties were assessed using cytotoxicity assays on cancer cell lines (MDA‐MB‐231, HT‐1197, and 5637), while biocompatibility was evaluated on skin cell monolayers and reconstructed human epidermis in accordance with OECD guidelines.

Results : The atelocollagen hydrogel formulation containing miRNA‐modified EVs demonstrated enhanced stability and sustained‐release properties. Biocompatibility tests confirmed safety, and in vitro assays showed accelerated keratinocyte migration and improved angiogenesis. Molecular analyses indicated modulation of chronic inflammation pathways, and cytotoxicity assays demonstrated reduced viability in cancer cells, indicating a dual therapeutic potential for wound healing and tumour inhibition.

Summary/Conclusion : This integrative approach, utilizing miRNA‐loaded, MSC‐derived EVs within a sustained‐release atelocollagen hydrogel, offers a promising therapeutic strategy for malignant wound management. These findings support further preclinical evaluation in animal models.

Funding : The programme is funded by the European Union Recovery and Resilience Facility of the NextGenerationEU instrument, through the Research and Innovation Foundation (ENTERPRISES/0223/Sub‐Call1/0212).

Treatment of lupus nephritis using CD38 targeting peptide‐modified extracellular vesicles derived from mesenchymal stem cells

Presenter: Yali Yu

Huazhong University of Science and Technology, China (People's Republic)

Introduction : Lupus nephritis (LN) is the most common and severe refractory complication of systemic lupus erythematosus (SLE). There is an urgent clinical need for targeted and effective therapeutic strategies to repair the damaged nephron and prevent the progression of renal fibrosis. While extracellular vesicles derived from mesenchymal stem cells (MSC‐EVs) have achieved great focus as a cell‐free therapy for LN, their rapid in vivo metabolism and suboptimal target site enrichment present significant limitations. Given the high expression of CD38 in diverse immune cells and senescent cells in LN pathogenesis, we hypothesized that modification of MSC‐EVs with CD38‐targeted polypeptides could target CD38‐high immune cells and senescent cells and repair the inflammatory injured kidney.

Methods : EVs were isolated from the human umbilical cord MSCs utilizing differential centrifugation and characterized following MISEV 2023 guidelines. CD38‐targeting peptides were conjugated to EV membranes via PEG‐2000 (CD38‐EVs). MRL/lpr mice, developing LN at 15 weeks, served as the experimental model. Biodistribution was assessed through immunofluorescence analysis of organ sections. LN mice received EVs or CD38‐EVs treatments at 48‐h intervals for 2 weeks. Renal function and autoantibody profiles were quantified by enzyme‐linked immunosorbent assay. Histological analyses evaluated renal inflammatory infiltration and fibrosis. Gene expression patterns associated with cellular senescence were characterized through immunofluorescence staining.

Results : Our findings showed that CD38‐EVs more efficiently targeted the kidney and predominantly accumulated in renal tubular structures. Both EV and CD38‐EV treatments showed an amelioration of renal function and autoantibody levels, and a reduction of inflammatory infiltration and interstitial fibrosis. These therapeutic effects correlated with reversed stress‐induced senescence in renal tubular cells. Notably, the CD38‐EV cohort exhibited significantly superior outcomes across all parameters compared to the EV group.

Summary/Conclusion : We have developed novel kidney‐targeting MSC‐EVs that demonstrate therapeutic efficacy in LN, potentially operating through the reversal of tubular cell stress‐induced senescence. While further mechanistic studies are warranted, this approach represents a promising therapeutic strategy for LN management.

Funding : This research was supported by the National Key Research and Development Program of China (No. 2021YFA1101500).

Evaluation of apple‐derived vesicles for the treatment of inflammatory bowel disease

Wei Heng Chng 1 , Yixiang Gao 1,2 , Mona Belaid 1,3 , Ram Pravin Kumar Muthuramalingam 1 , Nichakan Khunkitchai 1 , Wei Jiang Goh 4 , Bertrand Czarny 5 , Giorgia Pastorin 1

1 Department of Pharmacy and Pharmaceutical Sciences, National University of Singapore, Singapore; 2 Department of Biological Sciences, National University of Singapore, Singapore; 3 Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, King's College London, London, UK; 4 Craft Health Private Limited, Singapore; 5 School of Materials Science and Engineering, Nanyang Technological University, Singapore

Introduction : Inflammatory bowel disease (IBD) includes chronic inflammatory conditions affecting the gastrointestinal tract. Current therapeutic options focus on reducing inflammation, providing symptomatic relief and preventing remission. Recently, extracellular vesicles isolated from plants (termed as plant‐derived vesicles [PDVs]) have gained attention due to their anti‐inflammatory and antioxidant effects, combined with biocompatibility and scalability. Malus domestica (commonly known as apple) has been reported to have intrinsic therapeutic properties towards the treatment of IBD. However, it remains unclear if vesicles isolated from apple exhibit similar pharmacological effects.

Methods : Apple‐derived vesicles (APDVs) were isolated by differential ultracentrifugation. The storage stability and gastrointestinal tract stability of APDVs were characterized by dynamic light scattering to determine any changes in physical size. The pharmacological activities of APDVs, such as anti‐inflammatory effects, were evaluated in vitro. Colitis was induced in mice by adding 3% dextran sodium sulphate into drinking water. APDVs were administered orally to mice at a dose of 1E10 particles daily for 5 days. To determine the therapeutic activity towards IBD, the mice were monitored daily in terms of their weight, stool consistency and the presence of blood in stools. The in vivo animal work was approved by the Nanyang Technological University Institutional Animal Care and Use Committee in Singapore.

Results : APDVs were successfully isolated by differential ultracentrifugation with a yield of 3.4 ± 1.6 E10 particles per 100 g apple. APDVs displayed good storage stability at 4°C over a period of 7 days, with sizes remaining relatively consistent at around 200 nm. These APDVs exhibited stability under gastric conditions while undergoing some aggregation under intestinal conditions. APDVs did not show any significant toxicity towards RAW264.7 macrophages and Caco‐2 epithelial cells. These APDVs exhibited anti‐inflammatory effects in vitro, reducing the relative gene expression of TNF‐α and IL‐1β within 24 h. APDVs improved the disease activity index in vivo, with a particular improvement in stool consistency and reduction in stool occult blood.

Summary/Conclusion : APDVs at a daily oral dose of 1E10 particles over 5 days proved beneficial in the treatment of IBD. APDVs have the potential to reduce rectal bleeding, improve stool consistency, and maintain colon health.

Funding : This work is supported by a NAMIC grant (A‐8001601‐00‐00).

Bioengineering matrix vesicles as a nanotherapeutic approach to endochondral bone regeneration

Presenter: Flurina Staubli

University Medical Center Utrecht, The Netherlands

Introduction : Extracellular matrix vesicles (MtVs) are essential mediators of endochondral ossification (EO), a pivotal process in bone development and repair. These nanovesicles, derived from hypertrophic chondrocytes, play critical roles in extracellular matrix remodelling, chondrocyte communication, and growth plate development. The manufacture of stem cell‐derived cartilage tissues in vitro is a promising approach to generate MtVs; however, obtaining MtVs which exhibit a hypertrophic phenotype has remained elusive. In this study, we aim to bioengineer hypertrophic stem cell‐derived cartilage microtissues in vitro to generate EO‐mimetic MtVs as a novel nanotherapeutic strategy for bone regeneration.

Methods : Human bone marrow‐derived stromal cells (hBMSCs) microtissues were differentiated in chondrogenic medium with/without bone morphogenic protein 2 (BMP2) for 21 days. Following this, constructs were either maintained in chondrogenic medium or switched to hypertrophic medium till Day 28. The expression of chondrogenic and hypertrophic markers was assessed by biochemical and histological analysis. MtVs were isolated via enzymatic digestion, followed by ultracentrifugation. MtV size, morphology and concentration were assessed via nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and CD63 ELISA. MtVs therapeutic efficacy was assessed by evaluating their effects on hBMSCs recruitment, osteogenic differentiation, and human endothelial colony‐forming cells (hECFCs) angiogenesis. MtVs cytokine contents were determined via ELISA.

Results : Our findings revealed that BMP2 and hypertrophic medium conditioning significantly upregulated microtissue chondrogenic (glycosaminoglycans, collagen type II) and hypertrophic (vascular endothelial growth factor (VEGF), alkaline phosphatase (ALP), type I‐ and type X collagen) marker expression. NTA and TEM confirmed the nano‐sized nature of the MtVs (∼50–200 nm). BMP2 and hypertrophic conditioning significantly increased MtV yield, ALP activity and calcium binding compared to MtVs from chondrogenic microtissues. Notably, MtVs derived from BMP2/hypertrophic conditioned microtissues substantially promoted recipient hBMSCs proliferation, migration and mineralization, in addition to improving the angiogenic tube formation of hECFCs. Cytokine profiling confirmed that MtVs obtained from hypertrophic microtissues were enriched with BMP2 and VEGF.

Summary/Conclusion : Taken together, we showcase that synergistic BMP2 and hypertrophic conditioning significantly improve the yield and therapeutic potential of MtVs. These findings highlight the considerable potential of manufacturing EO‐mimetic MtVs as a nanotherapeutic approach for promoting bone regeneration.

Umbilical cord mesenchymal stem cell‐derived small extracellular vesicles as a cocktail therapy to ameliorate sepsis‐induced acute kidney injury

Presenter: Feng Chen

Tsinghua University; Southeast University, China (People's Republic)

Introduction : Sepsis is an infection‐induced immune imbalance that induces acute kidney injury with high morbidity and mortality in critically ill patients. However, specific therapeutic agents are lacking. Mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEV) are expected to provide an innovative and effective solution as a cell‐free immunomodulatory therapy.

Methods : MSC‐sEV was obtained using differential ultracentrifugation (dUC) combined with size exclusion chromatography (SEC) and evaluated for its renoprotective effects against sepsis‐induced acute kidney injury (S‐AKI). mRNA profiles of renal tissues and miRNA profiles of MSC‐sEV were characterized by RNA sequencing to explore the potential protective mechanisms. In addition, MSC‐sEV was modified by Kim‐1 targeting peptide and ExoMotif to precisely target and enhance renal therapy.

Results : In S‐AKI, MSC‐sEV homed and accumulated in renal tubules, effectively exerted antioxidant and anti‐inflammatory effects, protected tubular cells from pyroptosis, and improved renal function and survival rate. Mechanistically, we identified a key target centred on TNFR2. Similar to cocktail therapy, MSC‐sEV co‐targeted the inhibition of TNFR2 expression by delivering let‐7a‐5p and miR‐125a‐5p, thereby regulating the NLRP3/Caspase‐1/GSDMD signalling pathway. In addition, ExoMotif (GGAG) and Kim‐1 targeting peptide‐modified MSC‐sEV enable precise targeting of the injured kidneys, helping to optimize therapeutic dose and therapeutic effect.

Summary/Conclusion : MSC‐sEV has superior anti‐inflammatory, antioxidant and cytoprotective effects. This cell‐free therapy utilizing the strong immunomodulatory ability of MSC is expected to be an innovative strategy for the treatment of S‐AKI.

High‐yield production of EVs from mesenchymal stem cells using a hollow fibre bioreactor

Carlos Jesus 1,2,3 , Miguel Lino 2,3 , Hugo Fernandes 1,2,3,4 , Lino Ferreira 1,2,3

1 Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 3 Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal; 4 Multidisciplinary Institute of Aging (MIA‐Portugal), University of Coimbra, Coimbra, Portugal

Introduction : The full regenerative/therapeutic potential of extracellular vesicles (EVs) isolated from mesenchymal stromal/stem cells has been demonstrated in the setting of several diseases, including cardiovascular diseases. However, the limited scalability of production of therapeutically relevant EVs hinders its clinical translation. Here, we used a hollow fibre bioreactor to improve the yield of native EVs from MSC whilst maintaining their therapeutic potential.

Methods : We used human Wharton's Jelly MSC (WJ‐MSC)—upon informed consent—due to its easy isolation from the umbilical cord, retention of primitive stem cell characteristics and well‐known therapeutic capacity. A minimum of 100 million WJ‐MSC were inoculated, and conditioned media was collected every day. EV isolation was done by differential ultracentrifugation followed by purification by size exclusion chromatography. EVs were characterized according to their concentration, size, protein amount, surface charge and tetraspanins expression. We further validated EVs bioactivity in a scratch assay using endothelial cells.

Results : Compared to EVs secreted by cells cultured in 2D systems (T‐175 flasks), our results showed that EVs produced by the bioreactor are slightly bigger (203.2 vs. 161.9 nm), showed high expression of CD63 (93.0% vs. 97.8%) and similar surface charge (−24.3 vs. −20.9 mV). In addition, we showed a 3.3‐fold increase of EV collection per day, as compared to a single isolation using 10–12 T‐175 flasks. We were able to isolate over 2.0 × 10 12 EVs in total (after purification) in a 15‐day period. Our results showed that the bioactivity of EVs generated in a bioreactor is stronger (73.4% vs. 40.8%) compared to EV from 2D systems.

Summary/Conclusion : Overall, hollow fibre bioreactors are a suitable method for scalable production of EVs, maintaining their therapeutic properties.

Funding : The authors would like to acknowledge the funding by the FCT PhD Studentship (SFRH/BD/144092/2019), Programa Operacional Competividade e Internacionalização (POCI) na sua componente FEDER e pelo orçamento da Fundação para a Ciência e a Tecnologia na sua componente OE (Project 2022.03308.PTDC; 2022.07615.PTDC; 2022.02803.PTDC); EC projects REBORN (Ref. 101091852); PRR project HfPT—Health from Portugal (Ref: 02/C05‐i01.01/2022.PC644937233‐00000047); and ‘Project RESET_BONE_AGING_2’ (Ref: COMPETE2030‐FEDER‐01175000). MIA‐Portugal (European Union's Horizon 2020 No. 857524).

Polydopamine‐modified turmeric‐derived nanovesicles biomimetic hydrogel system for effective management of diabetic wound infections

Presenter: Bodeng Wu

Southern Medical University, China (People's Republic)

Introduction : Plant‐derived nanovesicles are gaining interest for their role in interspecies communication, yet challenges persist in distinguishing them from their source plants and understanding their regulatory mechanisms and clinical applications. Our previous studies identified turmeric‐derived nanovesicles (TDNVs) as pharmacological complexes that modulate the Nrf2/HO‐1 pathway in fibroblasts and the TLR4/MyD88 pathway in macrophages, enhancing the diabetic wound microenvironment. To address the frequent occurrence of multidrug‐resistant infections in diabetic wounds, this study developed an engineered TDNVs@PDA hydrogel system as a potential therapeutic strategy.

Methods : TDNVs were modified with polydopamine to create near‐infrared (NIR) responsive vesicles TDNVs@PDA (TP). Characterization of TP involved transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering for assessing morphology, size, and zeta potential. A PLGA‐PEG‐PLGA hydrogel was used as the matrix to load TP, forming TP@Gel. The photothermal effect, uptake efficiency, and antibacterial activity of TP@Gel against multidrug‐resistant Pseudomonas aeruginosa (MDRPA) were evaluated in vitro. The therapeutic efficacy of TP@Gel in diabetic wound infections was investigated, and transcriptomic analysis assessed changes in the tissue microenvironment.

Results : TP was synthesized with a uniform cup‐shaped structure, displaying a particle size of 100–200 nm. The TP@Gel system demonstrated effective TP release and significant uptake in MDRPA, enhancing antibacterial efficacy under NIR irradiation. In vitro studies showed that TP@Gel reduced reactive oxygen species levels in fibroblasts, enhanced Nrf2/HO‐1 signalling, and promoted macrophage polarization to the M2 phenotype, resulting in decreased pro‐inflammatory cytokines TNF‐α and IL‐1β. Gelation of TP@Gel at physiological temperatures effectively cleared wound infections and facilitated healing. Transcriptomic analysis revealed modulation of inflammation, antioxidant responses, and extracellular matrix remodelling pathways compared to controls.

Summary/Conclusion : TP@Gel serves as a multifunctional therapeutic approach that addresses bacterial infections while enhancing antioxidant defence and reducing inflammation in diabetic wounds. This study underscores the potential of TDNVs in regenerative medicine and their applicability as promising solutions for diabetic wound healing in clinical settings.

Funding : National Science Fund for Distinguished Young Scholars of China (82025024). National Key R&D Program of China (2021YFA1300604).

Novel biological therapies for pancreatic ductal adenocarcinoma

Isaiah Davis 1,2 , Karthik Gourishetti 1,2 , Deepak Bhere 1,2

1 Biotherapeutics Laboratory, University of South Carolina School of Medicine, Columbia, SC 29209, USA; 2 Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209, USA

Introduction : In the United States, pancreatic cancer is the third leading cause of cancer‐related deaths, with pancreatic ductal adenocarcinoma (PDAC) accounting for 80% of such tumours. At diagnosis, 51% of pancreatic cancers are already metastasized, contributing to poor prognosis and a reported 5‐year survival rate of 10%. Current treatment includes chemotherapy and/or radiation. Owing to the complexity of the malignancy, surgery is often not possible. Targeted combination therapies could serve as a promising potential treatment. Exosomes are extracellular vesicles used to deliver microRNA (miR) to treat cancer. miRs are small, minimally‐toxic, non‐coding RNA that can affect the transcription of specific genes in tumours, silencing oncogenic genes and enhancing expression of tumour suppressor genes. miR‐7 is a tumour suppressor that is often downregulated in PDAC, contributing to its poor prognosis. Oncolytic viruses (OVs) are naturally occurring or genetically modified viruses that target tumour cells, cause tumour cell lysis, and activate the immune system. Oncolytic herpes simplex virus (oHSV) can replicate within PDAC tumour cells and cause lysis without affecting healthy cells. oHSV tumour infection will also activate innate and adaptive immune responses, further contributing to tumour reduction. The aim of this study is to explore PDAC cell viability after a combination approach of exosome delivered miR‐7 and oHSV treatment.

Methods : Human adipose stem cells (ADSC) were transduced with miR‐7 plasmid and cultured in exosome‐depleted FBS. The miR‐7‐enriched exosomes were then isolated, purified, quantified, and characterized to be used for subsequent studies. Human and murine PDAC cells in vitro were treated with miR‐7‐enriched exosomes and oHSV to determine the combination treatment's impact on cell viability.

Results : Our data demonstrated a successful landing of the mIR‐7 and oHSV cargo into exosomes. Preliminary findings show that increasing miR‐7 expression in PDAC cells alongside oHSV treatment significantly impacts cell viability in vitro. Mechanistic studies revealed a time‐dependent activation of the cascade in vitro following treatment.

Summary/Conclusion : Enriching PDAC cells with miR‐7 and treating the same cells with oHSV has been shown to greatly affect cell viability. After confirmation, this method could lead to the development of more effective treatment strategies for patients with PDAC.

Microalgae‐derived extracellular vesicles as a next‐generation platform for bone health treatments

Presenter: Sabrina Picciotto

National Research Council of Italy, Italy

Introduction : Extracellular vesicles (EVs) represent an innovative advance in nanotechnology for targeted therapeutic delivery, efficiently transporting endogenous or exogenous bioactive compounds across tissue barriers. EVs derived from microalgae, known as (nano)Algosomes (Alg), offer a natural, sustainable platform for developing pharmaceutical formulations. Our previous studies have revealed that Alg possesses impressive antioxidant and anti‐inflammatory capabilities without causing allergic‐like responses. Gene expression analysis demonstrated no genetic alterations following Alg treatment, with no DNA damage observed. Additionally, our biodistribution research in mice showcased a distinctive organotropism, particularly observed in the bone tissue. These findings emphasize Alg's promising potential for safe and effective therapeutic applications.

Methods : Alg are obtained by isolating from a suspension of Tetraselmis chui microalgal cells through tangential flow filtration. Our innovative platform has been developed and validated for the medium‐scale production, characterization, and bioengineering of Alg. This cutting‐edge system was specifically designed to address the limitations associated with current sources of EVs and existing EV‐based nano‐delivery systems.

Results : Our research investigates the properties of Alg in alignment with MISEV2023 guidelines, focusing on their bioactivity, biocompatibility, and biodistribution, particularly highlighting their tropism for bone. Based on these findings, we intend to capitalize on the inherent organ‐targeting capabilities of Alg for the development of innovative pharmaceutical applications for osteosarcoma treatment. Alg demonstrated a strong capacity for loading, significantly improving the cellular uptake of small molecules such as doxorubicin. Functional assays performed on osteosarcoma cells, including variants resistant to doxorubicin, revealed that the drug's effectiveness was markedly greater when delivered via Alg compared to conventional free drug administration.

Summary/Conclusion : Alg present a natural and highly effective delivery system for both naturally occurring compounds from microalgae and exogenous bioactive compounds or synthetic drugs, particularly targeted for bone health enhancement.

Clinical‐grade extracellular vesicles isolated from telomerized mesenchymal stromal cells maintain biological activity over time

Johanna Gamauf 1 , Alessia Brancolini 1 , Matthias Wieser 1 , Matthias Postl 1 , Giulia Corso 1 , Madhusudhan Reddy Bobbili 2,3,4 , Johannes Grillari 2,3,4 , Regina Grillari 1,4 , Marieke Roefs 1

1 Evercyte GmbH, Austria; 2 BOKU University, Austria; 3 Ludwig Boltzmann Institute for Traumatology, Austria; 4 Austrian Cluster for Tissue Regeneration, Austria

Introduction : Extracellular vesicles (EVs) derived from immortalized MSCs hold great therapeutical potential for a range of regenerative and age‐related diseases. To develop MSC‐EVs further towards a scalable clinical application, prolonged EV storage with maintained therapeutic potential is required. Therefore, more information about MSC‐EV physical and biological stability over time is needed. Here, we compared EVs produced by telomerized MSCs that were derived from different tissues, produced in 2D or 3D culture vessels, and isolated in different storage buffers, for physical and functional characteristics up to 1 year after isolation.

Methods : MSCs were isolated from bone‐marrow (BM‐MSC), Wharton's‐jelly (WJ‐MSC) and adipose tissue (ASC), and immortalized by non‐viral introduction of the catalytic subunit of human TERT (hTERT). EVs were isolated from serum‐free 2D or 3D hollow‐fibre cell cultures, enriched by 300 kDa tangential flow filtration (TFF), diafiltrated and stored in PBS, 20 mM HEPES or Ringer's lactate. Aliquoted EVs were stored at ‐80°C until further use. Particle and protein concentrations were measured directly after isolation and after thawing at different time‐points using nanoparticle tracking analysis and BCA. In vitro anti‐fibrotic activity was assessed by adding EVs to TGF‐β stimulated hTERT‐immortalized fibroblasts and evaluating the subsequent reduction in α‐SMA expression. Anti‐inflammatory activity was determined by adding EVs to LPS‐stimulated RAW264.7 cells and measuring the resulting decrease in NO levels.

Results : BM‐MSC, WJ‐MSC and ASC‐derived EV particle concentrations were not greatly influenced by one freeze thaw cycle and prolonged storage at −80°C. Storage over time did not influence total protein content. MSC‐EVs maintained anti‐fibrotic and anti‐inflammatory activity for up to 1 year after isolation, irrespective of the used storage buffer and 2D or 3D MSC culture conditions.

Summary/Conclusion : EVs isolated from different telomerized human‐derived MSCs maintained in vitro biological activity after long‐term storage at −80°C, demonstrating biological stability over time. Total particle and protein concentration did not markedly reduce over time, probably due to the high protein content in TFF‐enriched EV preparations. This observed stability supports the upscaling of EV production and long‐term storage, reducing the variability associated with different cell batches and isolation parameters, and advancing the development of MSC‐EV therapeutics.

Funding : EU‐Horizon‐2020 #101072766; #721975. Evercyte GmbH, TAmiRNA GmbH.

Plant‐derived nanovesicle screening and compatibility mechanism based on a glioma organoid chip

Presenter: Shan Lin

Southern Medical University, China (People's Republic)

Introduction : Plant‐Derived Nanovesicles (PDNVs) have attracted much attention due to their natural biological activity, immune regulation and other biological activities. Due to the large number of plant species, the current research on PDNVs has not formed a systematic application in tumour. Organochips can simulate the complex tumour microenvironment and provide a powerful tool for drug screening. To provide glioma patients with more targeted and personalized therapeutic options, this study aims to establish a glioma organoid‐based PDNVs screening platform to evaluate and elucidate the mechanisms of action of PDNVs, contributing to the standardization and systematic assessment of PDNVs in tumour applications.

Methods : PDNVs were collected by differential centrifugation, and vesicles were characterized by transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering to assess morphology, size, and zeta potential. To develop a three‐dimensional organoid chip containing glioma cells and immune cells, and construct a glioma organoid bank that can be revived by freeze‐thaw. Glioma organoid chips were used to evaluate the effects of different PDNVs on tumour cell growth and immune cell activation. To study the specific pathways and mechanisms of PDNVs effect on tumour cells and immune cells. To evaluate the efficacy of candidate PDNVs and build PDNVs efficacy database.

Results : Thirteen common PDNVswere collected, all exhibiting uniform cup‐like or spheroidal structures, with particle sizes ranging from 100 to 200 nm and no significant differences in morphology or size. Among these, Atractylodes lancea derived nanovesicles showed the most potent anti‐tumour effects on glioma cells, while Platycodon grandiflorus ‐derived nanovesicles exhibited the strongest immunomodulatory activity, significantly enhancing M1 polarization of macrophages as revealed by flow cytometry.

Summary/Conclusion : A glioma organoid‐based screening platform for PDNVs was successfully established. Preliminary results demonstrated that extracellular vesicles from various plant sources can inhibit tumour cell growth and modulate immune cell activity. Future studies will focus on elucidating the mechanisms by which different PDNVs suppress glioma, paving the way for more effective and personalized therapeutic strategies.

Funding : National Science Fund for Distinguished Young Scholars of China (82025024); National Natural Science Foundation of China General Program (82302639).

Lyophilized small extracellular vesicles (sEV) from glycolytically activated mesenchymal stem/stromal cells promote chondroprotection in vitro and cartilage regeneration in vivo

Presenter: Yeimi Herrera‐Luna

Universidad de los Andes, Chile

Introduction : Osteoarthritis (OA) affects the articular cartilage, causing an increase in oxidative stress leading to cartilage damage. It has been shown that mesenchymal stem/stromal cells (MSCs) therapeutic effects depend on their small extracellular vesicles (sEVs). However, for optimal clinical outcomes there is a need to enhance their therapeutic efficacy and to improve their storage for potential clinical application. We propose that sEVs derived from metabolically reprogrammed glycolytic‐MSC (Glyco‐sEVs) have a better therapeutic effect than those under basal conditions (Basal‐sEVs) and that after a lyophilized process they maintain their phenotype and function against OA progression.

Methods : Chondrocytes were isolated from the knee of OA patients. The chondroprotective effect of frozen or lyophilized Glyco‐sEVs was measured by an apoptotic assay with menadione through an annexin/PI kit and flow cytometry. To determine the clinical effect of frozen or freeze‐dried Glyco‐sEVs, both were injected intraarticularly in a collagenase murine model of OA (CIOA) and evaluated by microCT and histopathology.

Results : Our results showed that lyophilized Glyco‐sEVs do not change their sEV characteristics; therefore, they maintain their chondroprotective effect on chondrocytes when exposed to menadione as a source of oxidative stress. Moreover, CIOA mice treated with either frozen or lyophilized Glyco‐sEVs showed a significant reduction in bone mineral density and the clinical damage score obtained from joint histology.

Summary/Conclusion : Lyophilized Glyco‐sEVs maintain their phenotype by expressing CD63, CD81 and CD9 and their size and shape with a yield of 86.5%. Moreover, they do not change their chondroprotective effect in vitro since, as frozen Glyco‐sEVs, they display higher resistance to apoptosis induced by oxidative stress as compared to basal sEVs. Moreover, CIOA mice treated with both Glyco‐sEVs showed a regenerative effect with significance evidenced in the histomorphometry parameters and the clinical joint score by histology. These results show that lyophilized Glyco‐sEVs represent a potential new strategy for treating OA through their chondroprotective and regenerative properties.

Funding : ANID FONDECYT Regular N°1211353; ANID FONDECYT Iniciación N°11220549; ANID FONDEF N°21110194; and ANID Fondo Basal 210024 (IMPACT).

Unlocking the potential of exosomes generated from induced pluripotent stem cell‐derived skin organoids for wound healing

Presenter: I‐Fen Cheng

Biomedical Translation Research Center, Academia Sinica, Taiwan (Republic of China)

Introduction : Exosomes derived from keratinocytes contain a wide variety of biomolecules, including DNA, microRNA, mRNA, lipids, metabolites and proteins that can potentially regulate the function and behaviour of fibroblasts, melanocytes, and immune cells in skin. For example, exosomes released from keratinocytes can improve skin diseases by regulating pigmentation in melanocytes and controlling stimulation of immune cell activity. Therefore, several clinical studies have demonstrated the potential of keratinocyte‐derived exosomes in treating skin disorders. However, the inefficient production of exosomes has hampered their development for clinical translation. Here, we described an easy and optimized protocol to produce functional keratinocyte‐derived exosomes from human induced pluripotent stem cells (iPSCs).

Methods : Skin organoids were generated by a stepwise differentiation protocol via modulating TGFβ and FGF pathways under suspension culture conditions. Exosomes were isolated from concentrated conditioned medium and followed by size exclusion chromatography. The exosomes were characterized by immunofluorescence staining and transmission electron microscopy (TEM), and the purity of exosomes was assessed by nanoparticle tracking analysis (NTA).

Results : Self‐organized organoids generated from human iPSCs resemble complex skin tissues consisting of epidermis, dermis and pigmented hair follicles. After 10 weeks of differentiation, the CK14+ cells were seen in the basal layer, whereas CK10 staining confirmed the formation of supra‐basal layers. E‐cadherin+ epithelium was also detected. By day 120, CK17 staining indicates development of the outer root sheath of the hair follicles. The stimulatory effects on proliferation and migration of human dermal fibroblasts were observed after applying conditioned medium of iPSC‐derived skin organoids. The exosomes were further isolated from concentrated conditioned medium of iPSC‐derived skin organoids, followed by size exclusion chromatography. These purified exosomes assessed by NTA and characterized by TEM were positive for CD9, CD63 and CD81. We validated exosomes isolated from iPSC‐derived skin organoids exerted similar effects in wound healing assays. Further analysis of these purified exosomes revealed enrichment of signalling regulators associated with keratinocyte‐fibroblast communication and signal transduction.

Summary/Conclusion : The findings of the current study reinforce the potential of exosomes derived from iPSC‐derived skin organoids to be used to improve wound healing. We also anticipated exosomes produced by iPSC‐derived skin organoids can be used to treat multiple skin disorders.

Targeted extracellular vesicles and doxorubicin formulation potential for triple‐negative breast cancer

Erna María Jónsdóttir 1 , Jens Guðmundur Hjörleifsson 2 , Berglind Eva Benediktsdóttir 1

1 Faculty of Pharmaceutical Sciences, University of Iceland, Iceland; 2 Faculty of Life‐and Environmental Sciences, University of Iceland, Iceland

Introduction : Triple‐negative breast cancer (TNBC), known for its poor prognosis and lack of targeted therapies, overexpresses EGFR1 in approximately 30% of cases, suggesting potential for targeted treatment. This study focuses on using engineering extracellular vesicles (EVs) as nanodrug carriers, utilizing their stability and bioengineering capabilities to specifically target TNBC, thus enhancing the therapeutic potential of EVs. Additionally, to compare different strategies to load doxorubicin into EVs.

Methods : Engineered HEK293E cell lines (releasing EGFR1‐targeting EVs with GFP (eEV1), GE11 (eEV2) or GFP only control (eEV3) and original HEK293E) were cultured in serum‐free medium. After 25 mL from each culture was clarified, EVs were isolated using Strep‐Tactin CD81 Fab‐TACS affinity chromatography and characterized using NTA, western blot (ALIX, Synthenin‐1, Calnexin) and the Amplex Red Cholesterol assay. EGFR1+ MDA‐MB‐213 and EGFR1− MCF‐7 cell lines were used to assess EGFR1+ EV targeting. EVs were stained with MemGlow, purified and co‐cultured (1 × 10 9 particles/mL) for 6 h before PBS wash, fixation, DAPI staining and confocal imaging. Doxorubicin was loaded into original EVs via passive or active loading with ammonium sulphate (0.25–0.5 M), sonication and extrusion (0.1 µm, 1‐4 cycles). Dox concentration was quantified using a fluorescence spectrophotometer, and encapsulation efficiency (EE) was calculated.

Results : The average/mode size of all EVs was 140/115 nm, respectively, with ALIX and Synthenin‐1 present in all EVs. eEV1 and eEV2 showed higher affinity towards the MDA‐MB‐231 cells versus controls, with no difference observed the MCF‐7 cell line. Sonication/extrusion of Dox post‐EV isolation resulted in EE of 13.9‐39.6%, resulting in highly polydisperse EVs. When this method was carried out pre‐EV isolation, EE ranged from 1.8% to 64.6%, with better EV size monodispersity.

Summary/Conclusion : EGFR1 + ‐targeting EVs (eEV1 and eEV2) demonstrated superior affinity towards EGFR1 + ‐expressing cell line compared to controls. Approaches in Dox loading show benefits of loading prior to EV isolation; however, EE needs to be optimized. Further studies with additional EVs in other cell line models, containing Dox, are warranted to clarify the targeting specificity of the engineered EVs.

Funding : Funding from the Icelandic Cancer Institute Science Foundation (Vísindasjóður Krabbameinsfélags Íslands), the National Breast Cancer Awareness Fund (Göngum saman) and the University of Iceland Research Fund is gratefully acknowledged.

Innovative AIE‐engineered extracellular vesicles to remodel the microenvironment for infected wound healing

Presenter: Hang Zou

Southern Medical University, China (People's Republic)

Introduction : Wounds with drug‐resistant bacterial infections and impaired healing pose significant threats to public health due to limited treatment options and severe clinical outcomes, including amputation and death. Extracellular vesicles (EVs) from tissues have emerged as novel cell‐free therapeutic strategies to address the dearth of interventions for wound healing and skin regeneration. Yet, they may not fully resolve issues stemming from drug‐resistant bacterial infections. Probes with aggregation‐induced emission (AIE), characterized by excellent optical stability and a range of properties, play a significant role in biomedical applications. Herein, AIE‐active photosensitizers, engineered for diverse ROS‐generating capabilities, have been synthesized. Furthermore, tissue‐derived EV/AIE hybrid nano‐vesicles (AEV) have been successfully created for the treatment of drug‐resistant bacterial infections in wound healing.

Methods : EVs were isolated from healthy mice tissue and analysed using proteomics and metabolomics. The AEV was characterized by NTA and TEM. The biocompatibility of the AEV was investigated by blood biochemical safety evaluation experiments and cell experiments. Fluorescence confocal microscopy was used to investigate the ability of the AEV to visualize bacteria. The antibacterial activity of the AEV was evaluated by its inhibition of bacterial growth both in vitro and in vivo. It was applied to a mouse model with drug‐resistant bacteria‐infected full‐thickness skin excisional wounds. Haematoxylin‐eosin, Masson's trichrome, immunofluorescence stainings were performed to explore the effect on skin repair.

Results : Transmission electron microscopy showed that AEV were round particles with a double‐layer membrane structure, and NTA showed that the size of AEV was about 120 nm. The AEV had a high binding affinity with the bacteria, and bright red fluorescence was observed on the surface of the bacteria and displayed distinctive photodynamic antibacterial effects under white light irradiation. In vitro and in vivo antibacterial experiments showed that it could effectively inhibit the growth of Gram‐positive bacteria. Histological results showed increased neovascularization, reduced wound scar, and more sebaceous gland and hair follicle formation, suggesting that the AEV could promote wound closure.

Summary/Conclusion : In summary, we successfully fabricated novel AIE‐engineered EV hybrid nano‐vesicles (AEV) to realize efficient antibacterial application and microenvironment remodelling in drug‐resistant bacterial infected wound healing.

Funding : National Natural Science Foundation of China, 82302647.

The neuroprotective effect of a complex multi‐strain probiotic secreted formulation in Alzheimer's disease

Presenter: Hsueh‐Te Lee

National Yang Ming Chiao Tung University, Taiwan (Republic of China)

Abstract unavailable

Subtopic Disease/therapy relevant mechanism/s of action 1

Plant‐derived vesicles as potential therapy for neuroendocrine prostate cancer

Presenter: Sharanjot Saini

Augusta University, USA

Introduction : Neuroendocrine prostate cancer (NEPC) is a highly aggressive lethal variant of prostate cancer (PCa) with extremely poor survival times (< 1 year). Clinical management of NEPC is currently challenging, as there are limited treatment options for these patients. These patients are treated with platinum‐based chemotherapy that is associated with toxicity and relapses. This emphasizes the need for newer, more effective therapeutic interventions against this challenging disease. The overall objective of this study was to develop an effective, non‐toxic, cheap yet scalable and non‐invasive extracellular vesicle (EV)/nanovesicle‐based therapeutic intervention against NEPC.

Methods : We isolated intact nanovesicles from pomegranate juice, referred to as POM‐EVs, using a combination of ultracentrifugation and filtration. Vesicles were characterized by Nanosight Tracking Analyses and Electron Microscopy. Further, we examined the therapeutic potential of POM‐EVs in a panel of prostate cell lines in vitro. To understand the mechanistic basis of POM‐EV‐induced alterations in NCI‐H660 cells, we performed next generation RNA sequencing in control vs POM‐EV treated NCI‐H660 cells on the Illumina HiSeq platform. Next, we sought to determine the in vivo effects of POM‐EVs on NEPC using the PDX model LuCaP145.1. We established LuCaP 145.1 tumours followed by systemic administration of POM‐EVs.

Results : POM‐EVs led to a significant reduction in viability of NEPC cell lines, NCI‐H660 and LASCPC‐01, as assessed by MTT assay after 7 days of treatment. We further performed an apoptosis assay by Annexin‐V‐FITC/7‐AAD staining of control/POM‐EV treated NCI‐H660 cells and LASCPC‐01 cells. Our analyses showed that POM EV treatment led to a significant increase in early apoptotic and late apoptotic populations. POM‐EV treated mice showed significant regression of LuCaP145.1 tumours. Sequencing studies identified that genes involved in ECM‐receptor interaction, focal adhesion, proteoglycans in cancer and small cell lung cancer genes were significantly impacted by treatment with POM‐EVs. Remarkably, POM‐EVs led to significantly attenuated levels of key proneural/neuronal transcription factors, including ASCL1, BRN2 and SOX2, EZH2, MYCN and AURKA, key genes involved in neuronal trans‐differentiation.

Summary/Conclusion : We propose that systemic administration of POM‐EVs can be employed as a novel treatment modality in neuroendocrine prostate cancer.

Funding : Augusta University Startup Funds

microRNA boosted EVs for cardiovascular disease

Presenter: Eunji Chung

University of Southern California, USA

Introduction : Vascular smooth muscle cells (VSMCs) change phenotype during atherosclerosis. Although contractile VSMCs express calcification inhibitors, VSMCs during atherosclerosis turn osteochondrogenic and lead to calcifications that cause plaque rupture. We harnessed and engineered healthy VSMCs that can secrete therapeutic extracellular vesicles (EVs) and hypothesized that EVs can act as endogenous nanoparticle drug delivery carriers in atherosclerosis. To test our hypothesis, we transduced VSMCs with plasmids containing miR‐133 and miR‐145 with ExoMotifs, which enable high loading of microRNA into EVs. Then, we isolated and decorated VSMC‐EVs with targeting peptides and evaluated their therapeutic properties in vitro and in vivo.

Methods : EV size and concentration were determined using NTA and TEM. Exosomal markers (CD9, CD63, and TSG101) were characterized via western blot, and osteogenic function was evaluated. miR‐133 and miR‐145 content was determined by qRT‐PCR. After EVs were modified with DSPE‐PEG (2000)‐HABP by sonication, DIO‐labelled HABP‐EVs, mouse and human VSMC‐EVs, and HeLa‐EVs were incubated with HA particles, and binding was measured. Calcified VSMCs were evaluated for osteochondrogenic markers, calcium production, and biocompatibility of EVs assessed via MTS assays. In vivo, ApoE knockout mice were administered HABP‐EVs, and atheroprotection was evaluated by serum cholesterol levels, plaque size, and histology.

Results : EVs from healthy VSMCs were found to be 100–200 nm in diameter and enriched for exosomal markers. Atheroprotective cargo, including MGP and miR‐133/145, was found to be enriched in healthy EVs and further significantly increased when EVs were isolated from cells transduced with miR‐133/145 ExoMotifs. Importantly, modification of EVs with DSPE‐PEG(2000)‐HABP did not result in a loss of cargo. HABP‐EVs showed statistically significant binding to HA particles and calcified VSMCs compared to unmodified VSMC‐EVs without altering biocompatibility, and biocompatibility and enhanced targeting were also found in advanced atherogenic mice in vivo. HABP‐EVs showed statistically significant upregulation of myocardin compared to PBS controls, showing therapeutic potential, as well as plaque inhibition upon assessment in vivo.

Summary/Conclusion : Our studies show promise of engineering VSMCs with therapeutic microRNA for therapeutic purposes.

Funding : The authors would like to acknowledge the financial support from the NSF GRFP granted to NP and the AHA Transformational Project Award and Ming Hsieh Research Grant awarded to EJC.

Hypoxia‐driven CAIX: a potential target to reduce extracellular vesicle release in cancer

Presenter: Rawan Almasri

Trinity College, Dublin, Ireland

Introduction : Neratinib benefits many patients with HER2+ breast cancer, but innate or acquired resistance to such drugs undermines their benefit. Our group has previously discovered that expression of hypoxia‐inducible carbonic anhydrase 9 (CAIX), which is associated with poor outcome in breast cancer, is increased in neratinib‐resistant compared to neratinib‐sensitive HER2+ cells. Separately, in prostate cancer, our group also made the original discovery that extracellular vesicles (EVs) can transmit drug‐resistance to formerly drug‐sensitive cells. Thus, we aimed to evaluate (i) if EVs carry CAIX reflecting their cells of original and (ii) if blocking CAIX can block EVs release.

Methods : Neratinib‐sensitive (HCC1954, SKBR3, EFM192A) and neratinib‐resistant (HCC1954‐NR, SKBR3‐NR, EFM192A‐NR) cells, established in our Laboratory, were cultured in medium containing EV‐depleted FBS under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. EVs were collected from their conditioned media using a differential ultracentrifugation in a 70Ti fixed‐angle rotor at 120,000 × g at 4°C. EVs were fundamentally characterised using immunoblotting, NTA, and TEM. Cellular and EVs CAIX was quantified by ELISA. Any effect of the CAIX inhibition by S4 on EVs (CD81+ and CD63+) release was evaluated via Amnis image stream flow cytometry.

Results : Hypoxic compared to normoxic conditions significantly (1.5–4.6‐fold; p = 0.01–0.0001) increased the expression of CAIX in all cell line variants, as expected. Induction of CAIX was not substantially different whether the cells were neratinib‐resistant or neratinib‐sensitive. Interestingly, CAIX was detected as cargo of the corresponding EVs, significantly increased ( p = 0.03–0.004) under hypoxia versus normoxia. Under hypoxia, S4 treatment led to a significant reduction of EVs (CD81+ and CD63+) release ( p = 0.01–0.001). Treatment with S4 also reduced CAIX expression in both cell lysates and the cargo of their corresponding EVs ( p = 0.01–0.001).

Summary/Conclusion : The CAIX cargo of EVs may have potential as a biomarker reflecting the hypoxic environment of a tumour. S4 blocking of CAIX and its reduction of CD81+ and CD63+ EVs release presents a possible way of reducing EVs release in cancer and thus their unwanted influences on recipient cells.

Funding : This research was supported by the Research Council Advanced Laureate Award EVIC to LOD [IRCLA/2019/49].

Subtopic Immunotherapy 1

Advantages and disadvantages of siRNA‐carrier using autologous serum‐derived small EVs compared with other types of nanoparticles

Presenter: Mai Hazekawa

Fukuoka University, Japan

Introduction : The final goal of our study is to develop an siRNA therapeutic system for the cancer metastases using autologous serum‐derived small EVs as a cargo. Recently, some kinds of nanoparticles have been featured as a useful tool for siRNA or mRNA delivery. However, the differences between them have not been clarified. The purpose of this study is to show the advantages and disadvantages of small EVs derived from serum compared with other types of nanoparticles, for example, lipid nanoparticles and micelles.

Methods : The small EVs isolated from mouse serum were used. To identify it, the expression of surface markers (CD9, CD63, CD81) was confirmed by Western blotting. Lipid nanoparticles and polymeric micelles were used as the comparison groups. Particle size and surface charge were evaluated using a DLS (dynamic light scattering) instrument. For cancer cells, the cellular uptake, uptake rate, and uptake mechanism of each formulation were evaluated using flow cytometry and confocal fluorescence microscopy. For the therapeutic efficacy assessment of siRNA, a WST‐8 assay was performed.

Results : The particles of small EVs obtained in this study were approximately 100 nm in size and had a neutral charge. For cellular uptake, all formulations showed an improvement in uptake efficiency, with an intracellular introduction rate of 100%. However, differences were observed in the intensity and speed of cellular uptake. Particles composed of lipid membranes, including small EVs, showed earlier cellular uptake, whereas micelles demonstrated a slower uptake. Lipid nanoparticles showed the highest intensity of cellular uptake, but no correlation was observed between therapeutic efficacy and uptake intensity. Additionally, while micelles were more heavily influenced by endocytosis, small EVs were found to utilize various uptake pathways. All formulations exhibited siRNA efficacy, demonstrating enhanced therapeutic effects compared to siRNA alone.

Summary/Conclusion : This study clarified the usefulness of serum‐derived small EVs as siRNA carriers and their advantages over conventional technologies. While all particles possess utility as drug delivery systems, differences in uptake speed, anticipated half‐life, and uptake pathways suggest the importance of accurately understanding each advantage and limitation. By optimizing these carriers based on the target disease, it is anticipated that ideal nucleic acid therapeutics can be realized.

Specific lung tumour targeting by lung cancer small extracellular vesicles (sEVs)‐based antibody

Seonghyun Ryu, Maryam Firouzi, Dongin Kim

University of Oklahoma Health Sciences Center, USA

Introduction : Targeted drug delivery to non‐small cell lung cancer cells (NSCLC) is challenging due to the paucity of specific targeting ligands. Existing ligands directed to known targets have limits. For example, Arginine‐Glycine‐Aspartate (RGD) binds αVβ3 integrins, which are overly expressed in NSCLC. However, RGD lacks specificity because healthy cells also express αVβ3 integrins. Also, not many targeting receptors of NSCLC are available for targeted therapy. Thus, this study established a new targeting strategy for generating a specific targeting ligand for HCC2814, the NSCLC line. It uses NSCLC‐derived small extracellular vesicles (sEVs) based on their similar membrane structures to their mother cells.

Methods : HCC2814, human lung squamous cell carcinoma, was cultured, and sEVs were harvested. sEVs were characterized with nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). sEVs are intraperitoneally injected into female BALB/c mice. sEV injection (10 10 sEVs per injection) was done 3 times with 2 weeks between each injection. Mouse blood serum was tested with immunofluorescence (IF) on HCC2814 and healthy lung epithelial cells to see antibody specificity to HCC2814. Once the IF result showed specificity, B cells were harvested from the spleen of mice. B cells are then fused with myeloma cells (SP2/0‐Ag14) to form hybridomas for antibody production. Antibodies were purified with a protein G‐HP purification column and were conjugated into Anchor‐Spacer‐Ligand (Anchor‐BODIPY, Spacer‐PEG, Ligand‐antibody: ASL). CD8+ T cell‐derived sEvs were then decorated with ASL and were tested in vivo for tumour‐targeting efficacy. Infrared dyes were encapsulated in sEVs to locate sEVs with IVIS imaging.

Results : The IF study on HCC2814 confirmed the presence of antibodies in mice serum after two vaccinations. After the formation of hybridoma, antibody production in the culture media was confirmed with IF. After a single cell culture, monoclonal antibody production was confirmed with IF. IVIS imaging showed a significantly high localization of sEVs in tumours for sEVs decorated with ASLs. In contrast, non‐targeting sEVs showed high localization in other organs, such as the liver.

Summary/Conclusion : This research suggests that sEVs can be used to develop antibodies that can be used as a targeting ligand for NSCLC. sEVs have the potential to be used for personalized targeting chemotherapy.

Hydrogel‐encapsulated exosome vaccine as a novel immunotherapeutic approach and its role in enhancing immunotherapy for prostate cancer

Presenter: Qi Chen

Shanghai Jiao Tong University, China (People's Republic)

Introduction : Due to the low immune cell infiltration and the suppressive tumour immune microenvironment (TIME), the efficacy of immunotherapy for prostate cancer is currently quite limited. This study aims to design a cancer vaccine using supramolecular peptide hydrogel encapsulated with ex vivo tumour tissue‐derived exosomes (TEXs) and adjuvants to inhibit the growth and recurrence of prostate cancer, as well as improve the effect of immune checkpoint inhibitors.

Methods : Here, we design an amphiphilic self‐assembling peptide, FK12, capable of forming hydrogels in situ after subcutaneous injection. TEXs are extracted through the lysis and ultracentrifugation of tumour tissue. The positively charged self‐assembler FK12F can adsorb negatively charged TEXs, CDA (a STING agonist), and GM‐CSF through electrostatic interactions to create the Gel‐Vaccine system. We characterize the controlled release of the vaccine components through the gel by in vitro and in vivo experiments. We then assess the therapeutic effects and related molecular mechanisms of gel vaccines in different mouse models of prostate cancer.

Results : Significant tumour regression was observed in mice vaccinated with the gel vaccine across multiple mouse models of prostate cancer. This effect was even more pronounced in mice treated with the combination of aPD‐1. We detected a higher number of DC aggregates at the site of gel vaccination, with an increased presence of mature and migratory DCs. Furthermore, we observed an elevated count of mature DCs, along with an increased presence of CD4+ and CD8+ T lymphocytes in the lymph nodes. Additionally, a greater number of CD8+ T cells with cytotoxic capabilities and fewer regulatory T cell infiltrates were detected within the tumour tissue.

Summary/Conclusion : TEXs are enriched with tumour antigens, making them potential stimulators of anti‐tumour immune responses. After subcutaneous inoculation, the exosome‐gel scaffold can be triggered in situ to form an artificial secondary lymphoid organ, continuously recruiting antigen‐presenting cells through cytokines and achieving highly efficient antigen processing induced by adjuvants. This orchestrated process induces immune infiltration in the TIME. When combined with aPD‐1 immunotherapy, it triggers a potent and long‐lasting tumour‐specific immune response, effectively inhibiting prostate cancer growth and recurrence.

SMAC mimetics and type I interferon induce tumour cell death and boost immune response through small extracellular vesicles

Rostyslav Horbay 1,2,3 , Daniel Panting 1,2,3,4 , Mikaela van der Merwe 1,2,3,4 , Jordan Yin 1,2,3,4 , Martine Saint‐Jean 1,2,3 , Eric LaCasse 1,2,3 , Robert Korneluk 1,2,3,4 , Dylan Burger 5,6 , Shawn Beug 1,2,3,4

1 Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute 401 Smyth Rd, Ottawa, ON, K1H 8L1, Canada; 2 Centre for Infection, Immunity and Inflammation, University of Ottawa, 451 Smyth Rd, Ottawa, ON, K1H 8L1, Canada; 3 Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, 451 Smyth Rd, ON, K1H 8L1, Canada; 4 Department of Biochemistry, Microbiology and Immunology, University of Ottawa; 451 Smyth Rd, Ottawa, ON, K1H 8L1, Canada; 5 Kidney Research Centre, The Ottawa Hospital Research Institute and University of Ottawa, 451 Smyth Rd, Ottawa, ON, K1H 8L1, Canada; 6 University of Ottawa, Department of Cellular and Molecular Medicine and School of Pharmaceutical Sciences, 451 Smyth Rd, Ottawa, ON, K1H 8L1, Canada

Introduction : A class of drugs called SMAC mimetics (SMCs) has been developed to antagonise members of the Inhibitor of Apoptosis protein family. SMCs synergize with immune ligands and immunostimulatory agents to eradicate tumour cells (TCs). We recently discovered that type I IFNs potentiate the ability of SMCs to induce cell death. This synergism is mediated through the SMC‐induced release of 30–150 nm small extracellular vesicles (sEVs) from TCs and immune cells (ICs). We aim to determine how SMCs can eradicate TCs via sEVs in cooperation with ICs, particularly macrophages.

Methods : sEVs were isolated by differential ultracentrifugation and characterized per the latest ISEV recommendations via Zetaview NTA, ONI Nanoimager, and Western blotting. We used cell culture approaches to decipher the impact of cytokine and SMC‐induced sEVs on TCs and ICs, including viability assays, direct and transwell co‐culture conditions, ELISA, and flow cytometry.

Results : Treatment of macrophages with SMCs increased the rate of sEV release by ∼5‐fold ( t ‐test, p < 0.05, n = 7), but did not significantly alter sEV secretion rate in TCs. Macrophage‐ and TC‐derived sEVs were positive for Flotillin‐1 and HSP70. SMC treatment on macrophages led to packaging of proinflammatory cytokine into sEVs, particularly TNFα, in an ESCRT‐dependent manner as reflected by the presence of Syntenin and Alix on sEVs via Western blotting. sEVs from SMC treated TCs polarized macrophages into the pro‐inflammatory M1 subset (from a baseline level of ∼4% to ∼15%, n = 2). TCs released Syntenin‐positive sEVs only under cell death conditions. The treatment of macrophages with type I IFNs led to greater encapsulation of TNFα within these sEVs. The resultant TNFα‐bearing macrophage‐derived sEVs induced the death of TCs in the presence of an SMC (ANOVA, n = 4, p < 0.0001). Lastly, we observed packaging of RIG‐I within tumour‐derived sEVs from SMC‐treated TCs, indicating that such sEVs could stimulate anti‐tumour cell killing by cytotoxic ICs.

Summary/Conclusion : SMCs are able to kill TCs through an sEV‐dependent process. These results provide critical insights into how SMCs are able to kill TCs and thereby boost immune responses.

Funding : CIHR (#PJT‐169126) and CRS (#935202)

Engineered bacterial outer membrane vesicles enhance immunotherapy efficacy by remodelling the immune microenvironment through bidirectional ferroptosis induction

Xinxing Du 1 , Huan Chen 2 , Xinrui Wu 1 , Yanhao Dong 1 , Cong Hu 1 , Zehong Peng 1 , Zhicheng Zheng 1 , Jiahua Pan 1 , Jinrao Liu 2 , Liang Dong 1 , Wei Xue 1

Jinrao Liu, Liang Dong, and Wei Xue are co‐senior authors.

1 Department of Urology, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 Institute of Molecular Medicine, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

Introduction : Immunotherapy has demonstrated promising results across various cancers, yet its effectiveness remains limited in immune ‘cold’ tumours such as prostate cancer, characterized by minimal immune‐cell infiltration and reduced antitumour activity within the tumour microenvironment (TME). This study aims to address this limitation by developing iron sulphide‐modified bacterial outer membrane vesicles (OMVs) that respond to the acidic TME of prostate cancer to enhance immune activation and antitumour responses.

Methods : We engineered and characterized iron sulphide‐modified OMVs, confirming successful modification and assessing their response in acidic conditions. The release of ferrous ions (Fe 2 ⁺) and hydrogen sulphide (H 2 S) was measured to evaluate their ability to induce ferroptosis in tumour cells. We further examined whether ferroptosis induced by these OMVs could enhance antitumour immunity. High‐throughput sequencing was used to investigate the specific mechanisms by which ferroptosis modulates the TME. Finally, we tested the therapeutic effects of combining these engineered OMVs with immunotherapy across multiple tumour models, including organoid and metastatic prostate cancer models, to assess tumour inhibition and immune activation.

Results : In acidic conditions, the OMVs released Fe 2 ⁺ and H 2 S, which synergistically promoted enhanced ferroptosis in tumour cells by inducing lipid peroxidation and suppressing antioxidative defences. Lipopolysaccharides on the OMV surface further promoted immune‐cell recruitment within the TME via IL‐1β‐mediated activation of the non‐canonical caspase‐11 pathway, leading to increased CD8+ T‐cell infiltration and antitumour activity. High‐throughput sequencing provided insights into the mechanisms by which ferroptosis reshaped the TME to foster immune activation. In various prostate cancer models, the combination of OMVs with immunotherapy significantly enhanced tumour suppression and immune activation.

Summary/Conclusion : Iron sulphide‐modified OMVs provide an effective strategy for enhancing immunotherapy by inducing ferroptosis and remodelling the immune microenvironment in prostate cancer. These findings highlight a novel approach for overcoming the limitations of immune ‘cold’ tumours and suggest potential applications for other cancer types with similarly suppressed immune responses.

Funding : This research was funded by the National Natural Science Foundation of China (82227801, 82472142).

Engineering bispecific extracellular vesicles for cancer immunotherapy

Yu Xuan Gee, Phuong H. D. Nguyen, Minh T. N. Le

National University of Singapore, Singapore

Introduction : Immunotherapy has significantly improved the survival rate of patients with advanced‐stage cancer, but challenges including immune‐related adverse events and low treatment response rates persist. Previous studies on EV‐based drug delivery systems have shown potential to enhance anti‐tumour immunity through multifaceted approaches. However, these strategies often rely on EVs generated from immortal cell lines, which carry potential risks of gene transfer and tumourigenesis, as well as challenges in large‐scale production. Here, we develop a novel drug delivery system using red blood cell‐derived extracellular vesicles (RBCEVs) surface modified for targeted immunomodulation and recruitment of immune cells to the tumour sites. Our system possesses low immunogenicity and tumourigenicity while significantly enhancing the efficacy of immunotherapies at lower dosages.

Methods : We employed click chemistry tools to surface functionalize RBCEVs with immunomodulatory antibodies (anti‐CD3 and anti‐PD‐L1) to generate bispecific RBCEVs (BEVs) for the treatment of lung cancer. We monitored tumour progression using the Lumina II in vivo imaging system (IVIS). To elucidate the mechanism of action of BEVs, we performed immune profiling by immunohistochemistry and flow cytometry, as well as assessed the safety profile of the system.

Results : We demonstrated the capability of BEVs to activate and recruit T cells to the tumour sites in mouse models of lung cancer as evidenced by upregulations of activation markers and the reduced tumour burden. Notably, BEVs showed an enhanced immunostimulatory effect on T cells compared to the free form of antibodies, highlighting their potential in enhancing the efficiency of immunotherapy at a lower dosage, thereby reducing the associated toxicities. Overall, BEV treatment improved the overall survival rate of the tumour‐bearing mice without inducing significant toxicity or inflammatory responses throughout the treatment regimen.

Summary/Conclusion : We have developed an efficient and versatile therapeutic platform in which surface functionalized RBCEVs serve as a T cell engager for the treatment of cancer. With the capacity of specific targeting and recruitment of immune cells to the tumours, our platform can be developed into personalized medicine to achieve optimal therapeutic efficacy with fewer side effects. Moreover, our technology does not require cell culture or genetic modifications, thereby offering a practical EV‐based immunomodulatory platform that can be clinically translated.

Multi‐omic profiling of circulating extracellular vesicles to identify response signatures and resistance mechanisms in lung cancer patients treated with immune checkpoint inhibitors

Presenter: Catherine R. Taylor

Atlantic Cancer Research Institute, Canada

Introduction : Immune‐oncology treatment has revolutionized cancer treatment, but not all patients benefit due to complexities in the interplay between cancer and the immune system. Extracellular vesicles (EVs) are particles secreted by both tumour and immune cells that reflect the state of the cell of origin and participate in the interplay between cancer and immune cells. Our study used RNA sequencing and proteomic profiling of circulating EVs isolated from the plasma of NSCLC patients treated with anti‐PD1 therapy to identify novel biomarkers of response and EV‐associated immune evasion mechanisms. Since the cargo of circulating EVs reflects the state of the cell of origin, we hope to identify predictive biomarkers of response to anti‐PD1 therapy as well as targetable pathways of treatment resistance.

Methods : Plasma from NSCLC patients treated with anti‐PD1 therapy was obtained with informed consent. EVs were isolated using peptide‐affinity purification with Vn96 peptide to precipitate EVs from plasma for RNA extraction or protein lysate. RNA was sequenced using the ComboSeq mRNA/miRNA kit on a NovaSeq 6000. Proximal Extension Assay (Olink Inc.) was used to profile ∼3000 proteins in the EV protein lysate. The Bioconductor package edgeR and Olink Insight were used to identify differentially expressed RNAs and proteins between responders and non‐responders, respectively. Cox regression analysis was performed in the R environment to identify RNAs and EV proteins that correlate to progression‐free survival (PFS).

Results : EV RNA analysis revealed enrichment of neutrophil recruitment, VEGF signalling, and CXC chemokine receptor signalling associated with poor survival. Proteomics analysis identified EV proteins associated with shorter PFS, including TRAIL‐R2 and VMO1, and pathway analysis revealed enrichment of VEGF signalling, chemotaxis, and macrophage polarization associated with poor progression‐free survival.

Summary/Conclusion : Profiling circulating EVs has unveiled EV proteins and de‐regulated pathways that may improve our understanding of how EVs participate in immune evasion during immunotherapy treatment and may point to new treatment modalities that could improve responses to immune checkpoint blockade in lung cancer patients. Furthermore, the data suggests that circulating EVs provide RNA and protein signatures that reflect the tumour immune microenvironment and are predictive of anti‐PD1 response.

Funding : Canadian Cancer Society J. D. Iriving Ltd Excellence in Cancer Research Fund

Bdellovibrio bacteriovorus‐derived extracellular vesicles enhance the therapeutic effect of immune checkpoint inhibitor

Kentaro Jingushi, Atsunari Kawashima, Takuro Saito, Akinaru Yamamoto, Toshihiro Uemura, Sassi Nesrine, Yu Ishizuya, Yoshiyuki Yamamoto, Hiroaki Hase, Norio Nonomura, Kazutake Tsujikawa

University of Osaka, Japan

Introduction : The gut microbiota characteristic of ICI (immune checkpoint inhibitor) responders has been reported, but how it affects intratumour immune activity in distant tumours is unknown. We have found multiple BEV (bacterial EV) common in the blood of ICI responders of urologic cancer patients and hypothesized that BEV may act as a link between the gut‐tumour axis. In this study, we tested whether the BEV identified in the blood of ICI responders could enhance the therapeutic effect of ICI treatment.

Methods : BEVs isolation—Bdellovibrio bacteriovorus Stolp and Starr were grown predatorily on stationary‐phase E. coli prey (16 h, 29°C). Cultured media were centrifuged at 2000 × g for 30 min and filtered with a 0.2 um syringe filter. The filtered supernatant was centrifuged at 100,000 × g for 90 min to obtain BEVs pellets (Bdello EV). BEVs were subjected to nanoparticle analysis, western blot, and TEM. In vivo experiments: All animal experiments were approved by the Animal Experimentation Committee of the Graduate School of Pharmaceutical Science at Osaka University. Mice allografted with the murine renal carcinoma cell line RenCa received BEV intraperitoneally daily for 2 weeks (1 × 10 9 EV/mouse). In addition, PD‐1 antibody alone or in combination with PD‐1 antibody and BEV was administered along with antibiotics in drinking water.

Results : Administration of the Bdello EV to a renal cancer allograft mouse model resulted in antitumour effects. We also found that administration of Bdello EV markedly increased the activity of intratumoural T cells. Furthermore, antibiotic administration decreased gut bacteria and suppressed the antitumour effect of the PD‐1 antibody, whereas concomitant administration of Bdello EVs resulted in antitumour effect of mouse PD‐1 antibody.

Summary/Conclusion : Bdello EV found in ICI responder blood had the potential to enhance the therapeutic effect of ICI. And bacterial EVs may play a role in the gut‐tumour axis.

CAR‐T cell‐derived extracellular vesicles express high levels of CAR and carry pro‐apoptotic molecules, mimicking CAR‐T cell anti‐tumour activity

Paola Lanuti 1,2 , Francesco Guardalupi 1,2 , Giulia Corradi 1,2 , Rosalba Florio 3 , Davide Brocco 4 , Serena Veschi 3 , Domenico De Bellis 1,2 , Francesca D'Ascanio 1,2 , Anna Piro 3 , Laura De Lellis 3 , Pasquale Simeone 1,2 , Maria Concetta Cufaro 2,5 , Serena Pilato 3 , Isabella D'Amario 1,2 , Fabio Verginelli 3 , Damiana Pieragostino 2,5 , Antonella Fontana 3 , Piero Del Boccio 2,3 , Alessandro Cama 3 , Mauro Di Ianni 1,2

1 Department of Medicine and Aging Sciences, Italy; 2 Center for Advanced Studies and Technology (CAST), Italy; 3 Department of Pharmacy, Italy; 4 Department of Medical, Oral and Biotechnological Sciences, Italy; 5 Department of Innovative Technologies in Medicine and Dentistry, Italy

Introduction : CAR‐T cell therapy has revolutionized cancer treatment of haematological malignancies. However, many challenges remain to be addressed, including their severe adverse effects. Extracellular vesicles (EVs) have been pointed out as new therapeutic agents for their advantages in terms of safety. Given that CAR‐T cell‐derived EVs possibly mimic the anti‐tumour activity of CAR T cells, here we studied phenotypes and functions of CD19.CAR‐T cell‐derived EVs with the final aim to investigate their therapeutic potential.

Methods : EVs derived from CD19.CAR‐T cells in pre‐infusion bags ( n = 3) or circulating CD19.CAR‐T cells ( n = 3) from patients infused with CAR‐T cells were separated by cell sorting and co‐cultured with two different CD19+ cell lines (Raji and SUP‐B15), using EVs from untransduced T cells from the same donors as controls. The antitumour activity of B7H3 CAR‐T‐derived EVs was evaluated by both MTT and killing assays, using 7‐AAD incorporation. CD19.CAR‐T EV morphological (nanoparticle tracking analysis, NTA, transmission electron microscopy, TEM, and atomic force microscopy, AFM) and phenotypical analyses (flow cytometry and proteomics) were carried out.

Results : CD19.CAR‐T EVs characterization revealed that EVs displayed a globular shape and an average size of 133.1 ± 65.5 nm. CD19.CAR‐T EVs expressed EV markers (CD63 and flotillin‐1), and typical EV contaminants (i.e., cytochrome C and apolipoproteins) were undetectable. CD19.CAR‐T EVs expressed higher CAR levels than their parental cells and carried a pro‐apoptotic protein cargo possibly associated with their killing abilities. Furthermore, CD19.CAR‐T cell‐derived EVs significantly affected cell viability of CD19+ cell targets in a dose‐dependent manner. Using a dose of 0.015 µg EV proteins/target cell of blood circulating CD19.CAR‐T EVs, a killing of 20.3% (±3.8) and 37.8% (±7.0) was reached when Raji and SUP‐B15 were used as targets, respectively, while the killing percentage induced by pre‐infusion bag CD19.CAR‐T EVs, was 42.0% (±12.3) and 35.8% (±13.8) for Raji and SUP‐B15, respectively.

Summary/Conclusion : Overall, our data indicate that CD19.CAR‐T cell‐derived EVs may be proposed as a new CAR‐T cell product. Contributing to the direct killing of the leukaemic target, they may represent a better tolerated therapy tool that could be infused independently from CAR‐T cells.

Funding : PNC‐E3‐2022‐23683269.

Killer EVs: enhancing ex vivo production of cytotoxic extracellular vesicles from cord blood natural killer cells for effective cancer immunotherapy

Isabel Doutor 1,2 , Cristiana Ulpiano 1,2 , Gabriel Costa 1,2 , Cláudia Lobato da Silva 1,2 , Ana Azevedo 1,2 , Ana Fernandes‐Platzgummer 1,2

1 Department of Bioengineering and iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Portugal; 2 Associate Laboratory i4HB—Institute for Health and Bioeconomy, Portugal.

Introduction : Umbilical cord blood (CB)‐derived natural killer (NK) cell immunotherapy offers significant potential against several malignancies, including blood cancers. This potential arises from the cytotoxic nature of NK cells, coupled with the allogeneic properties, low immunogenicity, and easy accessibility of CB. Another promising immunotherapy approach involves using extracellular vesicles (EVs), naturally secreted by different cell types, including NK cells, as a safer cell‐free alternative. However, the persistent challenge lies in obtaining the therapeutic quantities needed of functional NK(CB) cells and their derived EVs.

Methods : To address this, CD56+ NK cells from CB samples underwent expansion in various interleukin‐2‐supplemented culture media, namely X‐VIVO 15, NK MACS (NKM) with 5% human serum (hS), CTS NK‐Xpander (NKX) with 5% hS, StemSpan SFEM II, PRIME‐XV NK Cell CDM, and GMP SCGM. NK(CB) cells were characterized by flow cytometry, and their activation was assessed through degranulation and cytotoxicity assays against the K562 cell line. A conditioning phase followed, utilizing NKX without hS or supplemented with exosome depleted human platelet lysate (hPL‐EVd). The culture medium was renewed every 24 or 48 h to evaluate the impact on cell viability and EV production. EVs were isolated by anion‐exchange chromatography, characterized according to MISEV2023 guidelines and tested in cytotoxicity assays.

Results : NKM and NKX were the most favourable culture media for NK cell expansion, each maintaining a CD3‐CD56+ NK cell percentage above 90%. Enhanced NK cell activation was observed, demonstrated by heightened degranulation and cytotoxicity. Notably, hPL‐EVd‐supplemented NKX led to increased particle production during conditioning, while maintaining consistent cell viability over 96 h. No significant difference was observed between the two conditioning schedules tested. The optimized downstream process showed an enrichment of particles smaller than 200 nm with adequate morphology. Key proteins required for cytotoxic activity against cancer cells, such as perforin and granzyme B, were confirmed, along with the cytotoxic effects of NK(CB)‐EVs on K562 cells.

Summary/Conclusion : These results are crucial for the optimal production of NK(CB) cells and NK(CB)‐EVs, laying the foundation for robust manufacturing processes. This study is the first to produce highly cytotoxic EVs from readily available NK(CB) cells for use in immunotherapy.

Anti‐cancer effect of extracellular vesicles‐derived from IL‐21 activated NK‐92 cells against breast cancer cell lines

Nil Kılıç 1 , Eylül Gülşen Yılmaz 1,2 , Fatih Inci 1,2

1 UNAM‐National Nanotechnology Research Center, Bilkent University, Ankara, Turkey; 2 Institute of Materials Science and Nanotechnology, Bilkent University Ankara, Turkey

Introduction : Natural killer (NK) cells and their anti‐cancer effects have been the focus of many researchers in cancer immunotherapy. Extracellular vesicles (EVs) play significant roles in functions such as cell differentiation, and cell communication by regulating gene expression in recipient cells. Research on cancer treatment using EVs has also become one of the focal points of today. In this study, the anti‐cancer effect of EVs from IL‐21‐activated and non‐activated NK‐92 cells on breast cancer cells was investigated.

Methods : After activation with IL‐21 cytokine, NK‐EVs isolation was performed using microfluidic chip‐based methods. The characterisation of EVs was conducted using three analytical techniques: nanoparticle tracking analysis (NTA), western blot and SEM imaging. The most effective concentration and time for breast cancer cells incubated with NK‐EVs were identified using a WST‐1 cytotoxicity assay. The expression of apoptotic genes BAX, BCL‐2, CASPASE3, CASPASE8 and CASPASE9 in breast cancer cells was analysed using qRT‐PCR. Additionally, the synthesis of proteins associated with apoptosis (BAX, CASPASE3, CASPASE8 and CASPASE9) in breast cancer cells was examined using western blot.

Results : EVs characterization was conducted on both IL‐21‐activated and non‐activated NK‐92 cell lines. It was established that the concentration of EVs was higher in the group of cells that had been activated. The results showed that EVs from IL‐21‐activated NK‐92 cells significantly increased the apoptotic effect compared to EVs from IL‐21 non‐activated cells. The expression and synthesis of BAX, BCL‐2, CASPASE3, CASPASE8 and CASPASE9 apoptotic genes and proteins were observed to be elevated in comparison to the control group that had not been stimulated by IL‐21.

Summary/Conclusion : EVs obtained from the activated NK cell group showed more cytotoxic effect. In the future, miRNA profiles of NK‐EVs obtained from two different groups will be analysed. Furthermore, miRNA profiles of NK‐derived EVs with apoptotic effect against breast cancer cells will be revealed.

Funding : This study is supported by the 2218 National Postdoctoral Research Fellowship Program from The Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project No: 123C139).

Anticancer activity of extracellular vesicles from natural killer cells cultured under different conditions against human oral squamous cell carcinoma

Anyapat Atipimonpat 1 , Kittiphum Meekarn 1 , Natthawut Sombunma 1 , Anucha Sacharoen 2 , Supitcha Pannengpetch 2 , Jarunya Ngamkham 3 , Aussara Panya 4 , Nitchawat Paiyabhroma 1 , Kovit Pattanapanyasat 2

1 Naresuan University, Thailand, 2 Mahidol University, Thailand, 3 National Cancer Institute, Thailand, 4 Chiang Mai University, Thailand

Introduction : Extracellular vesicles (EVs) are lipid membrane‐bound nanoparticles released from various cell types. They are attracting clinical interest as therapeutic tools for various diseases, including cancer. Previous studies have shown that EVs derived from natural killer cells (NK‐EVs) could inhibit the proliferation of various cancers. However, the inhibitory effect of NK‐EVs on oral squamous carcinoma cells (SCC25) remains underexplored. Recently, NK‐EVs were isolated from a variety of condition mediums. This led to the research question of under which conditions EVs obtained from NK cells have a potent inhibitory effect on oral cancer growth. This research aimed to study the effect of NK‐EVs obtained from four cell culture conditions on inhibiting SCC25 cell viability.

Methods : Natural killer cells (NK92) were cultured in four different conditions. EVs were isolated from the culture medium by differential centrifugation. The isolated EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot analysis. The entry of NK92‐EVs into SCC‐25 cells was investigated by a confocal microscope. Finally, the inhibitory effects of NK92‐EVs on SCC‐25 cell viability were studied.

Results : NK92‐EVs isolated from all four different culture mediums showed a similar morphology as a cup‐like shape. The size distribution of NK92‐EVs ranged from 73 to 681 nm, with the majority of EVs being between 100 and 200 nm in size. However, NK92 cells cultured in serum‐free media produced fewer EVs than the others. All isolated NK92‐EVs expressed EV protein markers, including CD81 and TSG101. NK92‐EVs also demonstrated the ability to enter the SCC‐25 cell lines. Interestingly, NK92‐EVs isolated from serum‐free cultured media showed significantly inhibited SCC‐25 cell viability.

Summary/Conclusion : This study suggested that cultured NK92 cells in serum‐free media could produce EVs with potent inhibitory effects on SCC25 cell proliferation. Contents of cytotoxic proteins of NK‐EVs isolated from these different culture media need to be further investigated.

Funding : The project is funded by the National Research Council of Thailand (NRCT): High‐Potential Research Team Grant Program Contract no. N42A650870 and partially supported by Global and Frontier Research University Fund, Naresuan University; Grant number R2566C053.

Subtopic EV in metabolic disease 1

Gut pathobiont‐derived outer membrane vesicles drive liver inflammation and fibrosis in PSC‐IBD

Presenter: Claudia Günther

UKER, Medizinische Klinik 1, Germany

Introduction : Primary sclerosing cholangitis (PSC), often associated with inflammatory bowel disease (IBD), presents a multifactorial aetiology involving genetic, immunologic, and environmental factors. Gut dysbiosis and bacterial translocation have been implicated in PSC‐IBD, yet the precise mechanisms underlying their pathogenesis remain elusive. Here, we describe the role of gut pathobionts in promoting liver inflammation and fibrosis due to the release of bacterial outer membrane vesicles (OMVs).

Methods : Preclinical mouse models, in addition to ductal organoids, were used to acquire mechanistic data. A proof‐of‐concept study including serum and liver biopsies of a patient cohort of PSC ( n = 22), PSC‐IBD ( n = 45), and control individuals ( n = 27) was performed to detect OMVs in the systemic circulation and liver.

Results : In both preclinical model systems and in patients with PSC‐IBD, the translocation of OMVs to the liver correlated with enhanced bacterial sensing and accumulation of the NLRP3 inflammasome. Using ductal organoids, we were able to precisely attribute the pro‐inflammatory and pro‐fibrogenic properties of OMVs to signalling pathways dependent on Toll‐like receptor 4 and NLRP3–gasdermin‐D. The immunostimulatory potential of OMVs could be confirmed in macrophages and hepatic stellate cells. Furthermore, when we administered gut pathobiont–derived OMVs to Mdr2 – / – mice, we observed a significant enhancement in liver inflammation and fibrosis. In a translational approach, we substantiated the presence of OMVs in the systemic circulation and hepatic regions of severe fibrosis using a PSC‐IBD patient cohort.

Summary/Conclusion : This study demonstrates the contribution of gut pathobionts in releasing OMVs that traverse the mucosal barrier and, thus, promote liver inflammation and fibrosis in PSC‐IBD. OMVs might represent a critical new environmental factor that interacts with other disease factors to cause inflammation and thus define potential new targets for fibrosis therapy.

Funding : This research has received funding from the Deutsche Forschungsgemeinschaft.

Dissect human islet‐derived extracellular vesicle heterogeneity for type 1 diabetes early diagnosis and pathogenesis

Presenter: Tongjian Zhao

Uppsala University, Sweden

Introduction : The pancreatic islets are composed of cells with distinct functions and are crucial to the pathogenesis of T1D. Islet cells can selectively and actively release EVs. However, due to the limited quantity of EVs that can be obtained from islets, currently it is only possible to perform bulk analysis on islet‐derived EVs rather than single‐EV analysis. The aim of the study is to use proximity barcoding assay (PBA) for single EV surface protein profiling to characterize the heterogeneity of human islet‐derived EVs.

Methods : EVs from cultured human islets with and without cytokines (IL‐1β and IFN‐γ for 24 or 48 h) or other relevant cells were isolated by a classic ultracentrifugation procedure. NTA, TEM, immunoblotting, PBA and nanoscale flow cytometry were applied for EV characterization and quantification.

Results : PBA detected distinct EV membrane profiles of EVs derived from human islets, the EndoC‐betaH1 cell line, islet stellate cells (ISC) and plasma, which highlights the possibility of using PBA to track EV origins. We observed a sub‐population of EVs that co‐expressed the combination of CD44, MUC1, and ICAM1 on the EV surface. The percentage of this population increased significantly (from 0.82% to 4.40%, n = 6, p = 0.01, paired t ‐test) after 24 h of cytokine exposure. The combination of single‐cell RNA‐seq data of islets and PBA EV data identified that CD44 and MUC1 were cell type‐specific EV markers of ductal cells. ICAM1 was a cytokine‐inducible EV marker. To further confirm this, we isolated EVs from the EndoC beta‐cell line and the ductal PANC1‐1 cell line and confirmed these markers by both PBA and nanoscale flow cytometry. Thus, this CD44, MUC1, and ICAM1 positive EV population was very likely secreted from cytokine‐stressed ductal cells. Changes of cytokine‐treated human islet EV membrane profiles were associated with faster uptake of EV by PBMC and increased secretion of pro‐inflammatory cytokines IL‐1beta, TNF alpha and IL‐6.

Summary/Conclusion : The study established a method to dissect the heterogeneity of human islet‐derived EVs through membrane protein profiling and highlighted the importance of EV membrane proteins on EV uptake and downstream immune response triggering in T1D. Future study is warranted to test the feasibility of using PBA for plasma EV analysis for T1D early diagnosis.

Funding : Swedish Children Diabetes Foundation

Skeletal muscle‐derived extracellular vesicles and their impact on glucose regulation in type 2 diabetes

Presenter: Hagit Shoyhet

Technion‐IIT, Israel

Introduction : Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by insulin resistance and impaired glucose homeostasis, resulting in systemic complications. In T2D, disrupted intercellular signalling exacerbates inflammation and metabolic dysregulation, driving disease progression. Current therapies primarily address symptom management rather than underlying cellular communication deficits. Recent studies suggest that extracellular vesicles (EVs) are instrumental in modulating these cellular interactions, as they carry molecular cargo that influences metabolic and inflammatory pathways. Tissue engineering, particularly of skeletal muscle, offers a promising approach to utilizing EVs for therapeutic purposes in T2D. Our previous work showed that engineered muscle tissue overexpressing GLUT4 positively impacts diabetic phenotypes in Diet‐Induced Obesity (DIO) mice, improving blood glucose levels, insulin response, and cytokine profiles. Here, we explore the role of EVs derived from engineered constructs in mediating these systemic effects.

Methods : EVs were isolated from the conditioned medium of 3D‐engineered skeletal muscle tissue overexpressing GLUT4 using an affinity column kit. The EVs were characterized via DLS, AFM and cryo‐TEM. EV functionality was tested using in vitro glucose uptake assay and in vivo experiments, including the glucose tolerance test and histology. We also analysed their protein and small RNA content via proteomics and MiR‐seq, respectively.

Results : In vitro assays indicate that EVs isolated from GLUT4 overexpressing 3D skeletal muscle tissue increase glucose uptake in WT myotubes compared to EVs isolated from WT 3D skeletal muscle tissue. In vivo experiments on diabetic mice indicated that the GLUT4 OE myotubes EVs improve response in the glucose tolerance test (GTT). MiR‐seq and proteomic analyses suggested a regulatory pattern affecting various metabolic pathways, such as IGF‐1 and insulin signalling through the delivery of IGF‐1 and downregulation of specific MiRs, additionally confirmed by in vitro molecular assays.

Summary/Conclusion : Myogenic EVs derived from GLUT4 OE skeletal muscle constructs can improve glucose metabolism of WT muscle in vitro and of DIO mice in vivo. The EVs' unique cargo can help explain their mechanism of action and the in vivo effect of a small implanted construct. Our work suggests that tissue engineering can be used to affect the host metabolic systems, reaching beyond the small‐sized implanted tissue.

Fourier‐transform infrared (FT‐IR) spectroscopy to show alterations in molecular composition of EVs subpopulations derived from beta‐pancreatic cells cultured in different D‐glucose condition

Martyna Durak‐Kozica 1,2 , Andrzej Wróbel 1,2 , Ewa Ł Stepień 1,2 ,

1 Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland; 2 Center for Theranostics, Jagiellonian University, Kraków, Poland

Introduction : A 2016 World Health Organization report on diabetes states that over 400 million people worldwide have diabetes mellitus (DM). Recent studies indicate that extracellular vesicles (EVs) act as new bio‐mediators in T2D. Research suggests that hyperglycaemia triggers increased secretion of EVs, with elevated levels of endothelial EVs observed in individuals with pre‐diabetes compared to those with normal glucose tolerance. In vitro studies further show that high glucose levels enhance EV release compared to normal glucose concentrations. However, there is limited experimental data on how high glucose impacts vesicular transport in insulin‐secreting pancreatic β‐cells.

Methods : 1.1B4 cells were cultured in RPMI1640 medium with supplements. Cells were grown in 2 glucose concentrations: normoglycemic (NG, 5 mM) and hyperglycaemic (HG, 25 mM). After 3 passages, cells were starved for 24 h in serum‐free medium, and EVs were isolated as previously described. EVs were visualized using CryoTEM and TEM microscopy. EV markers were analysed using spectral flow cytometry. ATR‐FTIR spectra were collected using a Nicolet 6700 spectrometer equipped with a diamond ATR accessory.

Results : CryoTEM, TEM, and spectral flow cytometry confirmed the presence of EVs in the isolated samples. PCA of infrared spectra showed no impact of hyperglycaemia on cells compared to normoglycaemia, with no significant differences in key spectral regions (900–3150, 900–1800, 2800–3105, 1484–1720). PCA effectively differentiated cells from EVs across all ranges. Based on literature data, we showed that the 1452/1656 region and its area can detect DM in EVs samples, whereas the 1540/1343 region enables detection in cells (HG vs. NG).

Summary/Conclusion : This study explored hyperglycaemia's effect on EV secretion in insulin‐secreting pancreatic β‐cells, crucial for understanding metabolic stress in DM. Our results align with studies suggesting glucose levels influence EV release, supporting EVs as potential biomarkers for T2D. PCA proved effective in distinguishing EVs from cells under different glycaemic conditions, highlighting spectral analysis's potential for detecting metabolic changes. Findings support using spectral markers for early T2D detection, as EV‐based biomarkers hold promise for non‐invasive diabetes diagnostics.

Funding : This study was financed from Polish NCN grants: 2019/33/B/NZ3/01004 and 2022/45/B/NZ7/01430 for E.S.

Three‐dimensional in vitro model of liver fibrosis to test the effects of extracellular vesicles released by mesenchymal stromal cells

Giulia Chiabotto 1 , Armina Semnani 1 , Elena Ceccotti 1 , Marco Guenza 1 , Giovanni Camussi 1 , Stefania Bruno 1

1 Department of Medical Sciences, University of Torino, Torino, Italy

Introduction : Hepatic fibrosis, a consequence of chronic liver damage, is characterized by excessive extracellular matrix deposition due to hepatic stellate cells (HSCs) activation. With their limitations, the current treatments underscore the urgent need for novel therapies. Mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) have shown potential to reverse fibrotic processes, but their efficacy in 3D liver models needs further investigation. Moreover, it is crucial to eliminate potential contamination from culture medium components for the safe translational application of EVs. This study examined the impact of EVs derived from MSCs grown in chemically defined media on activated HSCs and liver spheroids.

Methods : EVs were isolated from human bone marrow and umbilical cord MSCs using ultracentrifugation. EVs were characterized following ISEV guidelines, by nanoparticle tracking analyses, transmission electron microscopy (TEM), western blot and cytofluorimetric analyses. 3D liver spheroids, consisting of hepatocytes and HSCs, were generated using the Sphericalplate 5D system. Fibrosis was induced by transforming growth factor‐beta 1 (TGF‐β1) stimulation. MSC‐EVs were added at different concentrations, and their effects on fibrotic markers were assessed using quantitative PCR, immunofluorescence, and Western blot analysis.

Results : MSC‐EVs expressed exosomal and mesenchymal markers, and TEM observation confirmed the presence of intact nanoscale particles with double membranes in the samples. MSC‐EVs significantly downregulated the expression of profibrotic markers, including α‐smooth muscle actin (ACTA2) and collagen type I (COL1A1), in TGF‐β1‐stimulated liver spheroids. The highest dose of MSC‐EVs (1 × 10 9 EVs/well) yielded the most pronounced reduction in fibrotic markers at both the mRNA and protein levels. MSC‐EVs attenuated the activated phenotype of HSCs in the spheroid model, showing the potential to modulate fibrosis in a complex 3D environment.

Summary/Conclusion : Our findings underscore the potential of MSC‐EVs as a promising cell‐free therapeutic approach for hepatic fibrosis. The ability of these EVs, obtained under chemically defined conditions, to effectively reverse fibrosis in 3D liver spheroids by targeting HSCs and reducing key fibrotic markers is a significant step forward. This research also highlights the utility of 3D liver models for evaluating antifibrotic treatments.

An adiponectin receptor agonist protects against myocardial ischaemia‐reperfusion injury by promoting Rab8a‐mediated extracellular vesicle release

Jialing Tang 1 , Hye Kyoung Sung 1 , Yubin Lei 1 , Khang Nguyen 1 , Eddie Tam 1 , Jun Wu 2 , Ren‐Ke Li 2 , Dylan Burger 3 , Lina Antounians 4 , Augusto Zani 4 , Gary Sweeney 1

1 Department of Biology, York University, Toronto, Ontario, Canada; 2 Toronto General Hospital, University Health Network, Toronto, Ontario, Canada; 3 Kidney Research Center, The Ottawa Hospital Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; 4 Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, Ontario, Canada Jialing Tang and Hye Kyoung Sung contributed equally .

Gary Sweeneyis the corresponding author .

Introduction : Ischaemia reperfusion (I/R) injury is associated with significant morbidity and mortality and remains the clinical indication with the greatest therapeutic unmet need. Adiponectin exerts multiple cardioprotective effects, yet evidence for a role of extracellular vesicles (EV) is lacking. The objective of this study was to evaluate the cardioprotective effect of ALY688, an adiponectin receptor agonist, in a rat model of myocardial I/R injury and the mechanistic role of EV.

Methods : Myocardial ischaemia was surgically induced for 45 min, followed by reperfusion ± intravenous ALY688 administration during ischaemia followed by daily subcutaneous injection for 28 days. Echocardiography and histopathological measurements were used to evaluate functional and remodelling changes, respectively. Proteomics analysis in both heart tissue and EV isolated from plasma was used to identify the potential mechanisms in ALY688‐mediated cardioprotective effects, and in vitro analysis of the functional impact of EV was performed in rat cardiomyoblast H9c2 cells exposed to hypoxia reoxygenation (H/R).

Results : Echocardiography analysis indicated that after 14 and 28 days, ALY688‐treated animals had reduced cardiac dysfunction as well as reduced infarct size after I/R injury. Importantly, ALY688 significantly increased EV accumulation in the myocardium and in circulation. We isolated plasma EV from ALY688‐treated animals and upon testing functional effects found that these EV conferred anti‐ROS and anti‐apoptotic outcomes in H9c2 cells subjected to H/R. Discovery‐based proteomics identified an EV secretion factor, Rab8a, to be reduced in hearts after I/R injury, but it increased by ALY688. Increased EV secretion from H9c2 cells in response to ALY688 was attenuated by CRISPR‐mediated deletion of Rab8a.

Summary/Conclusion : We demonstrate for the first time that adiponectin signalling regulates EV secretion via Rab8a activation. Targeting ALY688‐mediated EV release may be of therapeutic potential in protecting against myocardial I/R injury.

Funding : Heart & Stroke Foundation of Canada Grant.

Bioengineered adiponectin EVs prevent obesity‐related metabolic complications by improving insulin sensitivity

Presenter: Alexia Blandin

Ciloa SAS, France

Introduction : Thousands of scientific articles have demonstrated that adiponectin (APN) is a highly promising insulin‐sensitizing adipokine for treating the cardiometabolic complications of type 2 diabetes (T2DM). But the clinical development of recombinant APN was hampered by the challenge of producing stable and active high‐molecular‐weight (HMW) APN. Previous works have revealed that, in the blood, HMW APN is associated with EVs, which are stable and exhibit insulin‐sensitizing properties. This association being transient, to stabilize it we decided to bioengineer EVs with APN and to assess their therapeutic activity in the treatment of metabolic diseases.

Methods : A chimeric gene was constructed encoding APN fused to a transmembrane domain and to a peptide (named pilot peptide, patented) which interacts with the ESCRT machinery to sort APN with EVs. A stable transfected HEK293T cell line secreting APN EVs was established. Several large‐scale APN EV batches were produced in cell factories and purified using tangential flow filtration and chromatography. EVs were characterized according to the MISEV guidelines by western blot and ELISA for EV markers and protein content and analysed by nanoflow cytometry for EV size and counting. The quantification of APN on EVs and its multimerization state were evaluated. The metabolic effects of APN EVs were assessed in DIO‐NASH mice after 12 weeks of injections of APN EVs alone or in combination with semaglutide.

Results : More than 1000 APN were stably anchored in the EV membrane as bouquets of HMW APN. Mice treated with APN EVs show less weight gain and a better insulin tolerance than control mice. Only APN EVs combined with semaglutide (i) reduce dramatically big adipocyte number while increasing small adipocyte number, (ii) restore full insulin sensitivity, and (iii) preserve muscle mass in contrast to semaglutide alone that reduces lean mass.

Summary/Conclusion : We demonstrate for the first time the obtention of a functional HMW APN and the reproducibility of large‐scale APN EV production. Our results on DIO‐NASH mice highlight the high insulin‐sensitizing potential of APN EVs and reveal their synergistic role in combination with semaglutide, opening up prospects for an innovative co‐agonist biotherapy for T2DM.

Funding : The research was performed by two companies, Ciloa and Physiogenex.

Subtopic EV therapeutics development 1

Injected extracellular vesicles hitchhike on erythrocytes and platelets from circulation towards organ clearance

André Görgens 1 , Svetlana Pavlova 1 , Doste Mamand 1 , Daniel W. Hagey 1 , Yesid Velasquez 1 , Wenyi Zheng 1 , Guannan Zhou 1 , Risul Amin 1 , Antje M. Zickler 1 , Manuela Gustafsson 1 , Samir EL Andaloussi 1

1 Karolinska University, Sweden

Introduction : The application of extracellular vesicles (EVs) for targeted delivery of drugs, proteins, and RNA to specific cell types or organs is a promising approach in molecular therapy. However, in order to achieve efficient and targeted delivery, it is crucial to improve our understanding of the fate of injected EVs in terms of tissue and cellular biodistribution. Data by us and others has demonstrated that EVs are rapidly cleared from the blood circulation to different organs following administration, with large fractions ending up in the liver and lungs within min after injection. In this study, we investigated the key steps and kinetics of this clearance process and particularly focused on studying how intravenously injected EVs would interact with circulating blood cells shortly after injection.

Methods : For this study, we used thoroughly characterized EV preparations from different stable producer cell lines, and EVs were prepared with different established EV isolation methods. Following the injection of fluorescently tagged EVs into mice, we utilized high‐sensitivity multicolour imaging flow cytometry to quantify the binding of EVs to different blood cell types in the circulation and their tissue resolution in terms of target cell type in the spleen and liver. Results were validated by single‐cell RNAseq of EV‐receiving cells in mouse models and by scanning electron microscopy (EM).

Results : We demonstrate that a significant proportion of injected EVs binds to blood cells within min after intravenous injection. EVs were particularly found to directly bind to erythrocytes and platelets in the circulation, which is highly relevant since those two blood cell types account for > 99% of all blood cells and have been largely neglected in this context up to now. We further show that EVs ‘hitchhike’ on both Erythrocytes and Platelets to different organs, where they are cleared by macrophages.

Summary/Conclusion : In summary, we report a highly relevant yet largely overlooked mechanism that significantly contributes to clearance of EVs bound to Erythrocytes and Platelets from the blood circulation to distant organs.

Acute toxicity testing of extracellular vesicles from umbilical cord mesenchymal stem cells: assessment in lungs and spleen as target organs

Cynthia Retna Sartika 1,2 , Sheila Fawziyya Jundan 2 , Angliana Chouw 2 , Geofanny Facicilia 2 , Annisa Nur Arofah 2 , Riska Agustina 2 , Sheila Mutia Rahmat 2 , Retina Shyallala Askandar 2 , Zulfa Maulidah 2 , Annisa Dwi Widya 2 , Yohana Shanny 2 , Mawar Subangkit 3 , Arief Boediono 3

1 Universitas Padjadjaran, Indonesia, 2 PT. Prodia StemCell Indonesia, Indonesia, 3 Institut Pertanian Bogor, Indonesia

Introduction : Understanding the safety profile of mesenchymal stem cell (MSC) extracellular vesicles (EVs) is crucial, especially as their therapeutic potential is increasingly recognized in the field of regenerative medicine. Acute toxicity testing is essential to identifying any immediate harmful effects following exposure of EVs, which helps ensure the safety of EVs in clinical applications. Therefore, this study investigates the acute toxicity of EVs derived from umbilical cord mesenchymal stem cells (UC‐MSCs), specifically focusing on the lungs and spleen as target organs.

Methods : The primary objectives of this research were to evaluate the intrinsic toxicity of these EVs and to gather information on potential hazards following acute toxicity testing. We used female Sprague‐Dawley rats aged 5–6 weeks. The EVs were administered through various routes: intravenously, intramuscularly, intraperitoneally, and intranasally. Each rat received a single dose at the beginning of the study, followed by close observation during the first 24 h and ongoing monitoring every day for 14 days.

Results : Clinical observations revealed no signs of toxicity across all treatment groups, with no fatalities recorded. However, it showed localized swelling in the paws of some rats within 24 h post‐treatment. Macroscopic examination of the lung and spleen tissues showed no abnormalities or signs of toxicity after administration with different routes. However, there are significant differences in the relative weight of the spleen in groups that received EVs intravenously, intraperitoneally, and intranasally compared to the control group. These findings suggest a possible reactive response of the spleen to the administered test substance.

Summary/Conclusion : In conclusion, while acute toxicity was not evident in the lungs, the notable changes in spleen weight indicate a need for further research into the biological effects of UC‐MSC‐derived EVs.

Regulatory considerations on characterization and quality control of therapeutic EV products

Akiko Ishii‐Watabe 1 , Yoji Sato 1 , Hirotaka Nishimura 1 , Tomofumi Yamamoto 1 , Noritaka Hashii 1 , Takumi Miura 1 , Yuchen Son 1 , Masato Kiyoshi 1 , Hiroko Shibata 1 , Yuki Takahashi 2 , Norimasa Nakamura 3 , Taro Akimori 4 , Hiroto Hanai 4 , Yu Moriguchi 4 Ryosuke Kuribayashi 5 , Anri Nishizawa 5 , Naohiro Otaki 5 , Akira Sakurai 5 , Yu Fujita 6 Takanori Ichiki 7 , Masahiko Kuroda 8 , Yoshiaki Okada 9 , Naohiro Seo 7 , Toshihide Takeuchi 10 , Kazuhisa Uchida 11 , Yusuke Yoshioka 8 , Takahiro Ochiya 8 , Rikinari Hanayama 12 , Yoshinobu Takakura 2

1 National Institute of Health Sciences, Japan; 2 Kyoto University, Japan; 3 Osaka Health Science University, Japan; 4 Osaka University, Japan; 5 Pharmaceuticals and Medical Devices Agency, Japan; 6 The Jikei University School of Medicine, Japan; 7 The University of Tokyo, Japan; 8 Tokyo Medical University, Japan; 9 Saitama Medical University, Japan; 10 Kindai University, Japan; 11 Kobe University, Japan; 12 Kanazawa University, Japan

Introduction : Development of therapeutic EV products is an attractive topic, while the regulatory guidelines for marketing authorization applications have not been established. In order to promote the efficient development of therapeutic EV products and to ensure their quality, safety and efficacy, showing the recommendation in the regulatory guideline will be quite useful. For the purpose of preparing the draft guideline that will be released for the public consultation, the contents were discussed and prepared in the research group founded by the Japanese government AMED (Japan Agency for Medical Research and Development).

Methods : Based on the general principles for quality control of biological products shown in ICH guidelines and experimental studies for EV characterization, fundamental issues for characterization and quality control of therapeutic EV products were discussed. The following two documents (1, 2) and MISEV 2023 were used as references. (1) Y. Takakura et al. Pharmaceutical Research 2024; 41, no. 8: 1573–1594. (2) “Report on therapeutic products based on extracellular vesicles (EVs) including exosomes,” released by the Subcommittee in PMDA. https://www.pmda.go.jp/files/000268368.pdf.

Results : The main scope of the draft guideline was defined as EV products produced from human allogenic cells. Since therapeutic EV products have complex structures with high heterogeneity and the quality attributes are variable depending on the manufacturing process, the quality risk management concept shown in the ICH Q9 guideline was thought to be suitable as a basic principle. As important issues in the establishment of the control strategy, elucidation of mode of action, identification of critical quality attributes, ensuring viral safety, and assessment of carcinogenicity of EVs produced from immortalized cells were identified. The recommendations in characterization and quality control of EV drug substance and drug product, manufacturing process, and comparability assessment that are required in case of process change were discussed, and a document was prepared.

Summary/Conclusion : Basic principles for ensuring the quality of therapeutic EV products were discussed, and an early draft of the guideline was prepared. This presentation is expected to provide an opportunity to exchange opinions among stakeholders in charge of the development and evaluation of therapeutic EV products.

Funding : AMED JP22mk0101218.

Track EV sources and basic biology

Subtopic Alternative EV sources 1

Milk extracellular vesicles for enhanced delivery of zeaxanthin: exploring bioavailability and aryl hydrocarbon receptor activation in immunomodulation

Presenter: Giorgia Manni

University of Perugiaa, Italy

Introduction : The growing demand for natural antioxidants, particularly carotenoids like zeaxanthin (Zea), highlights the importance of efficient delivery mechanisms to improve their solubility and bioavailability. Zea, a potent lipophilic compound, has been widely studied for its anti‐inflammatory and antioxidant properties, yet it faces challenges in traditional formulations due to its low solubility in aqueous environments. As interest rises in its potential as a functional food ingredient and dietary supplement, developing optimized delivery systems becomes essential. Milk extracellular vesicles (miEVs), with considerable nutritional benefits, are emerging as promising candidates for drug and nutrient delivery. miEVs are recognized for their intrinsic stability, biocompatibility, and potential to enhance the bioavailability of encapsulated compounds, thereby offering novel applications for health promotion. Additionally, dietary molecules like Zea can interact with the aryl hydrocarbon receptor (AhR), a critical mediator in inflammation, presenting an opportunity for miEVs to serve as modulators of immune response when combined with bioactive compounds.

Methods : miEVs were isolated from goat milk through ultracentrifugation, adhering to the MISEV 2023 guidelines. Their size distribution and particle count were analysed using Nanosight technology. Zea was loaded into the miEVs via saponin incubation, creating zeaxanthin‐loaded miEVs (Zx‐miEVs), which were then assessed for their physicochemical properties. The amount of Zea in the particles was determined through HPLC. The anti‐inflammatory effects of Zx‐miEVs were evaluated in vitro using macrophage cells stimulated with lipopolysaccharide (LPS), focusing on cytokine release. Additionally, Zx‐miEVs’ ability to activate the AhR pathway was measured through a luciferase‐based assay.

Results : Preliminary findings demonstrate that Zx‐miEVs significantly improve the solubilization of zeaxanthin compared to conventional solvents, suggesting a superior delivery platform for functional foods and nutraceuticals. Notably, pre‐treatment of macrophages with Zx‐miEVs significantly reduced IL‐6 secretion in inflammatory conditions, indicating potent anti‐inflammatory effects. Further analysis revealed effective AhR activation by Zx‐miEVs, shedding light on the molecular pathways involved in their immunomodulatory activity.

Summary/Conclusion : This innovative approach not only enhances the bioavailability and health benefits of zeaxanthin but also advances the development of natural product delivery systems. By leveraging miEVs, this study paves the way for improved dietary interventions and therapeutic applications in inflammation‐related health issues.

Agri‐food residues as a source of bioactive nanovesicles: a new perspective to valorize agricultural by‐products

R. Latella 1 , L. Urbanelli 1,5 , E. Chiaradia 2 , L. Mezzasoma 3,5 , R. Romani 3,5 , M. Gargaro 4 , R. Pellegrino 1 , H. B. R. Alabed 1 , C. Emiliani 1,5 , S. Buratta 1

1 Department Chemistry, Biology and Biotechnology, University of Perugia (Italy); 2 Department of Veterinary Medicine, University of Perugia (Italy); 3 Department of Medicine and Surgery, University of Perugia (Italy); 4 Department of Pharmaceutical Sciences, University of Perugia (Italy); 5 Extracellular Vesicles network (EV‐net) of the University of Perugia (Italy)

Introduction : The growing demand for food production due to the rapid increase in the world population generates a huge amount of agricultural by‐products that might be valorized through the extraction of biomolecules and preparation of value‐added products useful for various biomedical applications. Here, we evaluated the possibility that agricultural by‐products could represent a valuable source of bioactive nanovesicles (NVs), reinforcing the concept of waste valorization within the context of the circular economy. In particular, we successfully isolated NVs from olive vegetation water (OVW), a by‐product of the olive oil industry, and serum milk (SM) generated during the cheese‐making process. NVs have been biophysically and biochemically characterized before the evaluation of their biological effects on mammalian cell lines.

Methods : OVW‐NVs were isolated through differential centrifugation followed by density‐gradient centrifugation [1], whereas SM‐NVs were isolated by a protocol currently used to isolate vesicles from milk [2]. NVs were biophysically characterized through scanning electron microscopy, nanoparticle tracking analysis, cryo‐transmission electron microscopy and dynamic light scattering. Since information on biochemical composition is essential to rationalize the biological effects of NVs on mammalian cells, a complete characterization of NVs was obtained by integrated lipidomic, metabolomic, and proteomic procedures. Finally, biological effects (cytotoxicity, internalization ability, antioxidant and anti‐inflammatory effects) of these vesicles on mammalian cell lines (PC3 and THP‐1) have been explored.

Results : Biophysical data and information on lipid, metabolite and protein composition demonstrated that the features of OVW‐NVs and SM‐NVs overlap with that of bioactive NVs isolated from edible plants/fruits and from milk, respectively. Both NVs are internalized by mammalian cells and protected them from cytotoxicity and oxidative stress induced by H 2 O 2 exposure. Further, OVW‐NVs and SM‐NVs suppressed NLRP3 inflammasome activation, warranting further investigation as potential anti‐inflammatory tools.

Summary/Conclusion : Overall these results demonstrate, for the first time, that agricultural residues might represent a source of bioactive NVs that can be internalized by mammalian cells, protecting them by oxidative stress and inflammatory stimuli. This evidence strengthened the idea that agricultural by‐product‐derived NVs might become a promising nutraceutical tool.

Funding : European Union—NextGenerationEU under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041—VITALITY.

Exploring the haemostatic role of platelet‐derived EVs: comparative analysis of platelet lysates versus activation‐induced EV release

Presenter: HyunAh Sung

Korea University, Republic of Korea

Introduction : Platelets are small but abundant cell fragments in the bloodstream essential for haemostasis and thrombosis. Upon activation, platelets release extracellular vesicles (EVs) containing bioactive molecules, including specific microRNAs, proteins, and growth factors that contribute to haemostatic functions. This study evaluated the haemostatic potential of platelet‐derived EVs (pEVs) generated via platelet lysates and through activation using both physical and chemical stimuli.

Methods : Platelets were activated physically using shear stress in a microfluidic chip and chemically with an agonist. EVs were isolated with the LipoFilter to remove lipoproteins, followed by ExoTFF for purification and concentration. Characterization adhered to MISEV 2023 guidelines, using TEM, ELISA, western blotting, and NTA. Platelet function with pEV addition was assessed via Anysis Pro, and blood coagulation characteristics were analysed with TEG 5000 and GTX 10 m.

Results : Activation triggered membrane blebbing, a key event in EV release, with a notable enrichment of growth factors within these EVs. Co‐stimulation (combining physical and chemical methods) yielded the highest concentration of pEVs. When the resulting pEVs were added to autologous blood, we observed remarkable increases in platelet aggregation, as well as a substantial enhancement in both the strength and speed of blood coagulation.

Summary/Conclusion : These results provide insights into the modulation of platelet function and EV release, with implications for therapeutic interventions in clinical and biological applications. Notably, the quantity of EVs and growth factors significantly increases depending on the activation method, which appears to have a direct impact on platelet aggregation and blood coagulation strength. These findings suggest potential haemostatic benefits, indicating that this approach could be advantageous for therapeutic applications aimed at enhancing coagulation and controlling bleeding.

Funding : This project was conducted with the support of the Alchemist Project of the Korea Evaluation Institute of Industrial Technology (KEIT 20018560/NTIS 1415184668) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) and the National Research Foundation of Korea (NRF) Grant funded by the Korean Government, MSIP (RS‐2023‐00207833).

Pioneering the development of novel human platelet‐derived exosomes for improved therapeutic applications in the biomedical field

Yee‐Hsien Lin 1 , Han‐Tse Lin 1 , Wei‐Tung Wang 1 , Shih‐Hwei Liu 1

AventaCell BioMedical Corp., Kent, WA 98031, USA

Introduction : The therapeutic potential of extracellular vesicles (EVs) has emerged as a key focus in the biomedical field, owing to their crucial role in intercellular communication. EVs contribute to various disease processes, cancer progression, and tissue regeneration. Among them, exosomes, a specific subclass of EVs, are distinguished by their size and rich content of bioactive molecules and genetic material. These unique biological features have generated significant interest in using exosomes as advanced drug delivery systems for tissue repair and cancer therapies. Therefore, precise methodologies are vital to ensure the production of high‐quality exosomes, particularly for cancer treatment and regenerative medicine. Platelet‐derived exosomes, enriched from human platelets, demonstrate a remarkable capacity to promote cell proliferation and inhibit apoptosis. With their high biocompatibility, low cytotoxicity, minimal tumourigenicity, and reduced immunogenicity, these exosomes show significant therapeutic potential, especially for homologous administration in clinical settings. The main objective of this study is to provide a comprehensive molecular and morphological characterization of exosomes derived from human platelets. By isolating and purifying exosomes from human platelet lysate, we introduce them as a promising candidate for future therapeutic applications in exosome research and clinical use.

Methods : Human platelet‐derived exosomes were successfully isolated from human platelet lysate using a tangential‐flow filtration system equipped with a hollow fibre for efficient depletion. Following isolation and purification, key CD markers, including CD9, CD63, and CD81, were effectively characterized. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) confirmed the average particle size.

Results : Additionally, when these exosomes were introduced into a 72‐h cell culture with mesenchymal stem cells, no cytotoxicity was observed throughout the culture period. Consequently, human platelet‐derived exosomes present themselves as a promising therapeutic candidate, offering significant potential for both exosome research and various clinical applications.

Summary/Conclusion : Human platelet‐derived exosomes show great potential as therapeutic agents due to their biocompatibility, low immunogenicity, and ability to promote cell growth. This study provides a foundation for further research into their clinical applications in tissue regeneration and cancer treatment.

Cloudberry plant‐derived nanovesicles for oral drug delivery system: stability in gastrointestinal conditions and biodistribution in young and elder mice

Feby Wijaya Pratiwi, 1,†* Keerthanaa Balasubramanian Shanthi, 1 Florence Naillat, 1 Ramila Mammadova, 1 Marjaana Sarpola, 1 Si‐Han Wu, 3* Marko Suokas, 4 Soile Jokipii‐Lukkari, 4 Susanna Kaisto, 1 Anatoliy Samoylenko, 1 Henrikki Liimatainen, 5 Caglar Elbuken, 6,2 Seppo Juhani Vainio 1,2*

1 Laboratory of Developmental Biology, Disease Networks Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland; 2 Kvantum Institute, University of Oulu, Finland; 3 Graduate Institute of Nanomedicine and Medical Engineering, Taipei Medical University, Taiwan; 4 Ecology and Genetics Department, University of Oulu, Finland; 5 Fiber and Particle Engineering Research Unit, University of Oulu, Finland; 6 Micro‐/Nanofluidics and Biosensor Research Group, University of Oulu, Finland

Feby Wijaya Pratiwi and Keerthanaa Balasubramanian Shanthi are equal first coauthors and Florence Naillat and Ramila Mammadova are equal second coauthors .

Introduction : The phrase ‘A man is what he eats’ has taken on new significance as research reveals that food provides nutrients and bioactive information that can influence mammalian genes. This information is transmitted by plant‐derived nanovesicles (PDNVs). Although PDNVs have demonstrated therapeutic potential, their effects on ageing populations remain largely unexplored. The non‐invasive nature of oral delivery is particularly appealing for this demographic, though the digestive tract poses challenges for effective drug absorption. Cloudberries (Rubus chamaemorus), which are rich in bioactive polyphenols and demonstrate high stability in the digestive tract, provide a promising source of PDNVs for oral delivery vehicles for the elderly.

Methods : PDNVs from cloudberries (CNV) were isolated using differential and ultracentrifugation methods, and various properties were analysed, including protein content, particle concentration, size distribution, morphology, zeta potential, miRNA content, and antioxidant properties. Their stability in the gastrointestinal environment was evaluated using simulated digestive conditions, followed by analyses of particle concentration, morphology, and surface potential. In vitro experiments with Caco‐2 cells were used to assess cytotoxicity, cell proliferation, and CNVs uptake and transport. Additionally, in vivo imaging analysed CNVs distribution in young and aged mice after oral administration, with tissue samples and blood examined using immunohistochemistry and flow cytometry for CNVs localization and immune profiling.

Results : This study highlights the unique properties and biomolecular content of CNVs and their potential for mass production. Our results show that CNVs remain stable during gastrointestinal digestion while preserving their bioactivity and properties in vitro. Additionally, they exhibit favourable biodistribution, extended retention in the GI tract, efficient cellular uptake, successful intestinal barrier translocation, entry into the bloodstream, and low immunogenicity in both young and elderly mice.

Summary/Conclusion : CNVs that can withstand gastrointestinal digestion and exhibit favourable biodistribution, efficient cellular uptake, effective intestinal barrier crossing, successful bloodstream transfer, and low immunogenicity are promising candidates for stable oral delivery vehicles in both young and elderly mice. Future research should concentrate on elucidating the specific mechanisms of CNV interactions with target cells, optimizing delivery methods, and validating therapeutic efficacy in relevant disease models.

Funding : This work was supported by the Kvantum Institute BioEVEngine project (University of Oulu).

Milk‐derived extracellular vesicles as drug delivery vehicle for myricetin

M. Brattini, M. Mosaico, E. Butturini, S. Mariotto

Dept. of Neurosciences, Biomedicine and Movement Science, Biological Chemistry Section, University of Verona, Verona, Italy

Introduction : One of the main attractive characteristics of extracellular vesicles (EVs) is the possibility of using them as drug delivery vehicles thanks to their natural origin and the intrinsic ability to carry molecular cargo and to cross biological barriers. However, the translation of EVs as delivery vehicles into clinical studies is hampered by the difficulties in obtaining them in an efficient and rapid way. Milk could represent a good EV source, offering an accessible and cost‐effective option with high EV yield through a relatively easy protocol. To demonstrate the potential of mEVs as a drug delivery system, myricetin, a natural polyphenol with anti‐inflammatory activity, was chosen as a model drug, while saponin was used as a loading agent.

Methods : MEVs were isolated from whole pasteurized bovine milk through the differential ultracentrifugation method in combination with rennet pre‐treatment to remove casein micelles. Nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscopy (TEM) and western blot analyses were carried out to characterize the isolated particles. Moreover, cellular uptake of MEVs was evaluated by analysing treated BV2 cells by confocal microscopy. Finally, myricetin was encapsulated through a saponin‐assisted method, and the encapsulation efficiency (EE%) was assessed using HPLC analysis after removing free myricetin by ultrafiltration.

Results : NTA and DLS analysis revealed particles with a size in the range of 150 nm, a surface charge of −21.7 ± 2.67 mV and a concentration in the order of 10 13 particles/mL. TEM images displayed particles delimited by a lipid bilayer and with spherical morphology; moreover, western blot showed the presence of positive EVs markers CD81, Hsp70, and TSG101. MEVs were efficiently taken up by BV2 cells after 24 h, as proved by confocal images. Finally, HPLC analysis confirmed the optimization of myricetin encapsulation conditions with an EE% of 21% ± 8.5%.

Summary/Conclusion : Our work demonstrates the effectiveness of the adopted isolation protocol, which enabled the efficient separation of MEVs from bovine milk. Moreover, this study highlights the potential of mEVs as a tool for optimizing the encapsulation parameters of myricetin in EVs. However, further analysis is needed to verify the cellular response to the treatment with myricetin‐loaded MEVs.

Anticancer activity of fingerroot exosome‐like nanovesicles in atypical teratoid/rhabdoid tumour (AT/RT)

Pisitpong Jarurachadanon 1 , Wittaya Panvongsa 1 , Intiporn Mingsakul 1 , Boon‐ek Yingyongnarongkul 2 , Suradej Hongeng 1 , Arthit Chairoungdua 1

1 Mahidol University, Thailand; 2 Ramkhamhaeng University, Thailand

Introduction : Atypical teratoid/rhabdoid tumour (AT/RT) is an aggressive paediatric brain tumour with poor treatment outcomes, highlighting the need for alternative therapies. Previously, we isolated nanovesicles from Boesenbergia rotunda (fingerroot) and demonstrated their anticancer activity against colorectal cancer cells. However, the effects and mechanisms of fingerroot exosome‐like nanovesicles (FELNs) in AT/RT remain unclear.

Methods : FELNs were isolated from fingerroot juice via ultracentrifugation and purified using a qEV size‐exclusion chromatography (SEC) column. Their size and morphology were characterized by DLS and TEM. Cytotoxicity was assessed in BT12 and BT16 AT/RT cell lines in 2D and 3D cultures, while migration and invasion were evaluated via transwell assays. Proteomic analysis was performed using mass spectrometry.

Results : The isolated FELNs were round‐shaped, membrane‐bound vesicles with an average size of 100 nm. Metabolite profiling of FELNs identified the presence of pinostrobin, pinocembrin, and panduratin A. FELNs were taken up by BT12 and BT16 cells and exhibited dose‐ and time‐dependent cytotoxicity through apoptosis induction. Interestingly, human brain endothelial cells (hCMEC/D3) demonstrated lower sensitivity to FELNs. Notably, at non‐toxic concentrations, FELNs significantly suppressed BT12 and BT16 cell migration and invasion. Proteomic analysis further revealed that FELNs disrupted signalling pathways associated with AT/RT development, including the Wnt/β‐catenin signalling pathway. Treatment with FELNs suppressed β‐catenin protein levels and its target gene, cyclin D1. Additionally, FELNs reduced phospho‐GSK‐3β (Ser9), suggesting that FELNs promote β‐catenin degradation through a GSK‐3β‐dependent mechanism.

Summary/Conclusion : Collectively, our results indicate that FELNs exhibit anticancer activity in AT/RT cells by inducing apoptosis and suppressing cancer cell migration and invasion. Mechanistically, the anticancer effects in AT/RT cells are partly mediated through the GSK‐3β‐dependent β‐catenin degradation pathway. Furthermore, FELNs demonstrated lower cytotoxicity in normal brain endothelial cells, highlighting their potential as novel therapeutic agents for brain tumour treatment.

Funding : Mahidol University (Basic Research Fund: fiscal years 2022 to A.C.) and National Research Council of Thailand (NRCT) and Mahidol University (N42A660523 to A.C.).

A novel method for the isolation of plant‐derived exosome‐like nanovesicles from fruit juices by polyethylene glycol‐mediated preferential precipitation

Wonil Nam, Donghyeon Yoo, Wooram Um

Pukyong National University, Republic of Korea

Introduction : Plant‐derived exosome‐like nanovesicles (PENs) contain natural bioactive components such as lipids and small secondary metabolites from their distinct plants, presenting anti‐inflammatory, antioxidant, and anticancer activities. PENs have similar size and morphology to mammalian extracellular vesicles and can be internalized into mammalian cells, making PENs able to deliver their cargo and regulate cellular response. Therefore, it emerges as a promising alternative source of exosomes. However, the isolation of PENs from plant juices or homogenised plants has demonstrated suboptimal reproducibility with impurities, including multiple hydrophobic proteins and lipids. Consequently, novel methodologies are required to isolate PENs in high purity and yield.

Methods : Poly(ethylene glycol), a compound that has been deemed Generally Recognised as Safe (GRAS) by the FDA for its use in food and cosmetic products, was subjected to preferential precipitation of PENs in fruit juices. This study involved the deliberate addition of poly(ethylene glycol) to broccoli juices with 12 wt%. The solutions were mixed (60 rpm) at 4°C for 16 h, centrifuged at 8000 × g for 1 h at 4°C. Thereafter, the supernatants were removed and the PEN pellets were resuspended in Phosphate Buffered Saline (PBS). Unbound PEGs were then removed using PD‐10 desalting columns.

Results : We demonstrated the successful harvesting of PENs from fruit juices, with the precipitation of poly(ethylene glycol)‐precipitated PENs with larger sizes. Importantly, the yield of PENs from the same volume of fruit juices increased by 5.64 times. Furthermore, no significant difference in antioxidant and anti‐inflammatory activity was observed between the isolated PENs and poly(ethylene glycol)‐precipitated PENs. Furthermore, poly(ethylene glycol)‐precipitated PENs exhibit size stability of more than 24 h at 37°C, an improvement of more than 8‐fold compared to native PENs.

Summary/Conclusion : This study has successfully developed a novel method capable of efficiently harvesting PENs from fruit juices by preferential precipitation with poly(ethylene glycol). The results demonstrate that preferential precipitation with poly(ethylene glycol) is a method that can significantly increase the amount obtained without compromising the PENs. Furthermore, our results suggest that preferential precipitation of PENs using poly(ethylene glycol) may be a novel method to produce more stable PEN ingredients.

Microcarrier based platform process for adherent cells to produce extracellular vesicles

Presenter: Andrea Haid

FH Campus Wien Biomedical Science, USA

Introduction : Producing extracellular vesicles (EVs) from different cells is relevant because they possess unique compositions and functions reflecting their cell of origin, and producing EVs from various adherent cells allows researchers to study cell‐specific intercellular communication mechanisms and explore their potential therapeutic applications.

Methods : By using Cytodex 1 microcarrier, we developed a platform for adherent cells to produce EVs in middle scale (up to 50 L). Different cell types can be grown on Cytodex 1, like human dermal fibroblast cells, CACO‐2 and others. EV extraction was performed with trans flow filtration (TFF) or, in lower volumes, by Centricons (100 kDa cut‐off), followed by size exclusion chromatography with Sephacryl S‐500 HR). Identity of EVs was determined by western blot (CD63, CD9), integrity was tested by atomic force microscopy (AFM), as well as concentration determined by total protein and RNA measurements and dPCR.

Results : We were able to grow different cells on Cytodex 1 microcarriers. With one cell line we scaled the process up to a 10 L bioreactor. A Design of Experiments (DOE)‐based optimizing approach led to optimal process conditions for EV production of this cell line.

Summary/Conclusion : The ability to cultivate diverse cell types, including human dermal fibroblasts and CACO‐2 cells, in stirred‐tank bioreactors represents a significant advancement, particularly for cell lines previously unadapted to such systems. The use of TFF and size‐exclusion chromatography ensured high‐purity EV isolates, validated by CD63/CD9 markers and structural integrity via AFM. Implementing a DOE approach to optimize EV production underscores the potential for reproducible, large‐scale manufacturing, which is essential for therapeutic applications. This work establishes a robust, mid‐scale bioreactor platform for EV production from adherent cells, overcoming scalability challenges while maintaining EV integrity and functionality.

Funding : This work was funded by Anschubfinanzierung ASF—Etablierung von Produktion und Charakterisierung von extrazellulären Vesikeln “Pro‐CHAR*EV.”

Exosomes engineered for reprogramming tumour‐associated macrophages and blocking PD‐L1 immune checkpoint

Presenter: Hyunwoo Hwang

Kyungpook National University, Republic of Korea

Introduction : Exosomes are emerging as next‐generation nanocarriers in cancer immunotherapy, leveraging tumour tropism and precise cargo delivery. While PD‐L1 blockade has reshaped immunotherapy, its efficacy remains limited by immunosuppressive tumour‐associated macrophages (TAMs). TREM2, a key regulator of TAM‐mediated immunosuppression and a myeloid checkpoint receptor, sustains an immunosuppressive microenvironment and correlates with poor prognosis. Here, we surface‐engineered exosomes with a PD‐L1 blocking peptide and an endosome escape peptide using click chemistry and loaded siRNA against TREM2 to enhance tumour‐targeted immune modulation. The endosome escape peptide promotes intracellular siRNA delivery by helping the exosomes escape from endosomes.

Methods : HEK293 cells were treated with Ac4ManNAz to introduce surface azides (N 3 ) for bioorthogonal conjugation. Exosomes were isolated via differential ultracentrifugation and washed in phosphate‐buffered saline. TREM2 siRNA was loaded using the ExoFect kit. For surface modification, PD‐L1 blocking and endosome escape peptides were first conjugated to DBCO‐PEG‐maleimide via thiol‐maleimide. The peptide‐linker conjugate was then incubated with azide‐functionalized, siRNA‐loaded exosomes for click chemistry‐mediated conjugation.

Results : Engineered exosomes achieved efficient siRNA encapsulation and stable surface peptide conjugation. Immunofluorescence microscopy demonstrated efficient siRNA delivery, which was significantly enhanced by the endosomal escape peptide. Surface‐labelled PD‐L1 peptide remained stable for 120 h and effectively blocked PD‐L1, restoring T cell activity. TREM2 silencing reprogrammed TAMs into an M1‐like phenotype, altering marker expression and cytokine secretion.

Summary/Conclusion : The engineered exosomes deliver TREM2 siRNA, block PD‐L1, and reprogram TAMs, collectively enhancing antitumour immunity. This platform offers a promising strategy to overcome immune suppression and advance cancer immunotherapy.

Subtopic EV characteristics 1

Comprehensive identification and characterization of the biological properties of HEK293F cell‐derived small extracellular vesicles as promising drug delivery systems

Presenter: Zhi‐qing Chen

Southeast University, China (People's Republic)

Introduction : HEK293F‐derived extracellular vesicles (HEK293F‐EVs) have great potential as next‐generation drug delivery vehicles. A comprehensive understanding of their batch stability and in vivo safety is a prerequisite for clinical translation.

Methods : HEK293F‐EVs were purified using ultracentrifugation combined with size exclusion chromatography, and their physicochemical properties, such as morphology, size distribution, and biomarkers, were thoroughly characterized. Raman spectroscopy and multi‐omics analyses were employed to elabourate their molecular composition. Blood kinetics and biodistribution were assessed via IVIS spectrum imaging. Additionally, long‐term in vivo safety was evaluated following multiple‐dose administration through haematology, serum biochemistry, cytokine/chemokine profiling, and histopathology.

Results : HEK293F‐EVs exhibited stable yields, purity, physicochemical characteristics (morphology, size, zeta potential, and marker proteins), and chemical composition across different cell passages (P10, P20, P30), with no significant variations. Content profiling, including protein, microRNA, metabolite, and lipid, confirmed consistent molecular stability across five production batches. GO, reactome, and KEGG analyses revealed minimal enrichment in pathways related to acute immune response or cytotoxicity. Blood kinetics studies indicated rapid clearance of HEK293F‐EVs from circulation, though slightly slower than PEG‐liposomes. Organ biodistribution was comparable between HEK293F‐EVs and PEG‐liposomes, with HEK293F‐EVs potentially having longer retention times. Importantly, HEK293F‐EVs exhibited a favourable preclinical long‐term safety profile, showing low immunogenicity and fewer tissue lesions compared to PEG‐liposomes.

Summary/Conclusion : Our study demonstrates that HEK293F‐EVs maintain stable characteristics and compositions across batches and possess a superior safety profile, underscoring their significant promise as a safe and reliable drug delivery platform for clinical applications.

Effect of benzo[a]pyrene on the production by endothelial HMEC‐1 cells of large extracellular vesicles containing mitochondria

Joan Guillouzouic 1 , Camille Chauvin 1 , Valentine Brouard 1 , Agnès Burel 2 , Normand Podechard 1 , Lydie Sparfel 1 , Manon Le Goff 1 , Dominique Lagadic Gossmann 1 , Catherine Lavau 1 , Eric Le Ferrec 1

1 University of Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)—UMR_S 1085, France; 2 University of Rennes, Plate‐forme MRic‐TEM, Irset, Biosit—UMS 3480, France

Introduction : Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon classified as carcinogenic. We showed that this environmental contaminant exacerbates the production of extracellular vesicles (EVs). Among EVs, large‐EVs (L‐EVs) contain a sub‐population called mito‐L‐EVs used by cells to outsource mitochondria and mitochondria‐derived vesicles.

Methods : Using the human microvascular endothelial cell line (HMEC‐1), our work aims to understand the production and the role of mito‐L‐EVs in the context of B[a]P exposure (100 nM, 24 h).

Results : We first studied the ability of HMEC‐1 cells to produce Mito‐L‐EVs. We showed the presence of mitochondrial proteins in L‐EV lysate regardless of B[a]P exposure. Treatment with proteinase K confirmed the presence of these proteins within L‐EV. Transmission electron microscopy revealed structures with features of undamaged and damaged mitochondria within L‐EVs regardless of B[a]P exposure. Flow cytometry revealed that mitochondria within Mito‐L‐EVs are either polarized, in L‐EVs with a size > 900 nm, or totally depolarized, in L‐EVs smaller than 900 nm. The sizes of both populations are similarly upregulated by B[a]P. Nanotracking analysis was used to measure the number of Mito‐L‐EV and showed a ≈40% increase caused by B[a]P exposure. The ATP receptor P2X7 (rP2X7) is involved in the biogenesis of L‐EVs. We studied its role in the overproduction of mito‐L‐EVs in the presence of B[a]P. Brilliant Blue G, an inhibitor of rP2X7, either alone or with B[a]P, selectively induces a collapse in the production of mito‐L‐EVs containing polarized mitochondria. This indicates that Mito‐L‐EVs containing either polarized or depolarized mitochondria are regulated by distinct signalling pathways, and that B[a]P could promote the overproduction of mito‐L‐EVs with polarized mitochondria through rP2X7. In parallel, we showed by western blot that the main glycolytic enzymes, as well as pyruvate dehydrogenase, are present in L‐EVs. Exposure to B[a]P appears to increase the levels of these enzymes in L‐EVs. This suggests that L‐EVs produced by HMEC‐1 cells contain all the components necessary, at least through glycolysis, to produce ATP in the extracellular space, and that B[a]P could enhance this capacity.

Summary/Conclusion : Our results show that B[a]P is able to increase the production of Mito‐LEVs and modify the glycolytic enzyme content of L‐EVs.

Funding : ANR ENDOMITOPAH/AAPG2023/CES 34.

The analysis of eicosanoids contained in macrophage‐derived extracellular vesicles

Riko Asai, Haruka Sei, Fumie Nakashima, Takahiro Shibata

Nagoya University, Japan

Introduction : Eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), are arachidonic acid‐derived lipid mediators with a wide range of effects on various biological processes, including inflammation and immunity. Although many studies have investigated the mechanism of biosynthesis and the biological activities of eicosanoids, how lipophilic eicosanoids exist and are transported under the aqueous environment of in vivo systems hasn't been focused on. Therefore, we hypothesized that extracellular vesicles (EVs) could transport eicosanoids, similar to nucleic acids and proteins. In this study, we analysed eicosanoids in EVs derived from activated macrophage‐like cells, which produce eicosanoids in response to inflammation.

Methods : Murine macrophage‐like RAW264.7 cells were stimulated with lipopolysaccharide (LPS) for 24 h, and the resulting culture media were collected and subjected to ultracentrifugation to isolate EVs. Eicosanoids were extracted from the EVs using the Bligh‐Dyer method. To conduct a high sensitivity analysis, samples were analysed by LC‐MS/MS following derivatization with 1‐(4‐(aminomethyl)phenyl)pyridin‐1‐ium chloride (AMPP). The amounts of eicosanoid‐related enzymes in isolated EVs were evaluated by immunoblot analysis. To analyse the localization of those enzymes in EVs, proteinase K‐treated EVs were subjected to immunoblot analysis.

Results : LC‐MS/MS analysis revealed that PGE2 and its precursor, arachidonic acid, were contained in EVs derived from LPS‐stimulated cells. We also showed that phospholipase A2 and cyclooxygenase‐2, enzymes involved in eicosanoid biosynthesis, were contained in EVs derived from LSP‐stimulated macrophages. Furthermore, the proteinase K protection assay indicated that these enzymes were localized, at least partially, inside the vesicle.

Summary/Conclusion : Our results suggest that EVs not only transport eicosanoids but may also be involved in the biosynthesis of eicosanoids.

Why are EVs special? Demonstrating the carrier effects using designer EVs

Presenter: Brian S. Dobosh

Emory University, USA

Introduction : EVs reflect the state of the cell they originate from and exert significant effects on recipient cells due to their cargo. However, this cargo can sometimes also be found in a soluble (non‐EV) fraction. The ‘carrier effects’ of EVs describe how cargo function changes as a result of being associated with an EV. The carrier effects include (1) concentrating signalling molecules; (2) protecting cargo; (3) targeting specific cell subsets; and (4) altering the functional properties of the cargo. Here, we set up a demonstration using designer EVs with epidermal growth factor (EGF), green fluorescent protein (EGFP), EGFP mRNA, or neutrophil elastase protein (a protease) to illustrate the carrier effects of EVs isolated from HEK293T cells.

Methods : EVs were purified by 300 kDa tangential flow filtration. Structure, size, concentration, and contaminating factors were evaluated by transmission electron microscopy, ELISA, nanoparticle tracking analysis, western blotting, and qRT‐PCR following MISEV2023 guidelines.

Results : (1) To generate EVs with EGF in the protein corona, HEK293T cells were transfected with a plasmid expressing EGF fused to the N‐terminus of PTGFRN, a protein known to localize to EVs. Soluble EGF or EGF+ EVs were applied to EGFR reporter cells. Despite the same concentration of EGF being used, EGF+ EVs showed 2 to 13 times as much signalling as soluble EGF. (2) EVs were loaded with EGFP mRNA using a CD63‐L7Ae system with in vitro transcribed EGFP as a control. Following RNase treatment, the EVs protected up to 95% of the EGFP mRNA, but the non‐EV‐bound EGFP mRNA was not detectable. 3) Single‐chain variable fragments (scFv) targeting CD14 were fused to PTGFRN‐EGFP and given to neutrophils (CD14+). EVs with anti‐CD14 scFv showed at least double the delivery efficiency compared to EVs without anti‐CD14 or expressing a control scFv. (4) Neutrophil elastase (NE) was incubated with HEK293T EVs to place it on the corona or used in a soluble form. NE+ EVs had 3 times as much specific activity compared to soluble NE, demonstrating that the EV changed the kinetics of the proteins in its corona.

Summary/Conclusion : Studying these carrier effects is essential to elucidate the mechanisms by which EV‐associated biomarkers exert their effects.

Modulation of environmental and microbiome interactions by bacterial extracellular vesicles

Presenter: Meta Kuehn

Duke University Medical Center, USA

Introduction : As technology has improved, the complexity of natural bacterial habitats has been revealed, resulting in the appreciation of an ever‐increasing number of interactions within polymicrobial communities. In addition to containing vast numbers of diverse bacterial species, these natural bacterial environments also contain secreted bacterial products and viruses. Bacterial extracellular vesicles (bEVs) are constitutively secreted during bacterial growth. bEVs have roles in virulence, inflammation, and interkingdom communication in host‐pathogen and native environments.

Methods : We have quantitated the induction and characterized the functions of outer membrane vesicles (OMVs) by Gram‐negative bacteria as a bacterial mechanism for protection against changes in environmental conditions, stressors, antibiotics, and phage. OMVs were purified from a variety of organisms and coincubated with heterologous organisms in media containing lethal antibiotic concentrations. By monitoring bacterial survival, these investigations demonstrated functional offensive and defensive consequences of bEV production in polymicrobial environments. To investigate other environmental interactions, OMVs coincubated with phage were characterized and these complexes were mixed with heterologous bacteria to monitor interspecies effects. Further, the transport of bEVs through an environmental matrix was measured, and a theoretical model for bEV travel was deduced.

Results : Induction of OMVs occurred for several Gram‐negative bacterial cultures that had been challenged with cationic antimicrobial peptides (CAMPs). Low concentrations of heterologous OMVs increased the ability of several bacterial species to survive lethal concentrations of CAMPs. However, in some cases, the CAMP activity was potentiated, suggesting a novel, bEV‐mediated mechanism of antibiotic activation within a polymicrobial community. Phage‐OMVs are known to form stable complexes, inactivating the phage. Unexpectedly, the stable phage‐OMV complexes from one bacterial species induced a specific mechanism of lysis in heterologous bacteria. The data further demonstrate bEV stability in the environment and their ability to deliver content at a distance without deposition on environmental surfaces.

Summary/Conclusion : Our results point to the complex and long‐distance interplay of communication, deflection, and attack by bEVs in the natural, polymicrobial environment.

Funding : National Science Foundation (NSF 1931309); Duke University Medical Center.

Investigations into EV production from nitrogen‐fixing bacteria found naturally in soil

Gülce Güralp 1,2 , Stuart James Lucas 1,2

1 Sabancı University, Türkiye; 2 Sabancı University Nanotechnology Research and Application Centre (SUNUM), Türkiye

Introduction : Nitrogen is the most significant yield‐limiting nutrient for potato (Solanum tuberosum), which is the world's fourth most important crop for human nutrition. Nitrogen fixation is a significant process mediated by nitrogen‐fixing bacteria among beneficial soil microbes to convert nitrogen gas to ammonium, which is one of the most biologically available forms for plants. Thus, plant‐microbe interactions are important for plant nutrition, health and survival. Extracellular vesicles (EVs)‐released by microbes provide the communication between plants and microbes through their compositions of proteins, lipids, small RNAs (sRNAs) and nucleic acids. Several studies have demonstrated that EVs‐released from bacteria contribute to stress responses, quorum sensing, biofilm formation and symbiotic relationships with plants. The study purposes to explore roles of EVs in the effects of nitrogen‐fixing bacteria on potato plants, with the ultimate aim of developing biofertilizers from nitrogen‐fixing bacteria and their EVs.

Methods : Endophytic bacteria have been isolated from surface‐sterilized tubers and roots of potato plants, collected from high‐yielding potato fields of Türkiye. Individual colonies sub‐cultured on fresh media have been evaluated for nitrogen fixation capacity by inoculating into Nfb medium and then will be identified by 16S rRNA and nifK gene sequencing. EV production from the nitrogen‐fixing bacteria into liquid medium will be optimized and characterized by nanoparticle tracking analysis, transmission electron microscopy and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and so forth, to reveal potential roles of EVs‐produced by them in nitrogen fixation. Beneficial nitrogen‐fixing bacteria and their EVs will be investigated for their effects on potato nutrition in controlled conditions.

Results : Three promising isolates with nitrogen‐fixing ability have been selected, which will be identified by sequencing. We hypothesize that EVs‐released by nitrogen‐fixing bacteria may carry biomolecules or genes related to nitrogen fixation into potato plants, help to regulate interactions with other microbes in the soil, and mediate plant‐endophyte symbiosis.

Summary/Conclusion : Characterization of the nitrogen‐fixing and EV‐producing bacteria identified in this study is expected to shed new light on communication mechanisms during plant‐endophyte symbiosis.

Funding : This study has been supported by the Scientific and Technological Research Council of Turkey [TUBITAK, 124O159].

Subtopic EV biogenesis 1

The adaptor protein CD2AP controls the biogenesis of syntenin exosomes

Barnabas Irmer 1,2 , Christian Preußer 3 , Elke Pogge von Strandmann 3 , Guido David 4 , Pascale Zimmermann 4,5 , Kerstin Menck 1,2,4

1 University of Münster, Germany; 2 University Hospital Münster, Germany; 3 Philipps University Marburg, Germany; 4 Centre de Recherche en Cancérologie de Marseille, France; 5 KU Leuven, Belgium

Introduction : The syndecan binding protein syntenin‐1/SDCBP is a central regulator for the biogenesis of exosomes, a specific subclass of small extracellular vesicles (sEVs). Together with the ESCRT‐associated protein Alix, syntenin supports the budding of late endosomal membranes, resulting in the formation of intraluminal vesicles (ILVs), which are subsequently released as exosomes into the extracellular space. Proteomic studies have identified the intracellular adaptor protein CD2AP on exosomes. Although it is a known direct binding partner of several important regulators of the syntenin exosomal pathway, including Alix, its involvement in the biogenesis of exosomes has not been tested yet.

Methods : EVs were isolated by differential ultracentrifugation and comprehensively characterized by electron microscopy, immunoblots and NTA. Knockdown/overexpression approaches were used to modulate the expression of syntenin, CD2AP/CMS and its paralogue CIN85/SH3KBP1 in human MCF‐7 breast cancer cells. Effects on sEV number, size and protein cargo were tested by immunoblots and NTA, respectively. The overexpression of mCh‐tagged syntenin and mutant Rab5(Q79L), which induces the formation of enlarged endosomes, allowed analysing endosomal budding by confocal microscopy.

Results : CD2AP and CIN85 were expressed at high levels on sEVs and only barely detectable on large EVs. The expression of CD2AP, but not CIN85, on sEVs was dependent on syntenin. Vice versa, knockdown and overexpression of CD2AP identified it as a novel regulator of the syntenin levels on sEVs. Of note, vesicular alix and syndecan‐4 expression remained largely unaffected by CD2AP modulation. CD2AP co‐localized with syntenin in Rab5(Q79L)‐positive endosomes and controlled its endosomal budding dependent on Alix, Arf6 and Pld2. Using CD2AP serial deletion constructs, the C‐terminus of CD2AP, which contains four potential actin binding sites as well as a coiled‐coil domain, was observed to be essential for its effect on Syntenin endosomal budding and export to exosomes.

Summary/Conclusion : Taken together, our results shed further light on the biogenesis of exosomes and identified CD2AP as a novel regulator of Syntenin budding at late endosomal compartments and its exosomal export.

Funding : This project was supported by the DFG (projects 424252458, ME4573/1‐1), Else Kröner‐Fresenius‐Foundation (project 2019_A162), IMF of the University of Muenster (project ME121914 ) and Heidenreich‐von‐Siebold programme of the University Medical Center Goettingen.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion

Di‐Ao Liu 1 , Kai Tao 2 , Bin Wu 1 , Ziyan Yu 1 ,Malwina Szczepaniak 2 , Matthew Rames 2 , Changsong Yang 1 , Tatyana Svitkina 1 , Yueyao Zhu 1 , Fengyuan Xu 3 , Xiaolin Nan 2 , Wei Guo 1 *

1 Department of Biology, School of Arts & Sciences, University of Pennsylvania, Philadelphia, USA; 2 Program in Quantitative and Systems Biology, Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon, USA; 3 Department of Bioengineering, College of Engineering, University of Pennsylvania, Philadelphia, USA

Introduction : Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs can also fuse with lysosomes for degradation. How MVEs are transported to the plasma membrane rather than to lysosomes is unclear. Our study aims to understand the basic mechanism by which MVEs are directed to the plasma membrane for exosome secretion.

Methods : Cell culture, nanoparticle tracking (NTA), live cell microscopic imaging, super‐resolution fluorescence microscopic imaging, electron microscopy, and western blot.

Results : A conversion of phosphatidylinositol‐3‐phosphate (PI(3)P) to phosphatidylinositol‐4‐phosphate (PI(4)P) catalysed by myotubularin 1 (MTM1) and phosphatidylinositol 4‐kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation and exocyst function blocked exosomal secretion of programmed death‐ligand 1 (PD‐L1), a key immune checkpoint protein in tumour cells, and led to its accumulation in lysosomes.

Summary/Conclusion : Our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.

Funding : NIH R35 GM141832.

Full‐length tRNAs lacking a functional CCA tail are selectively sorted into the lumen of extracellular vesicles

Chantal Scheepbouwer 1,2,3,4 , Ernesto Aparicio 5 , Cristina Gómez‐Martin 1,4 , Monique A. J. van Eijndhoven 1,4 , Esther E. E. Drees 1,4 , Leontien Bosch 1,4 , Daphne de Jong 1 , Thomas Wurdinger 3 , Josée Zijlstra 6 , Michael Hackenberg 5 , Alan Gerber 2,3* and D. Michiel Pegtel 1,4*

1 Department of Pathology, Amsterdam UMC, VU University, Amsterdam, The Netherlands; 2 Cancer Center Amsterdam, Cancer Biology, the Netherlands; 3 Department of Neurosurgery, Amsterdam UMC, VU University, Amsterdam, The Netherlands; 4 Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, the Netherlands; 5 Faculty of Science, Department of Genetics, University of Granada, Granada, Spain; 6 Department of Hematology, Cancer Center Amsterdam, Amsterdam UMC, VU University, Amsterdam, The Netherlands Alan Gerber and D. Michiel Pegtel contributed equally to this study .

Introduction : High‐throughput sequencing has advanced our understanding of nucleic acid content in extracellular vesicles (EVs), mainly focusing on short non‐coding RNAs (ncRNAs) under 50 nucleotides. However, the composition and abundance of longer ncRNAs in EVs are less well characterized. To address this gap, tailored next‐generation sequencing (NGS) protocols have been developed to minimize length bias from conventional RNA sequencing. Our study used the ALL‐tRNAseq approach, allowing the detection of a diversity of structured full‐length RNA species, including transfer tRNA (tRNA), small nucleolar RNA (snoRNA), and Y RNA.

Methods : Small RNA in the 18–120 nt size range from four cell lines, including cellular, EV‐enriched, and non‐vesicular extracellular particle (NVEP) fractions, was sequenced using an adapted ALL‐tRNAseq approach. Data processing was performed using sRNAbench with normalization via the NormSeq web server.

Results : Our analysis confirmed a predominance of tRNA molecules, constituting 60%–85% of assigned reads across all samples. Sequencing and northern blotting confirmed the presence of mature, full‐length tRNAs in both cells and in the lumen of EVs. The tRNA modification profiles showed similar misincorporation signatures in cells and EVs, with no significant differences impacting EV secretion, suggesting that EV‐tRNAs primarily derive from the mature tRNA pool. We assessed 3′ CCA tails of tRNAs, finding that 98% of cellular tRNAs contained the essential CCA sequence, while EV‐tRNAs exhibited substantial truncation. We also analysed plasma EVs (pEVs), revealing that CCA‐truncated tRNAs remained the predominant component in both in vitro and in vivo.

Summary/Conclusion : Our findings challenge previous assumptions by demonstrating that tRNAs in EVs are primarily non‐functional full‐length molecules lacking the essential CCA tails required for aminoacylation. Despite their inability to participate in translation, these fully modified tRNAs resemble cellular counterparts and likely originate from previously functional cytoplasmic tRNAs. The consistent CCA truncation pattern across all tRNA types in EVs suggests a potential sorting mechanism for mature tRNAs. Altogether, this study highlights an important and underappreciated aspect of small RNA composition in EVs.

Funding : Stichting CCA, MRD Hodgkin Lymphoma, and AQrate TKI.

Secretion of Rab31 Play a Role Exosome Biogenesis Under Hypoxic Condition in Adipogenic Mesenchymal Stem Cells

H. Seda Vatansever 1,2 , Hilal Kabadayı Ensarioğlu 1 , Aslınur Aktas 1 , Burak Kutlu Aydın 1 , Nadire Kıyak 2 , Mehmet Vatansever 2

1 Manisa Celal Bayar University, Turkey; 2 Near East University, Turkey

Introduction : Microvesicular endosomes (MVEs) are characterized by containing intraluminal vesicles (ILVs), which can be controlled by multiple mechanisms. Exosome biogenesis is controlled by the ESCRT (endosomal sorting complex for transport)‐dependent and independent pathways. Rab31 plays a role in an ESCRT‐independent pathway. High intracellular levels of Rab31 are directed into the MVEs to form ILVs, which are then secreted as exosomes. Hypoxia modulates the secretion, composition, and function of exosomes, especially in various cancer. While hypoxia increased exosome release by upregulating Rab27a and reducing Rab7; however, the secretion of Rab31 by hypoxia remains unclear. Recent studies have demonstrated that the therapeutic benefits of MSCs are dependent on their extracellular vesicles (EVs). MSCs‐derived EVs can induce different immunosuppressive effects and contribute to the immunological tolerance. In our study, we investigated the detection of Rab31 during exosome secretion in adipogenic mesenchymal stem cells (AMSCs) in 2D and 3D culture conditions under normoxic and hypoxic environments.

Methods : AMSCs were obtained from the ATCC culture collection. They were cultured with 10% exososme‐free FBS, 1% pen‐strepromycin, 1% L‐glutamine included in DMEM. For the 2D condition, cells were cultured in a routine culture plate; for 3D, cells were cultured in Matrigel. A hypoxic condition was performed in a chamber containing 5% CO 2 , 5% O 2 , 90% N 2 . All cells were cultured for 48 h, and after fixation of cells with 4% paraformaldehyde, the presence of Rab31, Rab7, CD9, and CD63 proteins was evaluated using an indirect immunorexidase technique.

Results : Negative or very weak CD9 immunoreactivity was detected in both 2D and 3D hypoxic and normoxic conditions. While immunoreactivity of CD63 was moderate in 2D hypoxic and normoxic conditions, this immunoreactivity was strong and similar in both 3D hypoxic and normoxic conditions. Rab31 immunoreactivities were higher than Rab7 in all conditions. In addition, Rab31 immunoreactivity was higher in the 3D hypoxic condition than in the normoxic condition.

Summary/Conclusion : In our results demonstrated that exosome secretion from AMSCs was affected by culture conditions. Especially hypoxic conditions trigger secretion of Rab31, and co‐localization of CD63 and Rab31 under hypoxic conditions pointed out that exosome biogenesis is controlled by Rab31 in AMSC cells under hypoxic conditions.

Funding : This work was supported by the Research Fund of Near East University (Project number: SAG‐2023‐1‐026).

PCBP2‐dependent secretion of miRNAs via extracellular vesicles contributes to the EGFR‐driven angiogenesis

Presenter: Hou‐Fu Xia

Wuhan University, China (People's Republic)

Introduction : The epidermal growth factor receptor (EGFR)‐driven angiogenesis is a crucial process observed across various solid malignancies, facilitating tumour progression and therapy resistance, particularly by modulating extracellular vesicles (EVs)‐mediated intercellular communication. Previous studies, including our own, have highlighted EGFR's influence on miRNA production and the pro‐angiogenic effects of EV‐miRNAs. However, the precise mechanism by which EGFR controls miRNA content within EVs remains unclear.

Methods : Firstly, cell co‐culture and murine tumour models were used to investigate the effects of EGFR overexpression on the pro‐angiogenic ability and representative cargo composition of oral squamous cell carcinoma‐derived EVs (OSCC‐EVs). We then adopted small‐RNA sequencing to compare the differences in miRNAs carried by OSCC‐EVs with or without EGFR overexpression and conducted functional enrichment analysis and motif analysis on these differential miRNAs. Next, we used miRNA pull‐down experiments to identify candidate molecules involved in the sorting of these characteristic miRNAs. Finally, we validated the role of the candidate sorting protein in promoting the secretion of these characteristic miRNAs to regulate the EGFR‐driven tumour angiogenesis based on tissue specimens, cell lines, murine tumour models and reported data.

Results : EGFR overexpression significantly enhances the pro‐angiogenic effects of OSCC‐EVs, accompanied by a marked increase in the content of nucleic acid cargo in OSCC‐EVs. Small‐RNA sequencing identified a group of miRNAs that were significantly enriched in OSCC‐EVs due to EGFR overexpression, which primarily functioned in angiogenesis and shared a characteristic ‘GGGU’ motif. EGFR overexpression significantly enhanced the binding of PCBP2 with ‘GGGU’ motif‐containing miRNAs, thereby increasing their secretion through EVs to play a tumour angiogenesis promoting role. Mechanistically, EGFR overexpression upregulates PCBP2 through transcriptional regulation in OSCC cells. What's more, PCBP2‐depletion impaired the EGFR‐driven tumour angiogenesis by inhibiting the secretion of pro‐angiogenic miRNAs via sEVs.

Summary/Conclusion : EGFR overexpression upregulates PCBP2 through transcriptional regulation, followed by enhanced loading of angiogenesis‐associated miRNAs into sEVs by PCBP2 directly recognizing sEV motifs.

Funding : This work was supported by the National Natural Science Foundation of China (82101036, 82341023).

Caveolin‐1 overexpression induces extracellular vesicle release and protein cargo impairment in an in vitro model of rhabdomyosarcoma

Rachele Agostini 1 , Emanuela Polidori 1 , Paola Ceccaroli 1 , Laura Graciotti 2 , Stephanie Fondi 1 , Michela Battistelli 1 , Francesca Luchetti 1 , Silvia Codenotti 3 , Gabriella Pocsfalvi 4 , Massimiliano Bonafè 5 , Joao Ferreira 6 , Alessandro Fanzani 3 , Vilberto Stocchi 1 , Michele Guescini 1

1 Department of Biomolecular Sciences (DISB), Università degli Studi di Urbino Carlo Bo, Urbino, Italy; 2 Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy; 3 Department of Molecular and Translational Medicine (DMMT), Università degli Studi di Brescia, Brescia, Italy; 4 National Research Council (CNR), Napoli, Italy; 5 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Università di Bologna, Bologna, Italy; 6 Department of Proteostasis and Intercellular Communication, NOVA Medical School, Lisbon, Portugal

Introduction : Caveolin‐1 (CAV1) is a transmembrane protein enriched in caveolae and lipid rafts, which are involved in signal transduction and intracellular trafficking processes. In rhabdomyosarcoma (RD) CAV1‐overexpression promotes tumour growth and metastatic dissemination. Since extracellular vesicles (EVs), lipid‐bound vesicles secreted by all cells, have a well‐established role in cancer disease, the present work aims to investigate if CAV1‐overexpression impacts the EV machinery and if these EVs can contribute to their increased aggressiveness.

Methods : Three RD cell lines were employed for the study: RD‐Mock (transfected with an empty vector), RD‐CAV1‐F0 engineered for CAV1 overexpression, and RD‐CAV1‐F2 derived from the second generation of lung metastases after RD‐CAV1‐F0 injection in mice. EVs were isolated from conditioned media by sequential ultracentrifugation, and small‐EVs (sEVs) were further purified by density gradient centrifugation. For EV characterization, nanoparticle tracking analysis (NTA), western blot (WB), flow cytometry (FC) and proteomic analysis were employed. Intracellular trafficking was evaluated by immunofluorescence staining, WB analysis, endosome isolation and live cell imaging. Migration and proliferation assays were conducted on HUVECs. Results: The obtained data showed that RD‐CAV1 cells release more EVs compared to RD‐Mock. WB and FC analyses revealed that RD‐CAV1 sEVs exhibit TSG‐101 and Alix, but the expression of other typical exosomal markers CD63, CD81, and CD9 is much lower than in RD‐Mock. Proteomic analysis extended this alteration to other proteins, showing an overall reduction in protein loading and expression. These findings are combined with an increased intracellular trafficking in RD‐CAV1 cells, suggesting that CAV1‐overexpression induces an alteration of EV machinery and release. Moreover, RD‐CAV1 sEVs significantly increased HUVEC proliferation and migration compared to the control.

Summary/Conclusion : Taken together, these data demonstrate that CAV1‐overexpression critically affects RD‐intracellular trafficking and EV cargo and release, which might contribute to RD‐CAV1 increased aggressiveness. Future studies will focus on the characterization of RD‐EV lipid‐ and miRNA‐loading and on the evaluation of RD‐EV effects in other cell types typical of tumour niche.

Distinct signature proteins control the sEV biogenesis in cancer cells and stem cells

Yashvi Sharma, Sujata Mohanty

Stem Cell Facility, DBT‐Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India

Introduction : Mesenchymal stem cell (MSC)‐derived small extracellular vesicles (sEV) have emerged as a promising therapeutic candidate in regenerative medicine. However, challenges such as low yield and scalability hinder their broader clinical application. This study compared the biogenesis of MSC‐sEV with that of the more widely studied HeLa‐sEV, focusing on selected endosomal sorting complex required for transport (ESCRT) proteins (HRS, STAM, TSG101) and the ESCRT accessory protein ALIX. By identifying key regulators of sEV biogenesis in bone marrow (BM) MSCs, this research seeks to elucidate targets and strategies to enhance sEV yield for therapeutic use, revealing shared molecular markers to guide therapeutic strategies.

Methods : BM‐MSCs, obtained with informed consent (IC‐SCR/120/21(R)), were cultured in serum‐free media and characterized per ISCT 2006 guidelines. Both HeLa cells and MSCs were grown to 70% confluence for siRNA‐based lipofection. Transfection efficiency was confirmed using FITC‐tagged siRNA via fluorescence microscopy and flow cytometry. Key ESCRT proteins—HRS, STAM, TSG101, and ALIX—were silenced, with efficiency validated by qPCR and Western blot. sEVs were isolated via ultracentrifugation and analysed following MISEV 2023 guidelines using NTA, TEM, and Western blot to assess changes in sEV size, concentration, morphology, and protein markers. The effect of silencing on sEV markers CD63, CD81, and CD9 was evaluated by immunofluorescence and Western blot in both cell types, followed by assessments of regenerative miRNA packaging in MSC‐sEV.

Results : Results showed that ALIX silencing in BM‐MSCs led to a 3.65‐fold reduction in sEV concentration, whereas in HeLa cells, sEV yield remained nearly unchanged, as per both total protein and total number analysis. In MSCs, ALIX silencing significantly reduced tetraspanin expression in both cells and sEV, while STAM emerged as a primary regulator in HeLa cells. Moreover, ALIX silencing was found to significantly reduce the miRNA packaging within MSC‐sEV, thereby contributing to their therapeutic potential.

Summary/Conclusion : This study identifies ALIX as a crucial regulator for boosting MSC‐sEV yield, while in HeLa cells, ESCRT‐dependent pathway plays a key role. By investigating the mechanisms behind endosomal sEV biogenesis, it seeks to optimize production, bridging discovery with clinical translation to standardize MSC‐sEV as a platform for cell‐free regenerative therapies.

Evaluation of engineering tetraspanin knockout cells with tetraspanin fusion constructs

Presenter: Doste Rashid Mamand

Karolinska Institute, Sweden

Introduction : Extracellular vesicles (EVs) are lipid‐enclosed vesicles with diverse biomolecular content, including RNA and proteins. One such protein family which is highly enriched on the EV surface is tetraspanins, such as CD63, CD81, and CD9. However, their exact role in EV biogenesis and cargo sorting is still undetermined. In this study, we aimed to investigate the role of tetraspanins in EV biogenesis and cargo sorting of both endogenous and engineered protein cargo.

Methods : To understand the role of tetraspanins in EV biogenesis and cargo sorting, HEK293T knockouts (KO) of either CD9, CD63, CD81, or all of them together (PanKO) were generated by using CRISPR‐Cas9. Nanoparticle tracking analysis and single vesicle flow cytometry were used to characterise EVs. For determining the protein cargo in EVs, mass spectrometry proteomics was employed. For determining the role of tetraspanins on EV engineering, bioluminescent labelling of EVs were used as means for determining EV engineering.

Results : Interestingly, no differences were observed in EV numbers between CD9KO, CD63KO, CD81KO and HEK293T WT cells, whereas PanKO cells showed a slight decrease in EV number as compared to WT cells. Knockout of different tetraspanin proteins showed similar cargo enrichment with differential sorting into EVs. Transduction with thermoLuc (tLuc)‐CD63 generated a higher yield of engineered EVs in CD9KO, PanKO, CD63KO, and CD81KO cells compared with WT cells. Similarly, when expressing tLuc‐CD9 and tLuc‐CD81, CD9KO, PanKO, CD63KO and CD81KO cells compared to WT cells produced more engineered EVs than WT cells. In parallel, proteomic analysis revealed decreased levels of corresponding tetraspanins in the EVs from KO cells, indicating partial competition between endogenous and introduced tetraspanins.

Summary/Conclusion : The data emphasizes the role of tetraspanins in EV biogenesis and protein cargo sorting into EVs, which can be manipulated to optimize engineering.

Role of syntenin in autophagy‐dependent secretion

Presenter: Cristóbal A. Cerda‐Troncoso

KU Leuven, Belgium

Introduction : Non‐conventional secretion, including secretory autophagy or autophagy‐dependent secretion, but also the release of cargo through extracellular vesicles (EVs), is important for cell‐to‐cell communication. Yet, molecular mechanisms supporting autophagy‐dependent secretion and its relationship with EV release remain obscure. Previous studies indicate that syntenin, a protein supporting the production of small‐EVs of endosomal origin (exosomes), may be associated with classical degradative autophagy.

Methods : Utilizing loss‐ and gain‐of‐expression approaches for syntenin in MCF7 cells, we evaluated (i) the levels of autophagic proteins in EVs fractions and cell lysates, (ii) the formation and morphology of autophagosomes, and (iii) the colocalization of autophagy proteins with endosomal compartments.

Results : We found that syntenin controls the cellular levels and the exosomal secretion of proteins involved in different steps of autophagy. Our data also indicate that syntenin acts as a down‐regulator of autophagosome biogenesis but as a promoter of autophagosome‐lysosome and autophagosome‐endosome fusion.

Summary/Conclusion : The present study thus identifies a dual role for syntenin in autophagy and one piece of the molecular framework that governs the interplay between exosomes and autophagy‐dependent secretion.

Funding : KU Leuven Research Coordination Office. ANID, Becas Chile‐Postdoctorado No. 74240086.

Hypoxia regulates small extracellular vesicle biogenesis and cargo sorting through the HIF‐1α/HRS signalling pathway in head and neck squamous cell carcinoma

Presenter: Yiman Wang

Wuhan University, China (People's Republic)

Introduction : Small extracellular vesicles (sEVs) serve as crucial messengers within the hypoxic tumour microenvironment, significantly influencing cancer progression. Despite their importance, the precise mechanisms by which hypoxia affects sEV biogenesis remain poorly understood. This study aims to identify key regulators of sEV biogenesis and cargo sorting in head and neck squamous cell carcinoma (HNSCC) cells under hypoxic conditions.

Methods : Protein cargoes of normoxic or hypoxic HNSCC cells and their sEVs were detected by mass spectrometry. RNA sequencing and flow cytometry were conducted on macrophages exposed to specific sEVs. Furthermore, spatial transcriptomics and immunohistochemistry analyses were performed on clinical samples.

Results : Our findings indicate that hypoxia significantly increases sEV secretion and alters their cargo composition in HNSCC cells. We observed that hepatocyte growth factor‐regulated tyrosine kinase substrate (HRS, also known as HGS), a key subunit of ESCRT‐0, is upregulated under hypoxic conditions, leading to the activation of the endosomal system and reduced degradation of multivesicular bodies (MVBs). Depletion of HRS resulted in altered packaging of mitochondria‐related proteins into sEVs, which promoted a pro‐tumourigenic phenotype in macrophages. Additionally, we demonstrated that HRS is transcriptionally activated by hypoxia‐inducible factor‐1α (HIF‐1α). Spatial transcriptomics and immunohistochemistry revealed a positive correlation between HRS and HIF‐1α expression.

Summary/Conclusion : This study establishes a novel link between the hypoxic response, sEV biogenesis, and cargo packaging in HNSCC cells. Our findings enhance the understanding of how the hypoxic tumour microenvironment influences sEV biogenesis and their subsequent role in cancer progression.

Ageing‐induced extracellular vesicles from lung cells carry a specific SASP

Efthymios Fousekis‐Papakonstantinou 1 , Arghavan Gholamalipoortehrani 1 , Maria Camila Melo‐Narváez 1,2 , Amine Chahin 1 , Anna‐Lena Rupp 1 , Isabell‐Valerie Burhorst 1 , Thomas Heimerl 4 , Gert Bange 4 , Katrin Laakmaan 1 , Bernd Schmeck 1,3,5 , Anna Lena Jung 1,3 , Mareike Lehmann 1,2,5

1 Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps University Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany; 2 Comprehensive Pneumology Center (CPC), Institute of Lung Health and Immunity (LHI), Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Germany; 3 Core Facility‐Extracellular Vesicles, Philipps University Marburg, Marburg Germany; 4 Center for Synthetic Microbiology (SYNMIKRO), Philipps‐Universität Marburg, Marburg, Germany; 5 Institute for Lung Health (ILH), Giessen, Member of the German Center for Lung Research (DZL)

Introduction : Chronic lung diseases (CLDs) are among the leading causes of death worldwide, with incidence increasing with age. A major ageing hallmark is cellular senescence, a stress‐induced response reducing proliferative capacity. The Senescence‐Associated Secretory Phenotype (SASP) impacts CLD pathogenesis, with extracellular vesicles (EVs) playing a key role in SASP‐related cell communication. The lung's gas exchange unit includes multiple cell types, such as endothelial cells, fibroblasts, and alveolar epithelial cells, which show senescence markers in ageing‐associated chronic lung diseases. We hypothesize that ageing differentially alters EV protein cargo and secretion in those cells, thereby altering their SASP.

Methods : We induced senescence in alveolar epithelial cells (A549), fibroblasts (MRC5), and endothelial cells (HULEC) by different inducers (H 2 O 2 and doxorubicin) before EV isolation and characterization. We compared the quantity, size, protein cargo, and morphology of EVs from untreated and treated cells using NanoFCM, transmission electron microscopy (TEM), and flow cytometry. Furthermore, we isolated EVs from mouse bronchoalveolar lavage (BAL) fluid and examined differences between young and old mice regarding EV quantity, quality, protein cargo, and size using NanoFCM, TEM, ExoView, and flow cytometry. We also investigated the difference in pro‐inflammatory cytokine levels between soluble and EV‐bound fractions by flow cytometry.

Results : Treatment with H2O2 and doxorubicin induced senescence in a dose‐dependent manner. We detected senescence‐associated alterations in EV numbers and cargo. Senescent lung cells produced increased EV numbers compared to untreated cells, while EV size and shape remained unchanged. Moreover, we detected an age‐associated increase of EV surface proteins, including CD29 and CD81. We also observed an age‐associated size reduction on mouse BAL EVs. Finally, we detected an increase in proinflammatory cytokine concentration in BAL EVs compared to soluble BAL proteins.

Summary/Conclusion : Our results showed senescence‐dependent differences in EV production and protein cargo. Senescent lung cells secreted higher levels of EVs that carried specific cargo. Furthermore, we detected age‐dependent changes in EV size and protein cargo, including cytokines. Our findings suggest a specific EV‐SASP secreted by senescent lung cells and warrant further functional studies exploring their contribution to age‐associated chronic lung diseases.

Hypoxia promotes re‐localization of Rab17 into endolysosomal compartments, inducing the release of pro‐angiogenic small extracellular vesicles in A549 cancer cells

Belén Gaete‐Ramírez 1 , Claudio Retamal 1 , Vicente A. Torres 2,3,4*, Manuel Varas‐Godoy 1,3,5 *

1 Centro de Biología Celular y Biomedicina (CEBICEM), Facultad de Medicina y Ciencia, Universidad San Sebastián, Chile; 2 Institute for Research in Dental Sciences, Faculty of Dentistry, University of Chile, Chile; 3 Advanced Center for Chronic Diseases (ACCDiS), University of Chile, Chile. 4 Millennium Institute on Immunology and Immunotherapy, University of Chile, Chile; 5 Centro Ciencia & Vida, Fundación Ciencia & Vida, Chile

Introduction : Hypoxia is a common feature of the tumour microenvironment, which is associated with tumour angiogenesis and release of small extracellular vesicles (sEV). Our recent studies show that hypoxia induces the expression of the Rab5‐GEF ALS2, activating the endosomal trafficking. Additionally, we described that hypoxia promotes the secretion of pro‐angiogenic sEV in an ALS2‐dependent manner by mechanisms that remain unclear. Rab17, a Rab GTPase that participates in vesicular trafficking and fusion with the plasma membrane, has been described as an ALS2‐interacting protein. Therefore, we propose that in hypoxia, Rab17 changes its endosomal distribution in an ALS2‐dependent manner, thereby impacting the release of pro‐angiogenic EVs.

Methods : A549 lung carcinoma cells were cultured under hypoxic conditions (1% O 2 ) for 24 h. Subsequently, cells were processed for immunofluorescence with Rab17 and different endosomal markers (EEA1, Rab5, Rab11, Rab27a, and Rab7). Colocalization analyses were conducted using confocal microscopy. To evaluate the requirement of ALS2 in the localization of Rab17 in endolysosomal compartments, we knocked down ALS2 by shRNA. To analyse the contribution of Rab17 in the release of pro‐angiogenic sEV, we used a CRISPR interference system in our model, and we isolated sEV by size exclusion chromatography. The sEV were characterized with sEV markers by western blot, and size distribution and concentration were analysed by nanotracking particle analysis. Pro‐angiogenic properties of sEV were evaluated by tube formation assay.

Results : Hypoxia increased Rab17 colocalization with the early endosomal markers Rab5/EEA1 and multivesicular endosome (MVE) marker CD63. In this context, Rab17 shows higher colocalization with Rab27a (secretory MVE) and a decrease with Rab7 (degradative MVE). The distribution of Rab17 into the endolysosomal compartments is dependent on ALS2. Finally, knockdown Rab17 impacted the release of pro‐angiogenic sEV under hypoxic conditions.

Summary/Conclusion : These data suggest that under hypoxic conditions, ALS2 might be recruiting Rab17 to endolysosomal compartments, contributing to the secretion of a pro‐angiogenic sEV subpopulation in A549 cells. This could represent a novel mechanism by which tumour cells promote angiogenesis under hypoxic conditions.

Funding : Fondecyt‐1230983, ANID Fellowship 21230341, Fondecyt‐1220517, ANID/BASAL/ FB210008

A comparative study of inhibitors targeting extracellular vesicle biogenesis

Allegra Angenendt 1,2 , Janes Efing 1,2 , Uwe Hansen 3 , Jürgen Eirich 4 , Iris Finkemeier 4 , Witold Szymanski 5,6 , Johannes Graumann 5,6 , Annalen Bleckmann 1,2 , Kerstin Menck 1,2

1 Department of Medicine A, University Hospital Münster, Germany; 2 West German Cancer Centre, University Hospital Münster, Germany; 3 Institute for Musculoskeletal Medicine, University Hospital Münster, Germany; 4 Institute of Plant Physiology, University of Münster, Germany; 5 Philipps‐University Marburg, Institute of Translational Proteomics, Germany; 6 Philipps‐University Marburg, Core Facility Translational Proteomics, Germany

Introduction : Tumour cells release extracellular vesicles (EVs) that are vital for tumour progression. Several tumour EV subpopulations have been identified, including large oncosomes (LOs), large EVs (lEVs), and small EVs (sEVs). However, specific markers for lEVs and LOs, as well as the pathways regulating their biogenesis, remain largely unknown. The aim of this project, therefore, was to compare chemical inhibitors, targeting factors previously associated with LOs and/or lEVs, for their efficiency in blocking the release and cargo loading of the distinct EV subpopulations.

Methods : EVs were isolated from MCF‐7 (breast cancer), HT‐29 (colorectal cancer), and H1975 (lung cancer) cell lines using differential ultracentrifugation and were characterized by electron microscopy, nanoparticle tracking analysis (NTA), mass spectrometry, and immunoblotting. Six inhibitors targeting key factors involved in lEV biogenesis were selected, and their maximum tolerated doses were assessed in the three cancer cell lines using MTT and Annexin V/7‐AAD viability assays. Changes in EV secretion and cargo loading upon inhibitor treatment were evaluated using NTA and proteome analysis.

Results : Using viability assays, we selected inhibitor doses that did not affect cell viability or growth. The size of the secreted EVs upon inhibitor treatment remained likewise unchanged. The Arf6 inhibitor NAV‐2729 caused the strongest effect on lEV and LO, but not sEV, secretion rates, albeit in a cell line‐specific manner. In MCF‐7 cells, it not only reduced the number of secreted lEVs and LOs but also their loading with the marker proteins Rgap1 and CK18, or Arf6, respectively. In HT‐29 and H1975 cells, treatment with the RhoA inhibitor Rhosin resulted in the strongest decrease in LO and lEV protein cargo loading. The expression of selected marker proteins on sEVs remained largely unaffected in all three cell lines with any of the inhibitors.

Summary/Conclusion : Our results indicate key differences in the biogenesis of the three EV subpopulations. While inhibition of Arf6 and RhoA had the strongest effects on LO and/or lEV secretion in the tested cell lines, we observed strong cell line‐ and EV‐specific differences. Thus, the identification of a general inhibitor of EV secretion remains challenging.

Funding : This work was funded by the Wilhelm‐Sander Foundation (project 2022.139.1).

Regulatory mechanisms of sEV secretion via UBXN family proteins

Presenter: Jun Shirai

Tokyo Metropolitan University, Japan

Introduction : Valosin‐containing protein (VCP)/p97/CDC48 is an AAA+‐type ATPase that is crucial for endosomal sorting. Despite its importance in multivesicular body (MVB) biogenesis, the regulatory mechanisms of small extracellular vesicle (sEV) secretion mediated by VCP remain poorly understood. VCP demonstrates functional and spatial specificity through interactions with various adapters, including UBX domain‐containing proteins (UBXNs). Therefore, we investigated the potential roles of VCP and the UBXN family proteins in regulating sEV secretion in cultured cells.

Methods : sEVs were purified from HeLa cells using ultracentrifugation or the phosphatidylserine affinity method. Purified sEVs were validated by detecting multiple markers by Western blotting. Characterization of sEVs was conducted using nanoparticle tracking analysis and transmission electron microscopy. MVB imaging focused on the marker CD63, employing confocal microscopy.

Results : To determine whether VCP regulates sEV secretion, we analysed sEVs from HeLa cells treated with the VCP inhibitor NMS‐873 and found that VCP inhibition significantly enhances sEV secretion. To identify VCP adapters involved in sEV secretion, we performed a knockdown screening of UBXN family proteins. As a result, the depletion of UBXN3B increased sEV marker secretion. Importantly, there were no significant changes in particle size following UBXN3B knockdown, suggesting that UBXN3B regulates sEV secretion. Additionally, the knockdown of Rab27a decreased sEV secretion in UBXN3B‐depleted cells, indicating that UBXN3B regulates sEV secretion in a Rab27a‐dependent manner, which involves MVB trafficking. UBXN3B is known to function in endoplasmic reticulum‐associated degradation (ERAD). However, since the suppression of UBXN3B led to an increase in sEV secretion even under conditions of ER stress induced by tunicamycin, it is possible that UBXN3B regulates sEV secretion in an ERAD‐independent manner. Localization analysis revealed that endogenous UBXN3B is expressed at higher levels in close proximity to the MVB marker CD63, and its roles in MVB positioning and maturation are under investigation.

Summary/Conclusion : We demonstrate that VCP negatively regulates sEV secretion and identify UBXN3B, a VCP adapter, as a novel regulator of sEV secretion. In this presentation, we will report the latest findings regarding the regulatory mechanisms of MVB via UBXN3B and the role of other UBXN adapters in sEV secretion.

Funding : This work was supported by JSPS KAKENHI Grant Number JP24KJ1860.

ECM rigidity boosts the biogenesis of extracellular vesicles in breast cancer cells via the TSG101‐HSP90 axis

Pratiksha Mahadik 1 , Sejal Patwardhan 1,2

1 Advanced Centre for Treatment Research Education in Cancer (ACTREC) Tata Memorial Centre, India; 2 Homi Bhabha National Institute, India

Introduction : Introduction: Extracellular matrix (ECM) stiffening owing to excess deposition and crosslinking of collagen fibres is instrumental in breast cancer progression. ECM stiffness influences tumour behaviour and fate through direct and indirect effects. Recent reports highlight the profound role of ECM‐stiffness‐tuned exosomes in promoting tumour growth and invasion. However, the exact mechanism of ECM‐stiffness‐induced biogenesis and release of extracellular vesicles (EVs) remains largely elusive.

Methods : Breast cancer cells (MCF‐7 and MDA‐MB‐231) were cultured on stiffness‐tunable hydrogels of stiffness mimicking normal mammary stroma (soft ECM) and metastatic breast tumour (stiff ECM). EVs were isolated from culture supernatant by a kit‐based method and characterised by western blotting, scanning electron microscopy, and nanoparticle tracking analyser (NTA). The number and pattern of multivesicular bodies (MVBs) in cells were studied using transmission electron microscopy (TEM) and immunofluorescence for CD63, lysobisphosphatidic acid (LBPA). Specific inhibitors or shRNA, co‐immunoprecipation and proximity ligation assays were employed to gain mechanistic insights.

Results : The quality and purity of EV preparation were confirmed by the enrichment of EV‐specific markers (CD9, flotillin‐1) and the absence of cytosolic markers (cytochrome‐c, calreticulin) in both soft and stiff matrices. EV samples from both the substrate stiffness showed typical cup‐shaped morphology and size ranging from 70 to 130 nm. Interestingly, cells cultured on stiff matrices exhibited higher secretion of EVs as probed by NTA. Cells grown on stiff‐ECM displayed excess CD63 and LBPA puncta, suggesting a boost in EV biogenesis. TEM analysis revealed alterations in MVBs with increased intraluminal vesicles (ILVs) per MVB in stiff ECM conditions, which was abolished upon blebbistatin treatment. We mapped the role of various ESCRT pathways and associated proteins (e.g., Alix, STAM1, TSG101, VPS4, etc.), among which only TSG101 was found to be differentially expressed. Further investigation revealed the role of HSP90, since stiff ECM harboured high TSG101‐HSP90 interactions, and cells treated with 17AAG, a specific inhibitor of HSP90 abrogated ECM‐stiffness‐induced TSG101 levels with a concurrent decrease in LBPA spots, ILVs per MVBs and consequent reduction in EV secretion.

Summary/Conclusion : Taken together, our findings establish a link between mechanotransduction and EV secretion, which unveils the novel role of the TSG101‐HSP90 axis in ECM stiffness‐driven biogenesis of EVs.

Development of an optogenetic tool to modulate exosome secretion through inducible membrane contact sites

Ela Babursah 1,2 , Pierre‐Michaël Coly 1 , Thais de Avezedo‐Laplace 1 , Maribel Lara Corona 1 , Maarten Paul Bebelman 1,3 , Ismahan Abdirizak 1 , Matteo Tantucci 1 , Bogdan Sisca 1,4 , Marine Bretou 1 , Frederik J. Verweij 1,4 and Guillaume van Niel 1,4

1 Institut de Psychiatrie et Neurosciences de Paris, France; 2 Universiteit Utrecht, The Netherlands; 3 Amsterdam University, The Netherlands; 4 Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers (CRCI 2 NA) France Marine Bretou and Frederik J. Verweij both senior authors contributed equally to this study .

Introduction : Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and late endosomes (LE) are specialized subcellular compartments that regulate endosome maturation through lipid exchange, calcium homeostasis, or organelle remodelling. Previous work by our team suggests that MCSs regulate the secretion of extracellular vesicles (EVs) of endosomal origin (exosomes) by affecting MVB/LE motility and maturation.

Methods : Taking advantage of this mechanism, we developed a probe called ‘EV‐switch,’ where photodimerizers that associate in a fast and reversible fashion upon illumination at a 470 nm wavelength were fused with transmembrane resident proteins of MVB (CD63) and ER (VAP‐A), respectively. This genetically encoded probe, by promoting a light‐inducible anchoring of MVB to the ER, enables a spatio‐temporal regulation of EV secretion.

Results : We found that stimulating EV‐switch‐expressing HeLa cells with blue light leads to a rapid (< 5 min) increase in colocalization between CD63 and VAP‐A. Preliminary data obtained using fluorescently tagged cholesterol suggest that these induced MCS are functional, as they accelerate cholesterol egress from the endosomal system. Interestingly, dynamic light scattering experiments coupled with Western blots for tetraspanins indicate that inducing MCS results in a 2‐3‐fold elevation in EV levels found in the conditioned media, probably implying increased MVB secretion. Accordingly, we also observed reduced colocalization of CD63 with degradative compartments and increased colocalization with Arl8b, a mediator of anterograde transport.

Summary/Conclusion : Our findings suggest that light‐inducible MCS can effectively control EV secretion by modulating MVB‐ER interactions and influencing cholesterol trafficking and MVB maturation. The EV‐switch probe offers a novel tool for studying the regulatory mechanisms governing EV release and could have broader implications for modulating vesicle secretion in therapeutic contexts.

Subtopic EV cell interaction 1

Monocyte differentiation and function are immunoregulated by exosomes derived from the synovial fluid of osteoarthritis patients

Presenter: Nur Azira Mohd Noor,

Universiti Sains Malaysia, Malaysia

Introduction : Monocytes and macrophages play pivotal roles in immune activity during inflammation associated with osteoarthritis (OA). An imbalanced polarisation of M1 and M2 macrophages has been implicated in exacerbating OA. However, the interaction of monocytes with exosomes in OA conditions remains unexplored. This study intended to investigate the immunomodulatory role of exosomes derived from synovial fluid of OA patients on monocyte differentiation and function.

Methods : Exosomes were isolated from the synovial fluid of OA patients using ultracentrifugation and characterised using nanoparticle tracking analysis, scanning electron microscopy, and Western blotting. To assess the exosomal influence on monocyte biology, monocytes were cultured with exosomes prior to differentiation into M1 macrophages, M2 macrophages, and monocyte‐derived dendritic cells (MoDC). The phenotypic profiles and gene expressions of all monocyte‐derived cells were evaluated using flow cytometry and real‐time PCR, respectively. Functional validation included assessing phagocytosis activity, and the ability to promote the proliferation of naïve CD4+ T cells was determined by flow cytometry.

Results : Exosomes were found in a round spherical shape with sizes ranging from 30 to 150 nm and enriched with CD9, CD63, CD81, and HSP70. The presence of exosomes in monocyte culture induced monocyte differentiation into proinflammatory profiles characterized by increased CD86 expression and decreased CD206 expression on both M1 and M2 macrophages. The expression of CD80 and HLA‐DR was increased on MoDC. Gene expression analyses revealed significant upregulation of IL1A and downregulation of CD64 and AMPK on M1 macrophages. Similarly, IL1A was significantly upregulated, while CD64 was downregulated in M2 macrophages. In MoDC, HLA‐DR, MMP7, and AMPK were upregulated. The phagocytosis activity of M2 macrophages and MoDC increased in the presence of exosomes. The proliferation of naïve CD4+ T cells was markedly impaired, indicating dampened MoDC‐naïve CD4+ T cell interaction.

Summary/Conclusion : These findings suggest the immunomodulatory role of synovial fluid‐derived exosomes on monocytes, thus potentially contributing to the alteration of monocyte biology and influencing the inflammatory process in OA.

Funding : Grant number: FRGS/1/2020/SKK06/USM/03/6.

Cancer‐derived EVs elicit immunesuppressive effects in healthy immune cells

Chintan Bhavsar 1 , Rui Chen 1 , Trent Neilson 1 , Gowri Irgam 1 , Kaltin Ferguson 2 , John Hooper 3 , Sherry Y. Wu 1

1 School of Biomedical Sciences, The University of Queensland, Brisbane, Australia; 2 Mater Research, Anatomical Pathology Centre, Brisbane, Australia; 3 Mater Research, Translational Research Institute, Brisbane, Australia

Introduction : Extracellular vesicles (EVs) represent a highly heterogenous class of particles secreted by all the cells in the body. Studies to date have highlighted the role of tumour cell‐derived EVs in cancer progression. These studies specifically employ immortalized tumour cell‐derived EVs and delineate their mechanistic role in cancer progression. However, several studies at the multi‐omics level have highlighted that immortalized cells do not often recapitulate tumour cells obtained from cancer patients. As EVs are highly heterogeneous, it is vital that their source recapitulates the disease pathophysiology. Therefore, our knowledge on how EVs modulate tumour progression in cancer patients is still limited. To address this, we isolated cells from peritoneal fluid of ovarian cancer patients (PF‐OvCa) and isolated EVs directly from these cells.

Methods : Ethical approval and informed consent were obtained for all human subjects as per the Human Research Ethics Committee. Mass spectrometry (MS) method optimization was carried out using EVs derived from PF‐OvCa and healthy volunteer peripheral blood mononuclear cells (PBMCs). Flow cytometry was performed to isolate cells from PF‐OvCa. These cells were cultured in serum‐free media for 48‐h to obtain conditioned media. EVs from conditioned media and peritoneal fluid were isolated using ultracentrifugation and/or qEV columns. The downstream effect of EVs was determined by incubating tumour‐derived EVs with PBMCs for 24‐h. RNA‐seq was performed on PBMCs to determine EVs’ downstream biological effect.

Results : MS sample preparation revealed that the traditional single‐pot, solid‐phase‐enhanced (SP3) and in‐solution digestion methods resulted in unpredictable peptide chromatograms yielding low protein discovery. However, the SP2 method resulted in a more predictable chromatogram and improved protein discovery. Further profiling of EVs using this SP2 method revealed a significantly unique proteome profile for EVs derived from PF‐OvCa. More importantly, RNA‐seq analyses revealed that these EVs significantly altered a unique set of genes depending on the EV source and the recipient cell.

Summary/Conclusion : Our bioinformatic data suggests that cancer‐derived EVs can elicit immunesuppressive effects in healthy immune cells. However, further investigations on delineating the functional implications of EVs are critical to devising therapies that improve the treatment outcome.

Funding : The study is supported by the University of Queensland international PhD scholarship and internal funds.

Exosome release as a key modulator of cell survival and death in TSC‐null cells under glucose deprivation

Presenter: Jong Hyun Kim

Daegu Catholic University, Republic of Korea

Introduction : Exosome release plays a critical role in physiological and pathological processes, reflecting cellular health, disease states, and nutrient stress. Tuberous sclerosis complex (TSC) regulates cell viability by inhibiting the mammalian target of rapamycin complex 1 (mTORC1) under glucose deprivation. However, the regulation of TSC‐null cell viability by mTORC1 inhibition under such conditions remains unclear.

Methods : This study investigated the role of exosome release in glucose deprivation‐induced cell death in TSC‐null cells. The effect of mTORC1 inhibition using rapamycin on exosome biogenesis, secretion, and cell viability was examined. Additionally, the impact of exosome release inhibitors on cell viability under glucose deprivation was evaluated.

Results : mTORC1 inhibition via rapamycin significantly enhanced exosome biogenesis, secretion, and cell viability in TSC‐null cells. Conversely, the use of exosome release inhibitors reduced cell viability under glucose deprivation, indicating the critical role of exosome release in regulating cell survival.

Summary/Conclusion : These findings suggest that exosome release plays a pivotal role in glucose deprivation‐induced cell death and survival in TSC‐null cells. Targeting exosome release could provide a novel therapeutic strategy for controlling cell growth in TSC‐null cells resistant to mTORC1 inhibition.

Funding : This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government, Ministry of Science and ICT (MSIT) (RS‐2024‐00344834).

Extracellular vesicles from L. paracasei improve neuroinflammation, GABA neurotransmission and motor incoordination in hyperammonemic rats

Yaiza Arenas Ortiz 1,2,3,4 , Gaspar Perez‐Martinez 4 , Carmina montoliu, 2,3 Marta Llansola 1 , Vicente Felipo 1

1 Laboratory of Neurobiology, Centro de Investigación Príncipe Felipe, Valencia, Spain; 2 Departamento de Patología, Facultad de Medicina, Universidad de Valencia, Spain; 3 INCLIVA Instituto de Investigación Sanitaria, Valencia, Spain; 4 Laboratory of Lactic Acid Bacteria and Probiotics, Department of Biotechnology, Instituto de Agroquímica y Tecnología de Alimentos C.S.I.C, Valencia, Spain

Introduction : Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with motor incoordination and cognitive impairment that reduce life quality and span. Motor incoordination is due to neuroinflammation and enhanced GABAergic neurotransmission in the cerebellum. Recent reports support that probiotics, including L. casei , may improve cognitive function in different pathologies and MHE in cirrhotic patients. Extracellular vesicles (EV) are small cell‐derived membrane vesicles that carry bioactive molecules released from cells, including bacteria.

Methods : The study design included in vivo and ex vivo experiments. In the in vivo experiments, control and hyperammonemic rats were intravenously injected in the tail vein with 50 µg of EVs from L. paracasei or with PBS as the vehicle. Injections started after 2 weeks in the ammonium containing diet and were repeated once per week during 4 weeks. Behavioural parameters were assessed at Weeks 4, 5 and 6 of the HA diet, and the rats were sacrificed at Week 7 for neuroinflammation and neurotransmission analysis. In the ex vivo experiments, control and hyperammonemic rats were sacrificed at 4–5 weeks of diet, and cerebellar slices were obtained and incubated with 10 µg of the EVs to investigate the mechanisms.

Results : We show that LC‐EV treatment reverses glial activation and neuroinflammation in the cerebellum and restores motor coordination in hyperammonemic rats. Moreover, ex vivo treatment of cerebellar slices from hyperammonemic rats with LC‐EV also reverses neuroinflammation and the enhancement of the TNFR1‐S1PR2‐BDNF‐TrkB and TNFR1‐TrkB‐pAKT‐NFκBglutaminase‐GAT3 pathways and of GABAergic neurotransmission.

Summary/Conclusion : The results reported support that LC‐EV may be used as a therapeutic tool to improve motor incoordination in patients with MHE.

Funding : This work was supported in part by Ministerio de Ciencia e Innovación Spain (PID2020‐113388RB‐I00; AEI/ https://doi.org/10.13039/501100011033 ); Consellería Educación, Universidades y Ocupaci´on, Generalitat Valenciana (CIPROM 2021/082); European Regional Development Funds/ERDF (PID2020‐113388RB‐I00 and CIPROM 2021/082); Part of the equipment employed in this work has been funded by Generalitat Valenciana and co‐financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014–2020). Instituto de Salud Carlos III (PI23/00062), co‐funded by ERDF funds. This work was also supported by Spanish Ministry of Science and Innovation Grant PID2022‐136874OB‐C33 funded by MCIN/AEI (doi https://doi.org/10.13039/501100011033 ), and by the Severo Ochoa Accreditation CEX2021‐001189‐S.

Small extracellular vesicles from obese‐adipose tissue promote dysfunction in distal organ

Presenter: Jung Jae Lee

Seoul National University, Republic of Korea

Introduction : Obesity is associated with various metabolic diseases, including insulin resistance and inflammation. In addition to its critical roles in energy storage, adipose tissue secretes various bioactive molecules, including exosome, that participate in immune responses and metabolic regulation through their endocrine or paracrine actions. Small extracellular vesicles (exosome), with a diameter of 30–150 nm, are secreted by adipose tissue under pathological conditions such as metabolic diseases. It is well known that exosomes are released by the fusion of the multivesicular bodies with the plasma membrane and play a major role in intercellular communication by transferring proteins, lipids, and miRNA. These exosomes mediate intercellular communication and cross‐talk between adipose tissue and other distal organs.

Methods : In this study, we isolated exosomes from human adipose tissue from obese individuals who underwent bariatric surgery and from 3T3‐L1 cells induced into a glucolipotoxicity condition via classic ultracentrifugation and characterized by NTA; immunoblotting confirmed exosome‐specific markers. All human adipose tissue and exosomes were prepared using the miRNeasy kit, the libraries were sequenced on the HiSeq2500 platform, and high‐resolution mass spectrometry was used for proteomic and metabolomic analysis.

Results : We investigated the effects of obese adipose tissue‐derived exosomes on endothelial cells and pancreas β cells. It decreases cell viability and increases ROS in endothelial cells, and increases ROS and dysregulates insulin secretion in pancreatic β cells when treated with exosomes derived from obese‐adipose tissue. Moreobver adipose tissue‐derived exosomes promote TLR signalling in endothelial and pancreatic β cells. Importantly, we confirmed the inflammation phenotype in distal organs and hypothesized that this effect might be driven by specific exosomal cargo. To identify the responsible components, we conducted comprehensive analyses of exosomal miRNAs, metabolites, and proteins. We validated miRNA levels in exosomes and analysed miR‐seq in adipose tissue to identify which miRNA promote inflammation. Metabolomic analysis revealed significant increases in ceramides and phosphatidylcholine, key metabolites in the Sphingolipid metabolism pathway.

Summary/Conclusion : Exosomes derived from obese adipose tissue promote dysfunction in endothelial and pancreas β cells by upregulating pro‐inflammatory cytokines significantly related to TLR signalling pathways and dysregulating the sphingolipid metabolism pathway. In further studies, we aim to identify the specific miRNAs, proteins, and metabolites contained in exosomes derived from obese‐adipose tissue and investigate their functional impact on distal organs.

Probiotic extracellular vesicles: isolation, characterization and evaluation of their immunomodulatory properties

Federica Dell'Atti 1,2 , Davide Raineri 1,2 , Giuseppe Cappellano 1,2 , Patrizia Malfa 3 , Annalisa Chiocchetti 1,2

1 Department of Health Sciences, Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), Università del Piemonte Orientale, Novara, Italy; 2 Center for Translational Research on Autoimmune and Allergic Disease (CAAD), Università del Piemonte Orientale, Novara, Italy; 3 R&D Department, SynBalance Srl, Origgio, Varese, Italy

Introduction : Probiotics are defined as microorganisms conferring health benefits on the host. In the presence of inflammatory diseases, the mechanism by which they exert their beneficial effects in distant organs from the gastrointestinal tract remains under investigation. Our hypothesis is that probiotics act through the release of extracellular vesicles (EVs) containing bioactive metabolites. Hence, our aim was to isolate, characterize and investigate the immunomodulatory properties of EVs derived from two probiotic strains.

Methods : Two probiotic‐EVs (P‐EVs1 and P‐EVs2) were isolated by ultracentrifugation, and their concentration and dimension were measured by nanoparticle tracking analysis (NTA) and confirmed by scanning electron microscopy (SEM). To characterize EVs, protein concentration and antioxidant potential were measured by MicroBCA and DPPH assay, respectively. To evaluate the cytotoxicity and immunomodulation (TNF‐α and IL‐10 production), PBMCs isolated from the fresh blood of five healthy donors were stimulated with probiotic EVs in the presence and absence of a pro‐inflammatory stimulus for 24 and 48 h.

Results : P‐EVs1 are bigger than P‐EVs2, as confirmed by SEM images. Furthermore, the antioxidant potential of P‐EVs1 was significantly higher (47%) compared to P‐EVs2 (35%). In in vitro experiments with PBMCs, EVs from both the strains showed very good cytocompatibility. Furthermore, P‐EVs2 can promote the production of both TNF‐α and IL‐10, whereas P‐EVs1 exclusively stimulate the production of TNF‐α.

Summary/Conclusion : We were able to isolate EVs from the two probiotic strains, and our findings suggest that P‐EVs are able to trigger an immunomodulatory response with different effects.

Funding : Roelmi HPC Srl partially funded the PhD salary of the presenting author, and SynBalance Srl provided the two probiotic strains.

Menstrual blood‐derived mesenchymal stromal cell extracellular vesicles stimulate stem cell and chondrocyte functions and diminish osteoarthritic cartilage degradation in vitro

Presenter: Ilona Uzieliene

Kaunas University of Technology, Lithuania

Introduction : Osteoarthritis (OA) is a degenerative joint disease that affects more than 500 million people worldwide. Human mesenchymal stromal cell (MSC) EVs are frequently studied for the development of novel technologies for preventing OA development and repairing cartilage tissue. Menstrual blood‐derived mesenchymal stromal cells (MenSCs) are less studied; however, they were shown to possess a strong therapeutic potential for different diseases but were never studied for OA. The aim of this study was to evaluate the effects of MenSCs EVs on bone marrow mesenchymal stromal cell (BMMSCs) and chondrocyte functions/chondrogenic capacity and OA cartilage explant ECM.

Methods : Human MenSCs EVs were concentrated from cell serum‐free supernatants and isolated using SEC. EVs were counted and characterized by sensitive flow cytometry, WB (CD63, CD9, CD81, TSG101) and TEM. MenSCs EV proteome cargo was analysed using Mass Spec. Human BMMSCs and chondrocytes were treated with MenSCs EVs for 3 days and analysed according to their proliferation/metabolic activity, surface marker expression (flow cytometry, ELISA), morphological differences (holomonitor) and chondrogenic differentiation capacity (histology, RT‐qPCR). Cartilage explants were treated with MenSCs EVs for ECM marker analysis (ELISA, RT‐qPCR). Bioethics permission no. 158200‐14‐741.

Results : MenSCs EVs possess EV marker/tetraspanin expression and are carrying growth factor/cell‐cell, cell‐matrix adhesion proteins and ECM proteins, according to GO analysis. EVs decreased the proliferation capacity of BMMSCs, but not chondrocytes, after 7 days in culture and significantly decreased BMMSC and chondrocyte migration capacity, while stimulating ECM synthesis and collagen type II/TGF‐b receptor gene expression in both cell types after 21 days of chondrogenic induction with TGF‐b3. EVs decreased ECM protein release from cartilage explants with/without stimulation with IL‐1b.

Summary/Conclusion : This study demonstrates positive effects of MenSCs EVs on BMMSCs and chondrocyte functions/differentiation and cartilage ECM, which may turn out to be a promising cell‐free therapy for OA.

Funding : (1) Lithuanian Research Council, funded project under the programme “Promotion of post‐doctoral traineeships,” P‐PD‐24‐167 (agreement No. S‐PD‐24‐114). Project title: “Load‐Responsive Polymer Scaffolds for Targeted Extracellular Vesicle Release in Cartilage Repair.” (2) European Social Fund, supported project under the activity HORIZON‐WIDERA‐2021‐ACCESS‐03, title: “Twinning for Promoting Excellence, Ability and Knowledge to develop novel approaches for targeting inflammatory and degenerative age‐related joint diseases.”

Polygonum cuspidatum ‐derived nanoparticle‐mediated miR‐5054 delivery reverses oxidative stress in HUVECs by the Keap1‐Nrf2 antioxidant pathway

Presenter: Qi Xiu

Southern Medical University, China (People's Republic)

Introduction : The therapeutic potential of herbs has been extensively studied, primarily focusing on small molecule metabolites. However, research on cross‐kingdom interactions between plants and humans, particularly involving plant nucleic acids, remains limited. Emerging evidence suggests that plant‐derived nanoparticles play a crucial role in regulating human health and diseases through interkingdom crosstalk. In this study, we demonstrate for the first time that polygonum cuspidatum‐derived nanoparticles (PDNs) effectively scavenge reactive oxygen species (ROS) and reverse oxidative stress in HUVECs by transferring incorporated miR5054, thereby activating the Keap1‐Nrf2 pathway.

Methods : In this study, polygonum cuspidatum‐derived nanoparticles (PDNs) were isolated from the plant's juice, with other disruptive components removed. The physical characteristics of PDNs were analysed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Flow cytometry, fluorescence staining, cell activity assay, transmission electron microscopy, western blotting and immunohistochemistry were performed to evaluate the biosafety, pro‐migration, and anti‐oxidation properties of PDNs in vitro and in vivo. Multi‐omics analysis and a double luciferase reporting experiment were also performed to elucidate the miR5054‐mediated activation of the Keap1‐Nrf2 antioxidant pathway.

Results : PDNs are slightly negatively charged spherical bilayer membrane structures with a diameter of 150 nm, capable of stable preservation at 4°C, −20°C, and −80°C for over a month. In vitro experiments showed that PDNs have significant antioxidant effects, promote the migration of vascular endothelial and keratinized epithelial cells, and induce M0 macrophages to differentiate into anti‐inflammatory M2 macrophages. In a murine model of deep second‐degree burns, PDNs significantly accelerated wound healing by reversing oxidative stress and promoting angiogenesis. Moreover, miR5054 derived from PDNs was identified as a target of human Keap1 mRNA, leading to the activation of the Keap1‐Nrf2 antioxidant pathway.

Summary/Conclusion : PDNs, enriched with plant‐derived biomacromolecules, can enter the human body and effectively modulate human cell signalling pathways through cross‐kingdom interactions. Specifically, PDNs activate the Keap1‐Nrf2 antioxidant pathway by delivering miR5054 to HUVECs, enhancing their antioxidant capacity and promoting rapid healing at the wound site.

Funding : This work was supported by grants from the National Science Fund for Distinguished Young Scholars (82025024), National Natural Science Foundation project of China (82472387), and the Guangzhou Municipal Science and Technology Program (SL2024B03J00976).

Genome‐wide CRISPR/Cas9 screening identifies the COMMANDER recycling complex as a key player in EV uptake

Miguel Palma‐Cobo 1 , Victor Toribio 1 , Carlos Enrich Bastús 2 , Albert Lu 2 , María Yáñez‐Mó 1

1 Department of Biología Molecular, Universidad Autónoma de Madrid, IUBM, Centro de Biología Molecular Severo Ochoa, IIS‐IP, Spain; 2 Universidad de Barcelona, Facultad de Medicina, IDIBAPS, Spain

Introduction : Extracellular vesicles (EVs) hold immense potential in therapeutic delivery, warranting a comprehensive investigation of the molecular mechanisms that regulate their uptake by target cells. To identify key molecular regulators of EV internalization, we conducted a genome‐wide CRISPR (GWC) screen utilizing flow cytometry sorting aimed to pinpoint candidate genes that influence EV uptake.

Methods : We employed a GWC library in K562 cells, which included 10 single‐guide RNAs (sgRNAs) per gene spanning the entire human genome, along with appropriate negative controls, for a total of 250,000 individual sgRNAs. EVs were isolated from the SKMEL147 human melanoma cell line using serial ultracentrifugation and were characterized through nanoparticle tracking analysis (NTA), electron microscopy, western blotting, and confocal microscopy. For the GWC screen, 3.6×10 1 2 EVs were labelled with Alexa633‐C5‐Maleimide, a fluorescent dye that covalently binds to sulfhydryl residues on surface proteins, and incubated for 2 h with 500 × 10⁶ K562 cells, providing a 2000× coverage of the library. The top 5% of high and low fluorescence populations were sorted. Genomic DNA was extracted, and sgRNA sequences were amplified by PCR with Illumina adaptors added as overhangs. Next‐generation sequencing (NGS) was performed to quantify sgRNA enrichment in the sorted populations compared to the unsorted control.

Results : Remarkably, several members of the COMMANDER complex emerged as significant hits in our screen. To validate these findings, we employed quantitative EV uptake assays developed in our Laboratory, utilizing luciferase or maleimide‐based readouts. We conducted validations in knockout (KO) cell lines of both K562 and HeLa cells using EVs derived either from melanoma or breast cancer cell lines and kinetic follow‐up of EV cargo, including surface proteins, luminal proteins, and RNA.

Summary/Conclusion : Our data suggest that the COMMANDER complex plays a pivotal role in the early stages of EV uptake and in the recycling of transmembrane EV components.

Extracellular vesicle transport across the vascular endothelium

Presenter: Steven C. George

UC Davis, USA

Introduction : Produced from all cells in the body, extracellular vesicles (EVs) orchestrate a host of biological functions, including cell proliferation, migration, and phenotype. In order to exert these effects, EVs must be transported across biological barriers, including the vascular endothelium. However, the precise mechanisms that enable such transendothelial transport remain underexplored.

Methods : We engineered a microfluidic device that simulates key physiological forces, including interstitial flow, to enable precise assessment of EV transport dynamics across a confluent endothelial layer derived from cord blood. We utilized EVs (1E11 EVs/mL), derived from a subclone of the HEK293T cell line, created by lentivirally transducing the HEK293T cells with the pLenti‐pHluorin_M153R‐CD63‐mScarlet plasmid (Addgene). The plasmid encodes a fusion protein combining pH‐sensitive pHluorin_M153R and pH‐insensitive mScarlet linked to CD63. Thus, under neutral pH, CD63+ EVs fluoresce in the red and green channels, but only in the red channel under acidic (pH < 6) conditions. Permeability (cm/s) of the EVs, 70 kDa fluorescent dextran (paracellular transport), and fluorescent transferrin (paracellular and receptor‐mediated endocytosis, RME) across the endothelium was determined by examining the fluorescence in a region of interest as a function of time in the presence and absence of a competitive inhibitor of dynamin, and thus RME.

Results : The permeability of dextran (2.8 ± 1.5 × 10 −6 cm/s) was not impacted by inhibition of dynamin, consistent with a confluent endothelium. The permeability of transferrin (2.7 ± 0.7 × 10 −5 cm/s) was significantly higher than dextran, consistent with augmentation by RME, and was significantly reduced by inhibiting dynamin (1.5 ± 0.7 × 10 −6 cm/s). Similarly, the permeability of EVs at baseline was significantly abrogated by inhibiting dynamin (8.9 ± 1.8 × 10 −6 cm/s and 1.4 ± 0.4 × 10 −6 cm/s, respectively). Notably, within endothelial cells, distinct red‐only fluorescence patterns signal that a subset of endocytosed EVs are trafficked to acidic compartments, consistent with lysosomal targeting.

Summary/Conclusion : Our study reveals that EV transport across vascular endothelium is facilitated by RME and highlights heterogeneous intracellular trafficking post‐endocytosis, with a subset of EVs directed to acidic, lysosomal compartments. These findings deepen our understanding of EV dynamics across endothelial barriers and suggest specific intracellular fates for EV subpopulations that may be harnessed in targeted therapeutic or diagnostic applications.

Funding : Edward Teller Endowed Chair.

Tumour extracellular vesicles educated platelet ribosomal protein‐mRNA degradation

Presenter: Gaoge Sun

Tsinghua University, China (People's Republic)

Introduction : In tumour progression, platelets play a dual role in haemostasis and coagulation while reflecting tumour malignancy, termed tumour‐educated platelets (TEPs). Tumour‐derived EVs serve as a critical link, mediating the exchange of substances and signal transduction between tumours and platelets by transporting proteins and nucleic acids. However, the mechanisms governing the interaction and intercellular communication between tumours and platelets remain largely unexplored, with the specific tumour EV contents that educate platelets still being elucidated.

Methods : EVs were isolated from 2‐day cultured supernatants via differential ultracentrifugation and characterized according to MISEV2023 guidelines, employing nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis. Platelet‐like particles (PLPs) were isolated from the Meg01 cell media, which underwent centrifugation at 500 × g for 10 min, filtration through a 5 µm filter, and subsequent pelleting at 3500 × g for 30 min. For the dsRNA pull‐down assay, total RNA was extracted using TRIzol reagent, serving as the input. dsRNA was co‐immunoprecipitated with antibody J2 and sequenced to assess dsRNA content.

Results : Analysis of bulk RNA‐seq data from various tumour patients revealed a decrease in RP‐mRNA levels in platelets and plasma, indicating tumour EVs as the primary inducer of platelet transcriptome alterations. EVs, but not NVEP or EV‐depleted supernatant, were found to activate the dsRNA‐sensing and ribosome rescue pathways in platelets, initiating the TEVEP process. Further, EV activated the dsRNA‐sensing and ribosome rescue pathway in platelets and led the TEVEP process. dsRNA pull‐down assays identified dsRNAs enriched in tumour EVs and taken up by platelets, demonstrating that dsRNAs are not part of the EV corona but are protected by the EV‐membrane from RNase degradation.

Summary/Conclusion : This research has identified the downregulation in ribosomal protein mRNA (RP‐mRNA) levels in both platelets and plasma of tumour patients. We identified dsRNA types from tumour EVs. Tumour EVs educated platelets (TEVEP) by carrying double‐stranded RNA (dsRNA) which activated the dsRNA‐sensing pathway and the ribosome rescue pathway, leading to RP‐mRNA decay in platelets. This discovery offers a novel mechanism of EVs in platelet transcriptome and a potential biomarker for non‐invasive tumour monitoring.

Funding : The work was funded by the National Natural Science Foundation of China (Grant No. 21825702 and 22137004), and the Beijing Outstanding Young Scientist Program (Grant No. BJJWZYJH01201910003013).

The impact of nucleus pulposus cell exosomes under cyclic mechanical tension on nucleus pulposus cells

Weiye Zhang, Jiawen Zhan, Yuxuan Du, Ke Zhao

China Academy of Chinese Medical Sciences Wangjing Hospital, China

Introduction : Nucleus pulposus cells (NPCs) are regulated by various factors within the microenvironment of the intervertebral disc, and further exploration is needed regarding the effects of microenvironmental changes induced by abnormal stress on NPCs. We aim to observe the secretion of exosomes (EXO) from NPCs under cyclic mechanical tension (CMT) conditions and the impact of these EXO on NPCs.

Methods : Lumbar spine tissues were obtained from patients undergoing surgery, and NPCs were isolated and cultured in vitro. After subjecting NPCs to mechanical loading using the FX‐5000T system, NPCs‐EXO were extracted. The EXO was identified using flow cytometry and western blot. The uptake of NPCs‐EXO by NPCs was observed, and the apoptosis and activity of NPCs were measured. The mRNA expression of collagen II, ADAMTS‐5, MMP3, BMP2, and β‐catenin in NPCs was detected using RT‐PCR. Differentially expressed miRNAs in NPCs‐EXO were screened using high‐throughput sequencing, and their regulatory mechanisms in the Wnt/β‐catenin pathway were investigated.

Results : CMT stimulation increased the secretion of EXO from NPCs, and NPCs‐EXO were taken up by CEPCs. Tension‐loaded NPCs‐EXO significantly decreased the activity ( p < 0.01) and increased the apoptosis rate ( p < 0.05) of CEPCs. Additionally, the expression of type II collagen in NPCs decreased significantly ( p < 0.05), while the expression of ADAMTS‐5, MMP3, BMP2, and β‐catenin increased significantly ( p < 0.05). Differentially expressed miRNAs were identified, demonstrating their regulatory mechanisms in the Wnt/β‐catenin pathway.

Summary/Conclusion : CMT promotes the secretion of EXO from NPCs, which can inhibit the activity of NPCs and disrupt the synthesis and degradation metabolism of NPCs' matrix. Furthermore, NPCs‐EXO influence the Wnt/β‐catenin pathway through miRNAs, thereby regulating NPCs and impacting intervertebral disc degeneration.

Enhancing cellular uptake of extracellular vesicles via genomic glycoengineering

Weihua Tian, Jiasi Chen, Anne Louise Blomberg, Steffen Goletz

Technical University of Denmark, Denmark

Introduction : Engineering small extracellular vesicles (sEVs) to enhance drug loading capacity and targeting specificity holds considerable therapeutic potential. Carbohydrates play a pivotal role in mediating interactions such as cell–cell communication, cell‐molecule recognition, and pathogen‐cell engagement. In our previous work, we employed a glycoengineering approach, called the ‘simple cell’ strategy, which uses CRISPR/Cas9 to precisely modify glycan structures on sEVs. These glycoengineered sEVs provide a platform for exploring the role of glycosylation in sEV biology and optimizing their therapeutic applications.

Methods : The cellular uptake of glycoengineered sEVs was quantified using a commercial fluorescent dye that selectively labels EV membranes, followed by flow cytometry analysis. Their drug delivery efficiency was further assessed by loading the sEVs with plasmid DNA, siRNA, and DNA oligos. Comparative analyses evaluated uptake efficiency across sEVs with distinct glycan modifications.

Results : Glycan profiles on both sEVs and recipient cells were found to significantly affect cellular uptake. Among the glycoengineered sEVs, the knockout of B4GALT7, which disrupts glycosaminoglycan biosynthesis and generates ΔGAG EV, resulted in the highest uptake levels. ΔGAG EV also demonstrated superior performance as nucleic acid carriers, effectively delivering plasmid DNA, siRNA, and DNA oligos.

Summary/Conclusion : This study underscores the crucial role of glycosylation in sEV biology and highlights the potential of glycan modifications to enhance sEV uptake and therapeutic performance. Disruption of glycosaminoglycan biosynthesis via B4GALT7 knockout produced ΔGAG EV with significantly improved cellular uptake and nucleic acid delivery efficiency. These findings demonstrate the promise of genomic glycoengineering strategies for tailoring sEVs as optimized drug delivery systems.

Funding : This work was supported by the Novo Nordisk Foundation Grants NNF20SA0066621, NNF19SA0056783, NNF21SA0072683, and NNF19SA0035474 (to Prof. Steffen Goletz).

Identifying molecular drivers of extracellular vesicle‐mediated RNA transfer

Maria Laura Tognoli 1 , Naomie Zhou 1 , Olivier G. de Jong 2 , Stavros Therapontos 1 , Omnia Elsharkasy 1 , Willemijn de Voogt 1 , Jerney Francois 1 , André Görgens 3 , Samir El Andaloussi 3 , Pieter Vader 1,4

1 CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; 2 Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; 3 Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden; 4 Department of Cardiology, Experimental Cardiology Laboratory, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

Introduction : Extracellular vesicles (EVs) mediate intercellular communication by transferring bioactive cargo from donor to recipient cells. The EV‐content delivery process to recipient cells, however, is still poorly characterized and the molecular players orchestrating the process at the donor EV/recipient cell interface have not been elucidated. Here, we identify EV surface proteins that are critical in functional cargo transfer to recipient cells and determine at which step of the delivery process (from EV uptake to cargo delivery) these proteins play a role.

Methods : MDA‐MB‐231‐ and hTERT‐MSC‐derived EVs were isolated from conditioned medium by size exclusion chromatography and further purified via density gradient ultracentrifugation. EVs were analysed via liquid chromatography‐mass spectrometry and surface proteins were identified via manual annotation (Protter and Uniprot). A siRNA screen library (ON‐TARGETplus SMARTpool, Horizon Discovery LTD) was designed, based on the EV surface proteins list. The siRNA screen was performed using our recently developed CROSS‐FIRE co‐culture system for sensitive measurement of functional EV‐cargo (RNA) transfer, via flow cytometry. Hits derived from the screen were validated for their involvement in EV‐cargo transfer, via blocking antibodies or siRNA knockdown, using the CROSS‐FIRE system as well as luminescence‐ and flow cytometry‐based EV uptake assays.

Results : We identified approximately 200 proteins that decorate the membrane of MDA‐MB‐231‐ and hTERT‐MSC‐derived EVs. We performed a siRNA screen selectively targeting each of the previously identified 200 genes on the donor cells of the CROSS‐FIRE system, which led to knockdown of protein expression on the derived EVs. We found 6 leading targets potentially involved in EV cargo transfer. We characterized several of these targets as part of the EV corona. Further, we uncovered vesicle uptake as the exact step of the EV‐content delivery process where these proteins play an important role. Additionally, we identified the receptors on recipient cells these EV proteins interact with in order to mediate vesicle internalization.

Summary/Conclusion : In this study, we mapped EV surface proteins. Upon siRNA screening, we identified several novel regulators of EV‐mediated cargo transfer as well as their interacting partners on recipient cells. This study provides new insights on the mechanisms of EV‐mediated content delivery.

Cross‐species communication via fungal extracellular vesicles

Presenter: Renan E. A. Piraine

University of São Paulo, Brazil

Introduction : Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigates the role of EVs in intra‐ and interspecies interactions among pathogenic fungi from the Candida and Cryptococcus genera.

Methods : We performed co‐incubation experiments with EVs from Candida albicans and Candida auris in planktonic cells and biofilms of C. albicans , as well as Cryptococcus neoformans and Cryptococcus gattii EVs in planktonic cells and biofilms of C. neoformans . Scanning electron microscopy confirmed the association of EVs with the cell surface of recipient cells, and further validation was achieved by labelling EVs and incubating them with fungal cells and human macrophages. The biological effects of EVs on intraspecies and interspecies interaction were assessed, including assays for biofilm adhesion, dispersion, and antifungal drug tolerance. Immunomodulatory effects on macrophages were analysed by assessing cell viability, gene expression, and cytokine production.

Results : EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression of Cap59, Lac1, Ure1, and Erg11 genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitate cross‐species communication, which enhances biofilm adhesion and dispersion, highlighting their role in fungal phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor‐specific gene expression changes, notably the upregulation of galectin‐3, along with a pro‐inflammatory phenotype characterized by increased expression of iNOS and elevated cytokine levels (IL‐1β, IL‐6, and IL‐8).

Summary/Conclusion : Collectively, these findings underscore the critical role of fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms.

Funding : This project is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant number 2023/05800‐7.

EV‐mediated cross‐kingdom RNA trafficking between plants and fungal pathogens

Baoye He 1 , Brisa Davila 1 , Qiang Cai 2 , Shumei Wang 3 , Hailing Jin 3

1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, USA; 2 State Key Laboratory of Hybrid Rice, College of Life Science, Wuhan University, Wuhan, China; 3 Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, California, USA

Introduction : Cross‐kingdom RNA trafficking between plant hosts and microbes is a crucial process that regulates gene expression in both interacting partners during infection. Despite its significance, the mechanism of RNA transport between plants and microbes has remained elusive. Here, we demonstrate that extracellular vesicles (EVs) serve as a means for both plants and fungal pathogens to deliver RNA to their respective interacting partners.

Methods : Arabidopsis EVs were isolated from the apoplastic washing fluid of Botrytis cinerea‐infected plants by sequential ultracentrifugation at 100,000 × g followed by sucrose gradient fractionations or immunoisolation. Fungal EVs were isolated from the liquid culture by sequential ultracentrifugation at 100,000 × g , followed by sucrose gradient fractionations. Both EVs were characterized by western blot, nanoparticle tracking analysis, and transmission electron microscopy.

Results : We performed RNA‐seq analysis and revealed that both small RNA (sRNA) and messenger RNA (mRNA) were found in plant EVs. These sRNAs can be transferred to the interacting fungal pathogen B. cinerea , where they can suppress fungal virulence‐related gene expression. Meanwhile, the mRNAs can also be transported to fungal cells, where they can be translated to modulate fungal virulence. Plant tetraspanin proteins, for example, TET8, are enriched in plant EVs and can be used as a plant EV biomarker. The fungal pathogen B. cinerea can also secrete EVs to deliver fungal sRNAs into plant cells to suppress plant immune‐related gene expression and facilitate its infection. B. cinere a EVs can be internalized by plants via clathrin‐mediated endocytosis. B. cinerea tetraspanin protein BcPLS1 serves as a biomarker for B. cinerea EVs and plays an essential role in fungal pathogenicity.

Summary/Conclusion : EVs are identified as a mechanism used by both plants and fungal pathogens to deliver RNAs to the interacting sides to modulate the plant‐pathogen interaction. These findings could have significant implications for the development of novel plant protection solutions.

Funding : NIH (R35GM136379), NSF (IOS 2020731), USDA (2021‐67013‐34258), USDA (2019‐70016‐29067) to H.J., and the Texas A&M startup funding to B.H.

EVs from PDAC cells harbouring mutant p53 can drive tumour microenvironment remodelling and fibroblast activation

Presenter: Maayan Azulay

Ben‐Gurion University of the Negev, Israel

Introduction : Pancreatic ductal adenocarcinoma (PDAC) has a poor survival rate due to late‐stage diagnosis. Over 75% of advanced cases carry mutations in TP53, which encodes the p53 protein. Tumours expressing p53 gain‐of‐function (GOF) mutations induce pro‐invasive alterations to the extracellular matrix (ECM) secreted by cancer‐associated fibroblasts (CAFs), which make up over 70% of the tumour mass. In PDAC, activated fibroblasts promote tumourigenic factors, cytoskeletal rearrangements, and ECM remodelling, modulating tumourigenic phenotypes and CAFs infiltration. We hypothesized that EVs from mutant p53 cells activate CAFs, altering their fibre secretion to form a desmoplastic ECM. Uncovering the role of these EVs may reveal a mechanism by which mutant p53 cancers drive tumour progression and resistance to therapies.

Methods : Human and mouse PDAC cells (WT, KO or mutant p53) were cultured, and EVs were isolated and characterized. The EVs were then incubated with primary fibroblasts in 3D cultures. Fibroblast activation and ECM changes were analysed using immunofluorescence staining for fibronectin, α‐SMA, and palladin. Additionally, labelled EVs were injected into the pancreas of mice, with subsequent histological and morphological analysis of pancreatic tissue.

Results : Fibroblasts treated with mutant p53‐derived EVs showed significantly higher expression of α‐SMA and palladin, indicating activation and ECM remodelling. These EV‐treated fibroblasts also displayed altered fibronectin fibre orientation and density. In vivo, we injected EVs that reached the pancreatic tissue, and pancreases treated with mutant p53 EVs exhibited observable changes in tissue morphology and a significant increase in weight, supporting the in vitro findings of TME modulation.

Summary/Conclusion : Mutant p53 is a driver mutation in advanced PDAC cases. EVs from PDAC cells carrying mutations in TP53 might play a crucial role in reshaping the TME by modifying ECM structure and activating fibroblasts. Targeting exosome‐mediated signalling pathways could be a promising approach to disrupt PDAC progression and improve therapeutic outcomes.

Investigating the uptake dynamics of hyaluronic acid‐decorated extracellular vesicles using high‐content screening

Heikki Kyykallio, Janne Capra, Kirsi Rilla

University of Eastern Finland, Finland

Introduction : Hyaluronic acid (HA) has been widely utilized as a surface decoration to improve the cellular targeting and uptake of nanoparticles, including extracellular vesicles (EVs), via adhesion receptor CD44. However, the mechanisms of HA‐decorated EV uptake are not well understood. We utilized high‐content screening to observe the time‐dependent uptake and endocytosis mechanisms of HA‐decorated and non‐decorated small EVs to CD44‐expressing and CD44‐negative gastric cancer cells.

Methods : HA‐decorated and non‐decorated EVs were isolated from MCF7 breast cancer cells with inducible GFP‐HAS3 expression and characterized using nanoparticle tracking analysis, western blot and transmission electron microscopy. EVs were stained with PKH26 membrane dye and administered to HA‐receptor CD44‐expressing and CD44‐negative MKN74 gastric cancer cell co‐cultures to follow time‐based uptake of EVs for up to 24 h. To study the endocytosis mechanisms of non‐decorated and HA‐decorated EVs, MKN74 co‐cultures were incubated with high molecular weight HA to block CD44‐receptor interactions, or with methyl‐β‐cyclodextrin, chlorpromazine, or 5‐(N‐Ethyl‐N‐isopropyl)amiloride (EIPA) to inhibit cholesterol‐dependent mechanisms, clathrin‐dependent endocytosis, and macropinocytosis, respectively. The cultures were imaged using a spinning disc confocal microscope high‐content screening system. EV uptake was quantified from 2‐dimensional maximum intensity projections and 3D‐rendered images, and spot/area‐based and object/volume‐based analysis pipelines were compared.

Results : HA‐decorated and non‐decorated EVs were found to be endocytosed at different rates over time, with HA‐decorated EVs exhibiting increased uptake to CD44‐expressing cells. Methyl‐β‐cyclodextrin increased, and high molecular weight HA, chlorpromazine and EIPA inhibited the endocytosis of both EV types, independent of cell type. The uptake of HA‐decorated EVs was more effectively inhibited by chlorpromazine and EIPA compared to non‐decorated EVs.

Summary/Conclusion : Using high‐content screening, we observed an increased uptake of HA‐decorated EVs in CD44‐expressing cells, indicating CD44‐mediated targeting. While the HA‐decorated EVs shared similar endocytosis mechanisms with non‐decorated EVs, our results suggest clathrin‐mediated endocytosis and macropinocytosis as major mechanisms of HA‐decorated EV internalization to MKN74 cells.

Impact of irradiation on extracellular vesicle release in medulloblastoma and its effects on neural progenitor cells

Šárka Jarošová 1,2 , Viktor Sinica 1 , Erik Vlčák 1 , Milan Bojić 1 , Martina Zíková 1

1 Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic; 2 Charles University, Czech Republic

Introduction : Medulloblastoma (MB) is the most common malignant paediatric brain tumour, divided into four subgroups (WNT, SHH, Group 3, and Group 4), each associated with distinct prognoses. While irradiation remains an effective standard treatment, it results in enduring cognitive and endocrine deficits. Additionally, irradiation alters the release and composition of extracellular vesicles (EVs) from tumour cells, potentially enhancing the formation of pre‐metastatic niches and impacting surrounding cells.

Methods : Vesicles from irradiated and non‐irradiated MB cells (Groups 3 and 4) were isolated using differential ultracentrifugation to obtain apoptotic bodies (2000 × g pellet), large vesicles (10,000 × g pellet), and small vesicles (120,000 × g pellet). These EV populations were characterized using western blotting for specific markers, electron microscopy, and concentration measurements via imaging flow cytometry. Isolated EVs were then transferred to mouse neural progenitor cells (NPCs), and their uptake was quantified by flow cytometry. To assess morphological changes in NPCs following EVs exposure, we employed cell painting assays and conducted mRNA sequencing to evaluate changes in gene expression.

Results : Preliminary findings indicate that MB‐derived EVs are internalized by NPCs differently across MB groups. mRNA sequencing of NPCs after EV exposure revealed changes in the expression of genes associated with cytoskeletal remodelling and extracellular matrix degradation, suggesting potential shifts in cell motility and structural integrity. These findings support a mechanistic pathway through which MB EVs may influence the tumour microenvironment and promote metastatic potential.

Summary/Conclusion : Profiling of NSCs cells treated with EVs isolated from different subgroups of medulloblastoma cells have different effects on the target NPC. These results underscore the need for subgroup‐specific therapeutic strategies to mitigate the risks in MB treatment.

Visualizing the EV‐mediated AMR transfer process of the ESBL plasmid derived from Escherichia coli O104:H4

Presenter: Stelios Chatzimichail

University of Oxford, UK

Introduction : The spread of antimicrobial resistance (AMR) via outer membrane vesicles (OMVs) is a significant concern in bacterial pathogenesis. Specifically, the OMV‐mediated transfer of extended‐spectrum beta‐lactamase (ESBL) plasmids from Escherichia coli O104:H4 contributes to the dissemination of AMR. This study utilizes the parS–parB visualization system to examine the mechanisms of ESBL plasmid transfer through OMVs to acceptor E. coli cells.

Methods : OMVs were harvested from donor E. coli cells carrying a parS‐tagged ESBL plasmid fused with ParB‐mNeonGreen (MNG) and enriched via a combination of ultracentrifugation and size‐exclusion chromatography. Donor OMVs were incubated with acceptor E. coli expressing ParB‐mKate and monitored. Single‐molecule localization microscopy was employed to track OMV‐cell interactions and monitor plasmid transfer dynamics through changes in fluorescence signals. The effects of polymyxin B and ampicillin (used off‐label) on OMV‐mediated transfer were also assessed.

Results : In our results we quantify OMV‐mediated AMR transfer in real time for the first time. Successful events are observed as yellow foci forming upon simultaneous binding of both MNG and mKate on the same plasmid, indicating plasmid uptake. We observe and quantify, over the course of 1‐h runs, the formation of new foci within acceptor cells, suggesting plasmid replication and expression. In our findings we note that despite donor OMVs fully encapsulating acceptor cells, successful transfer events were infrequent and occurred only in areas of high cell division rates. We finally investigated the effects of membrane‐targeting antibiotics such as polymyxin B and ampicillin, where we found both increased prevalence of OMV‐mediated transfer events as well as more uniform uptake spatially associated with successful transfers.

Summary/Conclusion : Although infrequent, OMV‐mediated transfer events of conjugative ESBL plasmids lead to widespread dissemination of AMR genes within bacterial communities through subsequent plasmid replication and horizontal gene transfer. Controlling these processes is crucial for mitigating the rapid spread of AMR. Targeting factors that facilitate OMV uptake may be strategic in limiting the proliferation of resistance plasmids.

Funding : This research was supported by an Oxford Martin School Programme on Antimicrobial Resistance Testing, by BBSRC grants BB/N018656/1 and BB/S008896/1 (to A.K.) and by Oxford's EPSRC/BBSRC Impact Acceleration Account EP/X525777/1 (0012379, to A.K. and S.C.).

Host immune cell membrane deformability governs the uptake route of malaria‐derived extracellular vesicles

Presenter: Daniel Alfandari

Weizmann Institute of Science, Israel

Introduction : The malaria parasite, Plasmodium falciparum (Pf), utilizes extracellular vesicles (EVs) to facilitate its growth and communicate with the external microenvironment, primarily targeting the host's immune cells. However, how parasitic EVs are able to enter specific immune cell types within this highly heterogeneous pool of cells remains largely unknown. Using a combination of imaging flow cytometry (IFC) and advanced fluorescence analysis, we here demonstrate that the uptake route for Pf‐derived EVs is markedly different between host T cells and monocytes. While the adaptive system's T cells internalize Pf‐derived EVs mainly through an interaction with the plasma membrane (‘capping pattern’), the innate system's monocytes uptake them via endocytosis. We further reveal that the membranal/endocytic balance of the EV internalization is driven mostly by the amount of endocytic incorporation. Integrating atomic force microscopy with data analysis methods revealed that this incorporation heavily depends on the cell's biophysical properties rather than solely on the molecular interaction. Moreover, we demonstrate that altering the cholesterol content in the cell membrane can tilt the balance in favour of one uptake route over another, thereby facilitating the ‘decision’ for the EV internalization path. Our results provide a novel mechanistic insight into how Pf‐derived EVs ensure their entry into diverse host cells, by adjusting their entry mode to the recipient cell's specific mechanical properties. This study highlights the sophisticated cell‐communication tactics evolved by the malaria parasite.

Methods : Imaging flow cytometry, spectral flow cytometry, confocal microscopy, atomic force microscopy, machine learning.

Results : Overall, our study suggests that despite the many differences in biomolecular profiles and metabolic activity between the immune cell types, the fundamental physical property of membrane stiffness is sufficient to dictate favourable entry route of malaria‐derived EVs to the cells.

Summary/Conclusion : We investigated the biophysical mechanisms behind the uptake of Pf‐derived EVs, focusing on differences between innate and adaptive immune system host cells. We show that innate and adaptive immune cells use different pathways to internalize EVs. Membrane stiffness (mechanical properties) significantly influences the EV uptake mechanism, and cholesterol in the plasma membrane shifts the uptake mechanism between endocytosis and direct membrane fusion.

Mesoangioblast extracellular vesicles modulate macrophage behaviour

Presenter: Fabiana Geraci

University of Palermo, Italy

Introduction : Mouse mesoangioblasts (C57) are vessel‐associated multipotent progenitor stem cells, which are able to differentiate into different mesodermal cell types and are able to shed membrane vesicles (EVs) in the extracellular environment. EVs contain several molecules, such as MMP2/9 and HSP70, as transmembrane protein. The aim of our work was to determine the effect of C57 EVs on murine macrophages (Raw264.7), as they play a central role in all stages of the inflammatory response. In damaged tissue macrophages migrate to carry out their function of identifying and removing dead cells, debris, and foreign particles via phagocytosis.

Methods : C57 EVs were collected from conditioned media by ultracentrifugation. Raw264.7 cells were cultured with or without C57 EVs to evaluate the effects on cell proliferation, cell migration, phagocytosis ability, and phenotype characterization. To this aim we performed FACS analysis, microscope observations and enzymatic activity quantification.

Results : We have analysed the immunomodulatory effect of C57 EVs on Raw264.7 cells. We have first demonstrated that EVs are able to interact with Raw264.7 cells by negatively influencing their proliferation index and positively influencing their migratory capability. We also proved that this enhanced migration is mediated by an elevated expression and activity of MMP2/9. Moreover, in vitro phagocytosis index calculation highlighted that EV treatment is able to improve Raw264.7 phagocytic ability, which is important at early stages of tissue repair. The use of neutralizing antibodies (i.e., anti‐Hsp70, anti‐TLR2, and anti‐TLR4 antibodies) demonstrated that C57‐EVs are able to increase Raw264.7 phagocytosis through Hsp70 and its surface receptors. To point out whether EVs modulate macrophage phenotype, we explored the presence of M1/M2 markers (i.e., iNOS and arginase) and NO synthesis. At the initial stage after EV treatment, Raw264.7 synthesized both iNOS and NO, whereas no arginase mRNA was detected. These data suggest an M1 phenotype. After a recovery following EV treatment, an increased release of anti‐inflammatory cytokines, typical of M2 macrophages, was observed.

Summary/Conclusion : These data suggest that C57‐EV could positively influence tissue repair through macrophage modulation.

Funding : This research was supported by grants from the University of Palermo (FFR 2024 to FG).

Subtopic EV heterogeneity 1

Decoding the versatile landscape of Bacillus cereus extracellular vesicles: A multi‐OMICS approach

Presenter: Tanja V. Edelbacher

University of Veterinary Medicine Vienna, Austria

Introduction : The pathogen Bacillus cereus is a Gram‐positive endospore‐forming rod causing food‐borne diseases and non‐GI‐related illnesses, including systemic and local infections. Despite an increasing focus on extracellular vesicles (EVs) from Gram‐positive bacteria, the exact mechanisms of EV secretion remain elusive. Recently, we have shown that B. cereus secretes biologically active EVs that exert pathogenic effects contingent on their cargo. To advance EV screening platforms and diagnostic tools, it is crucial to comprehensively study their plasticity to understand their role in infection and inflammation. Thus, this work aims to investigate the impact of external factors on EV secretion dynamics to determine the variable and inherent characteristics of B. cereus EVs.

Methods : We investigated the influence of culturing conditions on vesiculogenesis. Quantification and characterization of EVs was performed using nanoparticle tracking analysis, Fourier‐transform infrared (FTIR) spectroscopy, transmission electron microscopy and atomic force microscopy. The impact of media selection on the composition of bacterial EVs was examined through profiling of the EV‐lipidome, ‐proteome, and ‐transcriptome. Additionally, EVs were harvested at various time points during bacterial growth to explore media‐dependent variations in EV secretion dynamics. The metabolic state of the bacteria was assessed using RT‐qPCR targeting pathways potentially central to EV regulation.

Results : Multiple OMICS approaches all revealed distinct compositional profiles of EVs depending on the culturing conditions, emphasizing the impact of the bacterial metabolic state on vesiculogenesis. We also highlight the role of the lipid bilayer in EV‐mediated pathogenicity and EV‐stability. Furthermore, FTIR spectroscopy was identified as a suitable tool to obtain compositional insights into different EVs, aligning with multivariate analyses of comprehensive multi‐OMICS profiling.

Summary/Conclusion : Our study shows that the choice of media significantly affects both the secretion dynamics and the compositional profiles of EVs. We propose that bacteria release functionally diverse EVs because of varying metabolic states and their lipid metabolism. This emphasizes the importance of appropriate culturing conditions to study EV secretion and composition, ultimately enhancing our understanding of bacterial EVs and their role in infection.

miRNA content in subpopulations of NK cell‐derived extracellular vesicles connects to different functional activity

M. Aarsund 1 , M. T. Trøften Hagen 1 , R. E. Crossland 2 , K. J. Schell 2 , Amanda Sudworth 1 , Yunjie Wu 1 , M. Inngjerdingen 1

1 Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 2 Translational and Clinical Research Institute, Faculty of Medical Science, Newcastle University, Newcastle‐Upon‐Tyne, United Kingdom.

Introduction : NK cells release extracellular vesicles (EVs) in response to cytokine stimulation or receptor engagement. These vesicles contain cytolytic proteins and can induce apoptosis of a wide range of cancer cells. The EV secretome is heterogeneous, and we previously showed that NK cells release distinct vesicle subsets, including a subset that is putatively derived from cytolytic granules (a secretory lysosome). Only vesicles with putative intracellular origin induce apoptosis of cancer cells.

Methods : To further understand how these vesicles target and kill cancer cells, we profiled the miRNA expression in EV subpopulations derived from both primary human NK cells and the NK‐92 cell line via the NanoString hybridization platform and quantitative PCR. EVs were harvested from NK cells cultured in IL‐15 or a combination of IL‐12/IL‐15/IL‐18 for 48 h in serum‐free culture, or alternatively after activation through the Fc‐receptor CD16.

Results : We found that miR‐22‐3p, miR‐129‐5p, miR‐382‐3p, and miR‐3140‐5p appeared enriched in EVs putatively derived from the plasma membrane, while there was an enrichment of miR‐16‐5p, miR‐19b‐3p, miR‐20a/20b‐5p, miR‐23a‐5p, miR‐29b‐3p, miR‐142‐3p, miR‐181a‐5p, and miR‐630 in cytotoxic EVs. No major differences were observed when comparing the different cytokine treatments. Target prediction analysis suggested that miRNAs enriched in cytotoxic EVs converge on genes related to apoptosis and TGF‐β receptor signalling, and we further show that the cytotoxic EV subpopulation specifically reduced expression levels of BCL2, RICTOR and TGFBR1 in the HCT‐116 colon cancer cell line. Subjecting HCT‐116 cells to miR‐16‐5p and miR‐19b‐3p led to increased apoptosis compared to scrambled controls, as well as reduction of RICTOR.

Summary/Conclusion : To conclude, our data indicate that cytotoxic NK‐EVs can target cancer cells through miRNA‐mediated modulation of anti‐apoptotic pathways.

Funding : Research Council of Norway.

Bone‐derived matrix vesicles as a multi‐targeted therapeutic strategy for bone regeneration

M. Maggio 1 , M. Y. Brunet 1 , C. Gorgun 1,2 , R. Almasri 1 , L. O'Driscoll 1 , D. A. Hoey 1,2,3

1 Trinity College Dublin, Ireland; 2 Royal College of Surgeons in Ireland, Ireland; 3 Advanced Materials and Bioengineering Centre (AMBER), Ireland

Introduction : Matrix vesicles (MV) are membrane‐bound nanoparticles secreted by mature bone cells that are embedded in the extracellular matrix (ECM). As key mediators of biomineralization, they are essential for bone formation and repair. Moreover, there is emerging evidence for the presence and functional role of MV in ECM‐derived scaffold‐based tissue repair. MVs are also likely to be present in auto/allografts, which remain the gold standard for bone repair. Nevertheless, the multimodal activity of MVs, their mechanism of action, and their definition as an extracellular vesicle subpopulation remain unclear despite their initial discovery in the 1960s. Therefore, this study aimed to establish a reliable method to isolate MV from animal tissues and to investigate their pro‐regenerative potential for bone repair.

Methods : Tissues from 5‐month‐old male C57BL/6J mice were dissected to obtain long‐bone (lb) and kidney (kd)‐derived MV. Tissues pieces were digested with collagenase type 2 for 3 h at 37°C prior to filtration (100 µm) and differential ultracentrifugation with a swinging rotor (TH660) at 110,000 × g for 90 min to collect MV. MVs were then characterized including NTA and alkaline phosphatase (ALP) activity. Finally, MV anabolic and anti‐catabolic potential was evaluated by adding increasing doses of MV to human osteoblastic cells and human CD14+ monocytes in the presence of osteogenic or osteoclastogenic factors, respectively.

Results : Comparative MV characterisation revealed that lb‐MV were significantly smaller than kd‐MV ( p < 0.0001) with no yield variation observed upon g/tissue of origin. In terms of their intrinsic activity, all MV populations were found to express functional ALP, indicating their pro‐mineralising potential, with lb‐MV possessing significantly more ALP than kd‐MV ( p < 0.0001). Finally, lb‐MV displayed both anabolic and anti‐catabolic potential, eliciting increased mineralisation and decreased osteoclastogenesis in a dose‐dependent manner from 0.25 to 2.5 µg/mL.

Summary/Conclusion : MV were successfully obtained and characterized from multiple adult mouse tissues, offering a physiologically relevant model to investigate MV's roles in mature bone beyond in vitro cultures. Importantly, for what we believe to be the first time, lb‐MV demonstrated both anabolic and anti‐catabolic effects for the first time, highlighting their role in bone physiology and their potency as nanotherapeutics for bone repair.

Funding : SFI FFP Grant (19/FFP/6533).

Deciphering the heterogeneity landscape of mesenchymal stem/stromal cell‐derived extracellular vesicles for precise selection in translational medicine

Presenter: Ye Li

Xi'an Jiaotong University, China (People's Republic)

Introduction : Mesenchymal stem/stromal cell‐derived extracellular vesicles (MSC‐EVs) have been considered promising therapeutics for disease treatments. However, MSC‐EVs harvested from different tissues present unique biological features reflective of their origins. The heterogeneity of MSC‐EVs constitutes an important barrier to their precise application in clinical translation that may probably lead to uncertain therapeutic effects.

Methods : To give hints for future clinical translation, five MSCs are employed, whose derived EVs are most intensively utilized, namely bone marrow mesenchymal stem/stromal cells (BMMSCs), umbilical cord stem/stromal cells (UCSCs), adipose‐derived stem/stromal cells (ASCs), dermal stem/stromal cells (DSCs), and dental pulp stem/stromal cells (DPSCs) and the heterogeneity landscape of the corresponding MSC‐EVs is documented.

Results : Overall, the basic parameters, stability, and biosafety of different MSC‐EVs are indiscriminate. Strikingly, UCSC‐EVs exhibit distinguishing productivity. UCSC‐EVs as well as DPSC‐EVs present better drug loading/delivery capacity. In addition, the heterogeneity of different MSC‐EVs in cargo diversity, cellular affinity, organ biodistribution, and therapeutic effects may cue the rational selection in different disease treatments.

Summary/Conclusion : Through a combined assessment, a rational strategy is combined for selecting MSC‐EVs in future clinics. Offering a panoramic view of MSC‐EVs harvested from different tissues, the current study may provide guidelines for the precise selection of MSC‐EVs in next‐generation therapeutics.

Funding : China Postdoctoral Innovation Science Foundation (BX20200254), China Postdoctoral Science Foundation (2021M692474), Wuhan Knowledge Innovation Special Project (2022020801010500).

Analysis extracellular vesicles in CLL subtypes using direct stochastic optical reconstruction microscopy

Presenter: Joel J. R. Mccay

Barts Cancer Institute, UK

Introduction : Chronic lymphocytic leukaemia is the most common leukaemia of the western world. Determining which patients will need further treatment and catching earlier relapses remains a clinical challenge. Extracellular vesicles have been demonstrated as biomarkers of disease in other conditions, though limited data exists on newer ways to image and analyse their role in CLL. DSTORM imaging allows for more precise analysis of EV surface and intramural cargo. As well as bulk analysis, single EV analysis can be used to study 3‐colour tetraspanin or cargo markers in high resolution (to 20 nm). We aim to characterise the tetraspanin profiles of EVs across CLL subgroups and patient linear samples. We also aim to study the use of dSTORM in the discarded fractions from SEC, which still contain nanoparticles but are often limited by high protein concentration.

Methods : Multiple EV extraction methods were used (SEC, UF, UC, kits) across multiple biofluids (serum, plasma and cell culture). EV analysis was performed using dSTORM.

Results : (1) Large variation by single positive EV, though CD9 is most abundant across all samples. Dual positive events were highly variable across all patients, with D9/63 and CD9/81 present in 94% and 82% of patients, respectively. (2) CD9 expression was higher in good prognostic groups compared to poor prognostic groups ( p = 0.025), with CD63/81 3‐fold higher in poor prognostic CLL than good and 1.6 times higher than intermediate ( p = 0.09). (3) Linear samples from the same patients over time showed that patients who responded to chemotherapy had a total reduction in CD9‐positive events by 77% in patient A and 92% in patient B. This was compared to patient C, who did not respond to chemotherapy and had resistant disease whose CD9 expression increased by 154% over time. (4) Fractions 11–20, which are usually discarded due to protein content, were successfully used to isolate, analyse and image EVs comparable to normal fractions used (6–10).

Summary/Conclusion : EV tetraspanin profiles vary based on prognostic group and disease state. CD9+ EVs correspond to disease progression and remission across linear patient samples. All fractions could be successfully imaged for EVs using dSTORM.

Unbiased analysis of single extracellular vesicles by nano‐flow cytometry reliably unravels their population changes in different sources and upon different stimuli

Presenter: Nathalie Nevo

Institut Curie Research Center, INSERM, France

Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed particles released by most cell types. EVs are able to transfer signals and cargoes between donor and recipient cells and are thus instrumental in intercellular communication. However, EVs released by a single donor cell source are increasingly recognized as extremely heterogeneous in terms of size, intracellular origin, and cargo composition. EVs are routinely characterized by the presence of given proteins in bulk preparations. However, analysing large numbers of EVs at the single‐vesicle level is the only way to truly decipher their heterogeneity.

Methods : Here, we developed a reliable pipeline of single EV analysis by an EV‐dedicated flow cytometer (Flow NanoAnalyzer), which detects all particles and measures their size down to 55 nm in diameter without the need for immobilization or fluorescent labels. The necessity to remove unbound antibody and determine the optimal antibody concentration was demonstrated using EVs devoid of the analysed markers as negative controls. The pipeline was then developed to reliably quantify the proportion of EVs bearing none or any combination of two markers and their size.

Results : We thus observed, depending on the cell source (MDA‐MB‐231 versus HeLa), around 15% versus 20% of EVs bearing none of the CD9 and CD63 tetraspanins, often used to define EVs, 37% versus 23% of EVs bearing both CD9 and CD63, and either a similar proportion of single CD9+ and CD63+ EVs (MDA‐MB‐231) or 3 times more CD9+ than CD63+ EVs (HeLa). We also observed CD29 (ITGB1) as a protein present as frequently on EVs as the tetraspanins (MDA‐MB‐231). Other surface markers, by contrast, were present in less than 20% of EVs, and mostly of relatively large size. Finally, we confirmed that treatment of cells with homosalate, a drug previously proposed to increase the release of plasma membrane‐derived ectosomes (Grisard et al. JEV 2022 11, no. 7: e12242), increased the proportion of CD9+/CD63− EVs (likely ectosomes) to the detriment of CD63+/CD9‐ EVs (likely exosomes), and we identified two other drugs with the reverse effect.

Summary/Conclusion : Overall, nano‐flow cytometry implemented with the proper negative controls and validated antibodies allows us to reliably quantify proportions of EV subpopulations suggested by bulk analyses of EV markers.

Characterization and comparative analysis of extracellular vesicles (EV) from B cell lymphoma cultures and patient serum: highlighting methodological variations across EV isolates

Presenter: Md Nasir Uddin Badal

University of Turku, Finland

Introduction : B cell lymphomas (BCL) represent a heterogeneous group of diseases; the diagnostics to differentiate between them are labourious and therefore require additional methods. Extracellular vesicles (EVs) derived from BCL cells hold potential as biomarkers and therapeutic tools in immune‐related diseases. Despite their pivotal role in cellular communication, their specific characteristics within distinct cellular pools, for example, immune lymphoid cell subsets, remain poorly understood. This study aims to characterize and compare the protein profiles of EVs derived from either B cell lymphoma (BCL) cell cultures or patient serum, emphasizing the observed methodological differences to analyse these EV preparations comparatively.

Methods : EVs were isolated from BCL cell lines and patient serum using size exclusion chromatography (SEC) and characterized via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and immuno‐electron microscopy (iEM). Western blotting (WB) and mass spectrometry (MS) were employed to assess EV protein expression heterogeneity.

Results : Our data from mass spectrometry (MS) analysis confirms the presence of EV‐related proteins in patient serum samples. However, likely due to the more complex matrix, diverse protein contents, and surface protein masking, immune assay techniques such as ELISA, WB, or iEM could not detect these proteins on patient serum‐derived EVs as in cell line‐derived EVs without additional technical modifications. This highlights the need to adapt protocols from cell line EVs for the use of patient‐derived EVs when doing comparative studies. We optimized the marker detection for ELISA and WB assays for both BCL EV sources and could pinpoint the similarities and differences regarding physical characteristics and marker expression.

Summary/Conclusion : This study underscores the vast morphological and molecular diversity within EVs from BCL and patient serum and reinforces the importance of methodological streamLining to enable the comparison of cell line EV findings to patient serum.

Common properties of extracellular nanoparticles from various sources: utilizing chromatography for a comprehensive characterization of complex samples

Darja Božič 1* , Katja Vrabec 1 , Valentina Novak 1 , Ivana Petrović Koshmak 1 , Andrej Raspor 1 , Nastja Bizjak Ternik 1 , Ana Železnik 1 , Maja Leskovec 1 , Matevž Arko 2 , Anna Romolo 2 , Alenka Nemec Svete 2 , Vladimira Erjavec 2 , Martina Gobec 2 , Nika Strašek Benedik 2 , Melanie Reininger 3 , Ingrid Hartl 3 , Tobias Tertel 4 , Bernd Giebel 4 , Klaus Graumann 3 , Veronika Kralj‐Iglič 2 , Aleš Štrancar 1

1 Sartorius BIA Separations, Slovenia; 2 University of Ljubljana, Slovenia; 3 Phoenestra Gmbh, Austria; 4 University of Duisburg‐Essen, Germany

Introduction : The functional studies of extracellular vesicles (EVs) remain controversial due to the limited purity of preparations and inadequate analytics, which are typically focused on an isolated parameter (e.g., particle concentration, marker‐positive subpopulations, total protein/nucleic acid content). We believe that a better understanding of preparations’ composition is crucial for relating specific functionalities to different sample constituents.

Methods : Size exclusion (SEC) and ion‐exchange chromatography (IEC) for characterizing EV preparations were explored using a PATfix system with incorporated detectors for light absorbance, fluorescence, and multi‐angular light scattering.

Results : Strengths and weaknesses of SEC and iEC were evaluated in detail. Ultimately, a combined, two‐dimensional liquid chromatography (2D‐LC) method was developed, providing a detailed profile of sample components resolved by size and by charge. The approach is applicable to biological samples of varying complexity, such as cell culture conditioned media and various EV‐enriched preparations. Similar subpopulations were recognized in samples from very diverse sources, opening a new perspective on subclasses of EVs and non‐EV extracellular nanoparticles (EPs).

Summary/Conclusion : Comprehensive chromatographic characterization of EV preparations provides the insights that can pave the way towards better understanding of the relationship between the structure and function of EVs and other EPs.

Funding : The study was funded by Sartorius Bia Separations.

EV preparations from telomerized mesenchymal stromal cell lines elicit robust performance in therapeutically relevant models

Presenter: Claudia Lindner

Phoenestra GmbH, Austria

Introduction : Extracellular vesicles (EVs) from different cell sources, including mesenchymal stromal cells (MSC) offer great promise for many therapeutic applications. Consequently, MSC‐derived EVs are being investigated in a number of pre‐clinical and clinical trials. However, successful translation of EV into pre‐clinical and especially clinical products has been slow. Therefore, gaining a deeper understanding of the biological effects of well‐characterized EVs is crucial.

Methods : Phoenestra has developed a novel manufacturing setup based on a patented perfusion bioreactor assembly and stable, telomerized MSC (MSC/TERT). Conditioned media harvests were processed by depth filtration and ultra‐/diafiltration. The resulting EV preparations have been analysed in‐depth regarding particle number, lipid particle and EV marker content as well as miRNA profiling. To further explore the therapeutic potential, we tested selected EV preparations in different bioassays and pre‐clinical models investigating, for example, anti‐inflammation, anti‐fibrosis (2D and 3D), HUVEC vascularization (2D and 3D), as well as kidney cell proliferation.

Results : MSC/TERT‐EV produced from different manufacturing runs show consistent quality profiles; however, with distinct donor/tissue‐specific differences. Anti‐inflammatory and anti‐fibrotic activities were consistently dependent on the origin of the cell source. From a human 2D angiogenesis and a kidney cell proliferation model, it became obvious that selecting the right cell to EV ratio (2,000‐10,000 EVs/cell) was essential for assessing biological function in a dose‐dependent manner. The selected human 2D and 3D models showed distinct differences in the tested EV preparations regarding cell proliferation, tissue protection and regeneration, as well as vascularization potentials.

Summary/Conclusion : Beyond manufacturing and in‐depth analytical characterization, we have demonstrated the biological potential of MSC/TERT‐EV preparations in relevant biological 2D and 3D model systems. With this approach we are collecting additional insights into the biological functionalities and therapeutic potentials of MSC/TERT‐EV preparations towards (pre‐)clinical applications.

Funding : The authors acknowledge public funding support for this work from the Austrian research promotion agency FFG (FFG Basisprogramme and Innovationsscheck, awarded to Phoenestra GmbH).

Extracellular vesicles define discrete nano‐based niches within the human haematopoietic system

I. Grenier‐Pleau* 1 , C. Wells 1 , S. Holmes 1 , C. Hall 1 , M. Vermeulen 1 , J. Penders 3 , S. Dick 1 , E. Bonneil 2 , A. Craig 1 , D. Knapp 2 , P. Thibault 2 , M. Stevens 3 , S. Mann 4 , J. F. Rudan 4 ;S. A. Abraham 1

1 Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada; 2 Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada; 3 SPARTA Biodiscovery Ltd., Imperial College London, London, England; 4 Department of Surgery, Queen's University, Kingston, Ontario, Canada

Introduction : Stem cell niches are complex multi‐signalling networks comprised of molecular cues and physical interactions, orchestrated by niche‐resident cells and the extracellular factors they produce. The bone niche specifically houses haematopoietic stem cells (HSCs), a critical cell type responsible for producing all blood and immune cells throughout life. Currently, how stem cell niches facilitate ideal environments with simultaneously coordinating compartmentalized intrinsic and extrinsic cellular signals is unknown.

Methods : Enrichment of extracellular vesicles (EVs) was performed using a two‐step iodixanol density cushion and size exclusion chromatography protocol from human plasma, bone marrow, and trabecular interstitial plasma, a yet‐to‐be‐characterized EV biofluid. Unbiased proteomic mass spectroscopy and machine learning techniques were used to identify differences between niche‐sourced EVs within the haematopoietic system. Characterization of nanoparticles at a single‐vesicle resolution using Raman spectroscopy and mathematical modelling was also completed. Finally, stem cell assays, single‐cell transcriptomics and pathway analyses were implemented to elucidate functional effects of niche‐sourced EVs.

Results : Studies presented here identify the existence of unique EV‐defined niches within the haematopoietic system of healthy human individuals. Proteomic signatures, single EV characterization and machine learning techniques reveal that EVs can be grouped by blood, bone marrow and trabeculae within a human individual. Stem cell assays demonstrate that these niche‐defined EVs impart functional effects on stem cells/progenitors based on the location within the haematopoietic system. Finally, using single‐cell transcriptomic analyses, results identify for the first time how niche‐sourced EVs differentially affect the most primitive human haematopoietic stem cells, keeping them in a protected state within the trabecular niche.

Summary/Conclusion : This study highlights the significance of nanoparticles on human immunity and blood production while concurrently providing evidence for a new role for EVs, namely the demarcation of distinct nano‐niches within biological systems.

Funding : This study is funded by a Canadian Institutes of Health project grant (394568, 175484; S.A.A.), a Terry Fox New Frontiers Program Project Grant (6039065; S.A.A.) and a Terry Fox New Investigator Grant (1133; S.A.A.).

Context‐dependent roles of extracellular vesicle subtypes in tumour angiogenic signalling

Grace Richmond, Rose Nguyen, Alanna Sedgwick, Jeff Schorey, Crislyn D'Souza‐Schorey

University of Notre Dame, USA

Introduction : Extracellular vesicles (EVs) have emerged as an important conduit for intercellular communication within the tumour ecosystem. Different types of EVs have been identified based on cellular origin and biogenesis mechanisms and may be classified as ‘large EVs’ (> 200 nm) or ‘small EVs’ (< 200 nm). Angiogenesis, a key cellular process, is co‐opted during tumour growth and development and is markedly impacted by EV‐based paracrine signalling. Here we show that large EVs (lEVs) derived from highly invasive tumour cell lines promote pro‐angiogenic behaviours in endothelial cells through mechanisms that are distinct from those documented for small EVs (sEVs) or soluble growth factors.

Methods : Tumour EV subtypes were assessed for their effects on endothelial cells using tube‐forming assays. To this end, large and small EVs shed from tumour cell conditioned media were isolated and characterized for size and morphology per MISEV guidelines. Endothelial cytokine expression and release were determined by cytokine arrays and validated by qRT‐PCR and ELISA. EV angiogenic cargo was further studied via immunofluorescence microscopy, protein biochemistry and western blotting.

Results : Our findings reveal that lEVs from highly invasive tumour cell lines promote angiogenesis via unique mechanisms that are distinct from those reported for other EV subtypes. In response to lEV uptake, endothelial cells exhibit increased induction of pro‐angiogenic phenotypes, including increased tubulation and branching, altered cytokine expression and increased autocrine signalling by pro‐angiogenic factors. Our study also highlights differences in pro‐angiogenic cargos in tumour EV subtypes.

Summary/Conclusion : We suggest that the effects of EVs on angiogenesis are likely tumour‐type and EV subtype dependent. Of particular bearing to the findings described is that lEV production appears to be usurped as tumour cells transition to metastatic and more aggressive ‘amoeboid’ phenotypes.

Funding : This work was supported by NIH 1R01GM148708, NIH 1R03CA273598‐01, and the Boler Foundation.

Know your vesicles: how cell source shapes extracellular vesicle attributes

Melanie Reininger, Ingrid Hartl, Claudia Lindner, Roland Prielhofer, Klaus Graumann

Phoenestra GmbH, Austria

Introduction : Investigating the composition‐function relationship of mesenchymal stromal cell (MSC)‐derived extracellular vesicles (EVs) is complex due to their inherent heterogeneity and lack of process reproducibility. To address the challenge, we strive for process standardization to minimize product variability. Our approach involves the use of telomerized MSC lines, xeno‐ and hPL‐free media and a controlled, scalable stirred tank bioreactor setup. This strategy enables us to reproducibly establish a solid data foundation towards product definition and supports the identification of true cell line‐specific commonalities and differences in EV characteristics.

Methods : EVs were produced from five telomerized MSC lines derived from different human sources (adipose tissue, chorionic plate, Wharton's Jelly, bone marrow, and placenta) in our patented stirred tank bioreactor setup. Conditioned media was harvested, filtered, concentrated and washed via tangential flow filtration using a low‐shear membrane pump. These EV preparations were analysed in multiple ways: particle concentration and size distribution were determined by nanoparticle tracking analysis (NTA), protein content was measured by bicinchoninic acid assay (BCA), surface marker composition was analysed via nanoflow analysis, miRNA profiles were established by small RNA sequencing, and their biological function was assessed in several different cell‐based bioassays. The results from each analysis method were collectively correlated with biological activity.

Results : Several cell line‐specific differences in EV composition were found: although cultured in identical media conditions, the protein‐to‐particle ratio demonstrated to be highly cell line‐dependent, ranging between 3 and 23 mg/1E + 10 particles. Further, we were able to confirm the presence of relevant EV markers and lipids and show that a broader marker profile as well as the bioactivity in different assays are determined by cell source and cultivation time. In‐depth miRNA analysis showed that the 10 most prevalent miRNAs are mostly consistent in identity across different cell lines, though they vary in abundance and ranking.

Summary/Conclusion : Our reproducible process enables the generation of standardized EV preparations that facilitate comprehensive analysis and characterization. Thereby, we are paving the way for better product understanding and, eventually, definition of critical quality attributes and key contributors to biological functionality. Ultimately, it allows us to select and tailor EV preparations to meet specific (pre‐)clinical needs.

Probiotic extracellular vesicles as potential immune mediators

Agnieszka Razim 1,2 , Anna M. Schmid 1 , Magdalena E. Paschal 1 , Tamara Weinmayer 1 , Michael Thaler 1 , Paweł Migdał 2 , Magdalena E. Skalska 3 , Mattia Morandi 4 , Catherine Daniel 5 , Dagmar Srutkova 6 , Martin Schwarzer 6 , Ursula Wiedermann 1 , Sabina Górska 2 , Irma Schabussova 1

1 Medical University of Vienna, Austria; 2 Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Poland; 3 Jagiellonian University, Poland; 4 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Czech Republic; 5 Institut Pasteur de Lille, France; 6 Institute of Microbiology of the Czech Academy of Sciences, Czech Republic

Introduction : The immunomodulatory potential of probiotics is well known. Not long ago, we learnt that probiotic bacteria produce extracellular vesicles (EVs), the role of which is not yet fully understood. However, since EVs are non‐living and non‐replicating, they offer many advantages over live bacteria in clinical applications. Therefore, EVs can be used in immunocompromised individuals and appear to be ideal for intranasal administration.

Methods : We isolate postbiotics in the form of EVs from the probiotic Lactiplantibacillus plantarum NCIMB8826 (LpEVs). We purify these EVs with size exclusion chromatography and characterized them with respect to their physical characteristics (size, zeta potential, and stability studies), their composition (proteins, lipids, DNA, and RNA) and their immunomodulatory properties in vitro and in vivo. We use SDS‐PAGE, Bioanalyzer, proteomics, and lipidomics to analyse the cargo. In in vitro studies we analysed receptor recognition and cytokine induction using epithelial and immune cell lines.

Results : We used multiple methods to show that L. plantarum produces significant amounts of EVs with a size of about 70–80 nm in the MRS medium. High doses of LpEVs significantly reduced the number of recruited eosinophils in the lungs of allergic mice. Moreover, these LpEVs are resistant to long‐term storage, physiological pH, different salt concentrations and detergents (found in the digestive tract), making them perfect for intranasal or oral administration. However, EVs produced in MRS differed from those produced in MRS + 0.25% bile in size and protein content.

Summary/Conclusion : We have shown that LpEVs retain some immunomodulatory properties of the live strain, but at the same time their characteristics are highly dependent on the conditions under which they are produced. These data suggest that the bacterium actively responds to changing environmental conditions. The immunomodulatory potential of the ‘bile‐stressed’ EVs is currently being investigated.

Funding : This research was funded by HORIZON‐MSCA‐2021‐PF (project no. 101066450), START2022 by the Foundation for Polish Science, SONATA (2023/51/D/NZ7/02220) financed by the National Science Centre Poland and the Danube Allergy Research Cluster, mobility OEAD WTZ CZ 07/2023 and PL 03/2022 and FWF P 34867. We acknowledge CEITEC/Brno—Nanobiotechnology of CIISB, Instruct‐CZ Centre, supported by MEYS CR (LM2023042)) and European Regional Development Fund‐Project “UP CIISB” (No. CZ.02.1.01/0.0/0.0/18_046/0015974).

Culprit site EV protein signatures uncover molecular details of plaque rupture in acute myocardial infarction

Ulrike Resch 1,2 , Taras Afonyushkin 3 , Silvio Kau‐Strebinger 4 , Anna S. Ondracek 5 , Luisa M. Schmidt 2 , Marcus Krueger 2 , Christoph Binder 3 , Irene Lang 5

1 Center for Physiology and Pharmacology, Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria; 2 CECAD Cluster of Excellence for Aging Research, Institute for Genetics, University of Cologne, Germany; 3 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 4 Department of Biomedical Sciences and Pathobiology, Center of Pathobiology, Institute of Morphology, Vetmeduni, Vienna, Austria; 5 Department of Internal Medicine II, Cardiology, Medical University of Vienna, Vienna, Austria

Introduction : Acute myocardial infarction (AMI) is a leading cause of heart failure and death. Hypoxia reperfusion leads to damage and death of microvascular cells and cardiomyocytes and the release of DAMPs. Subsequent local immune cell infiltration not only contributes to the immediate damage but also determines cardiac remodelling. Extracellular vesicles (EVs) shed during this process reflect activation and death of different cells and are also actively involved in the pathogenesis of AMI. However, potential differences of the EV protein composition at the culprit site as compared to the peripheral circulation are unknown. Characterizing the proteome of culprit‐site plasma and EVs may provide more detailed molecular insights into culprit site plaque rupture and subsequent myocardial remodelling and allow for the development of new prognostic markers and therapies.

Methods : This study included a patient subset ( n = 7) of the ‘Strategic targeted temperature management’ study (STATIM, NCT01777750 ) recruited at the time of diagnosis of ST‐segment elevation myocardial infarction (STEMI). EVs were isolated from cell free citrated‐plasma aspirated from arterial culprit and peripheral sites after centrifugation at 18,000 × g . EV size, shape, and surface markers were analysed using NTA, TEM and flow cytometry in agreement with MISEV recommendations. Comparative proteome profiling of crude plasma, MagReSyn‐depleted plasma and EVs was realized using an EvoSep One‐30SPD‐timsTOF‐pro 2 setting operating in dia‐PASEF mode.

Results : We identified 2673, 933, and 1615 proteins in EVs, crude or depleted plasma. Eight hundred sixty‐one proteins were common to all three sample types, 10 and 21 proteins were unique to crude or depleted plasma, and 1627 proteins were unique to EVs, emphasizing the valuable gain of information obtained from the plasma EV‐compartment. We found that EV, but not whole plasma proteome signatures, shows distinct site‐specific patterns. Culprit EV proteomes contained predominant neutrophil and platelet degranulation signatures. In addition, we found selective enrichment of mitochondrial proteins in culprit EVs consistent with massive cardiomyocyte death.

Summary/Conclusion : We uncovered a unique acute phase EV proteome signature delineating molecular details of the STEMI culprit site that support a critical role for neutrophil activation.

Role of SERINC proteins on EV lipid content

Alix Zhou 1 , Julio Lopes‐Sampaio 2 , Clotilde Théry 1,3 , Alain Joliot 1,* , Lorena Martin‐Jaular 1,3,*

1 Institut Curie Research Center, INSERM U932, PSL Research University—Paris, France; 2 Institut Curie Research Center, CurieCoreTech Metabolomics and Lipidomics Technology Platform, Orsay, France; 3 Institut Curie Research Center, INSERM U932, CurieCoreTech Extracellular Vesicles, PSL Research University‐Paris, France

Introduction : Extracellular vesicles (EVs) are structures delimited by a lipid bilayer which are naturally released by cells. They are the subject of increasing interest due to their capacity to carry information and their numerous biological functions. A previous study in our group identified that SERINC3 was present on EVs and involved in their surface protein composition. SERINC proteins have been reported to facilitate the biosynthesis of sphingolipids (SP) and phosphatidylserine (PS). Moreover, SERINC3 and SERINC5 are known HIV‐1 antiviral factors, whose effect is counteracted by Nef by reducing their presence in the plasma membrane. The contribution of SERINCs to the EV lipidome is currently not well known.

Methods : We have used the CRISPR Cas9 system to reduce the levels of SERINC3 alone or in combination with SERINC5 reduction in Jurkat cells. In addition, Jurkat cell lines expressing Nef have been obtained. EVs from these cell lines have been isolated by size exclusion chromatography (SEC) and characterized in terms of number and size by the nanoparticles tracking analysis (NTA). Western blot has confirmed the presence of EV‐specific markers on the isolated material. Finally, mass spectrometry‐based shotgun lipidomics have been used to analysed the lipidome of both cells and EVs.

Results : We have established the conditions and proper controls to analyse EV lipidome. Our results show that the lipid compositions of cells and EVs differ. EVs are enriched in gangliosides, cholesterol, sphingolipids and LPS, while other phospholipids, lysolipids, DAG and HexCer are reduced compared to cells. When comparing the EVs released by the different cell lines, we have observed that the reduction of SERINC3 alone does not result in a significant change in the lipids studied. However, the levels of phosphatidic acid (PA) are slightly decreased when SERINC3 and 5 are reduced together or when Nef is expressed.

Summary/Conclusion : Our results show that SERINC3 reduction alone has no major impact on the lipid composition of cells or EVs. However, we cannot rule out that the SERINC family has a more subtle role in the lipid composition of EVs, which would require more targeted strategies.

Subtopic EV Cell Interaction 1

Exosomes are necessary and sufficient for fibronectin assembly

Bong Hwan Sung 1,2,3 , Merlyn Emmanuel 4 , Metti Gari 5 , Jorge Guerrero 5 , Evan Krystofiak 6 , Suzanne M. Ponik 5 , and Alissa M. Weaver 1,2,3,7

1 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA; 2 Program in Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA; 3 Center for Extracellular Vesicle Research, Vanderbilt University, Nashville, Tennessee, USA; 4 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada; 5 Department of Cell and Regenerative Biology, University of Wisconsin‐Madison, Madison, Wisconsin, USA; 6 Vanderbilt University Cell Imaging Shared Resource, Nashville, Tennessee, USA; 7 Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

Introduction : Extracellular matrix assembly is critical for tissue integrity and is deregulated in diverse diseases. Fibronectin is a key stromal matrix molecule whose assembly into fibrils is thought to require cells. Integrins and matrix molecules are frequently identified in EVs and have been implicated in driving cell migration and cancer metastasis. We previously found that fibronectin association with exosomes depends on the presence of α5 integrin and that exosome‐associated fibronectin drives nascent adhesion formation and cell migration by cancer cells. Those data suggest that not only is fibronectin a true EV cargo but also that it is likely to be in an assembled, adhesive form. Based on these data, we hypothesized that exosomes act as acellular molecular platforms that are specialized for inducing fibronectin assembly.

Methods : EVs were isolated by differential centrifugation and iodixanol density gradient and verified by nanoparticle tracking analysis and electron microscopy. Tandem mass tag proteomics quantitated protein cargo in EVs. A purified components assay for fibronectin assembly was developed, and in vitro fibronectin assembly by EVs was identified biochemically and microscopically. Integrin and syndecan‐1 presence in EVs was quantitated using super‐resolution microscopy.

Results : We identify small exosome‐type EVs as critical initiators of fibronectin assembly. Fibroblasts engineered to be deficient in exosome secretion showed greatly reduced assembly of fibronectin and other stromal matrix molecules in 2D, 3D, and in vivo environments and led to reduced lung metastasis by breast cancer cells. Consistent with a critical role for exosome secretion in stromal matrix assembly, bleomycin‐treated Rab27a/b double knockout mice exhibited greatly reduced lung fibrosis. Using a newly developed purified components assay, we find that the addition of purified small EVs to purified soluble cellular fibronectin is sufficient to induce fibronectin assembly—contradicting the dogma that fibronectin assembly requires cells. The EV‐induced fibronectin assembly requires the presence of fibronectin‐binding integrins and syndecan‐1 in the EVs.

Summary/Conclusion : Our data indicate that exosome secretion plays a critical and unsuspected role in the intrinsic ability of stromal cells to assemble fibronectin. We propose a model in which fibronectin‐binding integrins and syndecan‐1 clustered in exosomes synergistically produce robust fibronectin assembly.

Funding : R50CA238661, R01CA206458, and R01CA249424.

Suppression of senescence‐associated inflammation by secretome and EVs derived from hTSCs

Presenter: Kotb Abdelmohsen

NIH, USA

Introduction : Cellular senescence, a state of irreversible cell cycle arrest, plays a pivotal role in ageing and age‐related disorders. Understanding senescence and identifying novel therapeutic strategies are critical for mitigating its pathological effects. In this study, we investigated the secretome and extracellular vesicles (EVs) derived from human trophoblast stem cells (hTSCs) for their potential anti‐senescence properties. We found that the hTSC‐S and EVs significantly suppressed the expression of key SASP‐related mRNAs, including CXCL1, CXCL8, and GDF15. We found that the hTSC‐S and EVPs are enriched in proteins involved in extracellular matrix remodelling, cell adhesion, and tissue repair, underscoring their anti‐inflammatory impacts. The hTSC‐S TEMPTEMP effectively reduced both the expression of SASP‐related mRNAs and the secretion of their encoded proteins. These TEMPTEMP effects may be linked to decreased senescence‐associated β‐galactosidase (SA‐β‐Gal) activity and reduced DNA damage. Furthermore, hTSC‐S attenuated inflammatory signalling by inhibiting NF‐κB phosphorylation, a major SASP regulator. In vivo analysis indicated that hTSC‐S treatment decreased the levels of the pro‐inflammatory marker CXCL1 and the ageing marker GDF15. Our findings establish hTSC‐S and EVs as promising candidates for senescence‐targeted therapies, offering a novel strategy to address ageing and age‐related diseases.

Methods : Size exclusion chromatography (SEC), EVP isolation by ultraceltifugation, nanoflow cytometry (NFCM), ExoView tetraspanin analysis, transmission electron microscopy

Results : hTSC‐secretome suppresses SASP mRNAs, hTSC‐EVs suppress SASP mRNAs, hTSC‐secretome promotes growth and ECM remodelling in IR‐induced senescence, Potential restoration of key proteins in senescence by hTSC‐derived EVP hTSC‐secretome reduces DNA damage and NF‐κB signalling activity hTSC‐secretome reduces inflammatory SASP markers in aged mice.

Summary/Conclusion : hTSC‐Secretome suppresses SASP mRNAs; hTSC‐EVs suppress SASP mRNAs; hTSC‐secretome promotes growth and ECM remodelling in IR‐induced senescence; potential restoration of key proteins in senescence by hTSC‐derived EVPs; hTSC‐secretome reduces DNA damage and NF‐κB NF‐κB signalling activity; hTSC‐Secretome reduces inflammatory SASP markers in aged mice.

Funding : This research was supported by the National Institute on Aging Intramural Research Program of the National Institutes of Health. The TSC‐secretome was provided by Accelerated Biosciences.

Subtopic Functional cell state‐related EVs 1

Hypoxic stress mediates cardiac extracellular vesicle release and transcriptional changes in bystander cells

Presenter: Hargita Hegyesi,

Semmelweis University, Hungary

Introduction : Cardiomyocytes (CMs) comprise approximately 30% of cells in the heart and communicate with cells to maintain cardiac functions. Numerous cargoes originating from the mitochondria, cytosol, and the plasma membrane of cardiomyocytes have been identified in small and large EVs, which, once transferred to other cells, may influence their biological activity of the recipient cells. Ischaemic heart disease is associated with increased production of extracellular vesicles (EVs), and the molecular composition of EVs from hypoxic cells differs significantly from that of normoxic cells.

Objective : This study investigates how hypoxia‐reoxygenation (H/R) alters EV release from CMs and assesses whether cardiac EVs influence the gene expression profile of unstressed (bystander) CMs.

Methods : We cultured C57/Bl6 mouse‐derived primary neonatal cardiomyocytes. Hypoxia was induced for 4 h using Oxoid AnaeroGen paper sachets, followed by reoxygenation in an incubator for 16 h. Large EVs (lEVs) and small EVs (sEVs) were isolated via the combination of gravity driven size filtration and differential centrifugation. EV characterization was performed using flow cytometry, TEM, and nanoparticle tracking analysis (NTA). CM‐derived EV uptake was assessed by confocal microscopy and flow cytometry. The functional impact of CM‐EVs on bystander cells was analysed using the PAMM‐174ZA RT2 Profiler PCR Array (qPCR). KEGG pathway analysis was conducted using DAVID Functional Annotation Tools.

Results : CM cells expressed caveolin‐3 and α‐actinin. Compared with controls, H/R‐treated cells released a significantly increased number of lEVs, as indicated by Annexin V binding, suggesting elevated lEV release following H/R treatment. H/R treatment decreased CD81+ sEV levels, with CD9 and CD107α remaining undetectable. Treatment with H/R CM EVs significantly upregulated ADRA1A, ADRA1D, AEBP1, MYH10, TNNI3, CCL11, and COL1A1 in bystander cells after 24 h in the recipient CM cells. In contrast, the expression of CREM, CCN2, TNNT2, FRZB, and MAP3K5 was reduced. The genes modulated by H/R EVs are involved in a common molecular pathway implicated in hypertrophic cardiomyopathy development.

Summary/Conclusion : This study advances our understanding of the mechanisms of CM‐EV biogenesis under physiological and pathological conditions, contributing insights into potential pharmacological strategies for ischaemic heart failure.

Funding : NVKP_16‐1‐201 (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003 (NCLP), 2019‐2.1.7‐ERA‐NET‐2021‐00015, 2024‐1.2.5. TÉT and the EU's Horizon 2020 No. 739593.

Drugs impairing the autophagic flux point out a role for autophagic machinery in the release of extracellular vesicles

G. Cerrotti 1 , S. Buratta 1,2 , R. M. Pellegrino 1 , H. Alabed 1 , P. Gorello 1,3 , E. Calzoni 1 , C. Emiliani 1,2,3 , L. Urbanelli 1,2,3

1 Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; 2 Extracellular Vesicles network (EV‐net) of the University of Perugia, Italy; 3 CEMIN – Center of Excellence for Innovative Nanostructured Material, University of Perugia, Perugia, Italy

Introduction : The endolysosomal system plays an important role in the biogenesis and secretion of EVs. We analysed EVs release of cell treatment with drugs impairing the autophagic/lysosomal system by administering to cells (1) chloroquine (CQ), commonly used to impair autophagy by autophagosome‐lysosome fusion blockade; (2) bafilomycin A1 (BafA1), known to disrupt autophagic flux by independently inhibiting V‐ATPase‐dependent lysosomal acidification and Ca‐P60A/SERCA‐dependent ER calcium transport.

Methods : EVs were isolated from cell medium by differential ultracentrifugation and further separated by density gradient. Small EVs were collected, characterized morphologically by SEM, quantified by NTA, and their markers assessed by IB. Their lipid content was analysed by LC/MS. The biological anti‐inflammatory effects of these vesicles on THP‐1 were also assayed.

Results : BafA1 and CQ both induce the release of EVs enriched in autophagy markers in several cell types, but interestingly only BafA1 induced the release of a remarkably higher number of small EVs. Baf A1‐EVs were also less dense, and their lipid composition was consistent with an enrichment of the general lipid content. Therefore, lysosomal alkalinization, induced by both drugs, is associated with the release of EVs carrying autophagy markers, but only Baf A1, an inhibitor of the ER calcium pump SERCA, increases the number of EVs. We extended our search for autophagy markers associated with EVs to biological fluids. EVs from plasma of healthy subjects and individuals with sickle cell and thalassaemia traits revealed the presence of autophagy markers.

Summary/Conclusion : Although Baf A1 and CQ are drugs commonly used to inhibit autophagy, their role in prompting EVs release is usually not considered but may have implications in pathologies characterized by impairment of the autophagic/lysosomal system, such as age‐related neurodegenerative disorders.

Funding : This work has been funded by the European Union‐NextGenerationEU under the Italian MUR National Innovation Ecosystem grant ECS00000041 – VITALITY (to Carla Emiliani). The authors acknowledge Università degli Studi di Perugia and MUR for support within the project VITALITY. This manuscript reflects only the authors’ views and opinions; neither the European Union nor the European Commission can be considered responsible for them.

Human amniotic fluid‐derived EVs increase regenerative ability of early‐senescent human dermal fibroblasts

Martina Guzzetti 1 , Marilena Castelli 1 , Letizia Mezzasoma 1 , Lara Macchioni 1 , Magdalena Davidescu 1 , Virginia Boccardi 1 , Giuseppe Servillo 1 , Rita Romani 1,2 , Francesca Fallarino 1,2 , Ilaria Bellezza 1,2

1 University of Perugia, Italy; 2 Extracellular Vesicles Network (EV‐net) of the University of Perugia, Italy

Introduction : Ageing, the progressive decline of the physiological functions, is charachterized by several hallmarks, including telomere erosion, mitochondrial instability and replicative senescence. Senescent cell secretome includes pro‐inflammatory molecules and extracellular vesicles (EVs) which, by conditioning tissue microenvironment, can play a pivotal role in the development and progression of chronic age‐related diseases.

Methods : Human dermal fibroblasts (BJ cells) were subjected to serial passaging for approximately 3 months. Senescence was verified by analysing structural and morphological changes, cell cycle arrest, mitochondrial dysfunction, and an increase in senescence‐associated β‐galactosidase (SA‐βGal) activity. Telomerase‐immortalized human skin fibroblasts (BJ‐hTERT) were used as non‐aged control cells. EVs were isolated from FBS‐deprived culture media of BJ cells at different ageing stages and from human amniotic fluid (hAF‐EVs) by differential ultracentrifugation. EVs were characterized for size and concentration by nanoparticle tracking analysis and for morphology by scanning electron microscopy. Protein content was used to characterize EVs following MISEV 2023 guidelines. EVs immunomodulatory effect was tested by inflammasome activation assay in human monocytic THP‐1. The anti‐ageing effects of hAF‐EVs were analysed by scratch wound healing assay.

Results : Replicative senescence in BJ cells was obtained after approx. 40 serial passages, showing a senescence‐like morphology, the loss of replicative potential, an increased SA‐βgal activity, a decreased mitochondrial membrane potential (MMP), and an alteration of the senescence‐associated markers p16 and lamin B1. Proliferating SA‐βgal‐negative BJ cells, characterized by increased p16 and decreased MMP, were considered early‐senescent cells. Independently of the ageing stage, EVs isolated from BJ cells showed significant differences in size, concentration, or marker expression, nor in immunomodulatory activity, maintaining their ability to inhibit inflammasome activation. The anti‐ageing effects of hAF‐EVs were evaluated in BJ cells by wound healing assay. Long‐term exposure to hAF‐EVs increased the reparative ability of early senescent BJ cells, while they were ineffective in aged cells.

Summary/Conclusion : These preliminary results showed that EVs may exert an anti‐ageing effect by increasing the skin healing process. Although the composition and role of their cargo are not yet fully understood, EVs can be proposed as a novel anti‐ageing strategy to prevent age‐related skin diseases, including defective wound healing and skin malignancies.

Caspase inhibitors attenuate extracellular vesicle‐mediated cell death in microglia and astrocytes in people living with HIV

Erika G. Marques de Menezes 1,2 , Scott A. Bowler 3 , Thomas A. Premeaux 3 , Cecilia M. Shikuma 4 , Mohamed Abdel‐Mohsen 5 , Lishomwa C. Ndhlovu 3,6,4 , Philip J. Norris 1,2,7

1 Vitalant Research Institute, San Francisco, CA, USA; 2 Department of Laboratory Medicine; 3 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York City, New York, USA; 4 Department of Tropical Medicine, Weill Cornell Medicine, New York, New York, USA; 5 Northwestern University, Chicago, Illinois, USA; 6 Hawaii Center for AIDS, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA; 7 Department of Medicine, University of California, San Francisco, California, USA

Introduction : We have previously demonstrated that extracellular vesicles (EVs) expressing neuroinflammatory markers in people living with HIV (PLWH) are associated with HIV‐related neurocognitive impairment, which persists despite effective suppression with antiretroviral therapy. However, the mechanistic role of EVs in contributing to neuroinflammation in PLWH remains unclear. Given that microglial and astrocyte cell injury contributes to neurological impairment, we investigated the impact of EVs from PLWH and their effects on survival of brain resident cell types involved in neuropathology.

Methods : EVs were isolated by differential centrifugation from plasma samples obtained from 11 PLWH and 13 people without HIV (PWOH). The concentration and size of purified EVs were evaluated using a NanoSight instrument (Malvern). Primary human microglia and astrocytes were exposed to EVs in the presence or absence of caspase‐1, ‐10, and ‐12 inhibitors, and apoptosis and necrosis rates were quantified by staining with annexin V and propidium iodide (PI) using an Aurora spectral cytometer. Statistical analysis was performed using the Kruskal‐Wallis test and Dunn's multiple‐comparison post hoc test ( p < 0.05).

Results : The levels of necrosis (annexin V‐PI+) in microglia exposed to EVs from PLWH who were aviremic or viremic were significantly increased when compared with EVs from PWOH or to unexposed microglia. Necrosis was also significantly increased after astrocyte exposure to EVs, irrespective of HIV status, when compared with unexposed astrocytes. No significant difference in early apoptosis (annexin V+PI‐) or late apoptosis (annexin V+PI+) levels between the EV treatment groups was noted; however, a significant increase in late apoptosis was observed between all EV treatment groups and untreated cells. Inhibition of caspase‐1 in EV‐exposed microglia and inhibition of caspases‐1, ‐10, and ‐12 in EV‐treated astrocytes significantly reduced EV‐induced necrosis irrespective of HIV status.

Summary/Conclusion : A reduction in EV‐mediated cell death by caspase inhibitors, acting downstream of critical inflammatory pathways (TNF, Fas, calpain), highlights a potential targetable mechanism by which EVs may contribute to HIV‐associated neuroinflammation and neuronal injury in PLWH through the promotion of an inflammatory cell death in CNS resident cells. Our findings further suggest that targeting EVs holds promise for limiting apoptosis and mitigating HIV‐associated cognitive impairment even among those with viral suppression.

Phenotypic and functional changes of iron‐laden microglia: involvement of EVs in paracrine senescence

Presenter: Maria Teresa Golia

CNR Institute of Neuroscience, Italy

Introduction : Neurones are highly dependent on microglia. In several brain diseases microglia display a disease‐associated microglia (DAM) transcriptional state that initially protects neuronees but then becomes detrimental due to downregulation of homeostatic genes. Microglia normally lose their protective function during senescence. Senescent microglia exhibit cell cycle arrest, dystrophic morphology, deficits in phagocytic and lysosomal activities, impaired metabolism, and senescence‐associated secretory phenotype (SASP) with enhanced EV secretion. In various tissues, accumulation of intracellular iron and ferritin are key features of cellular senescence. Accordingly, immunopathological studies have identified dystrophic, iron/ferritin‐positive microglia in the aged and diseased human brain. However, RNAseq of iron‐laden microglia suggests enhanced activation, proliferation and metabolic activity. Therefore, how microglia respond to iron accumulation is still unclear. This study aimed to study the temporal response to iron of murine microglia in vitro and to explore the impact of their secretome/extracellular vesicles (EVs) on neighbouring cells.

Methods : The impact of iron (500 µM) on microglia functions and senescence was assessed by immunofluorescence assays. RNA‐Seq was used to define the most deregulated transcriptional pathways. Finally, metabolic alterations were evaluated using biochemical assays, western blot, RT‐PCR techniques, and metabolomic analyses.

Results : We found that short exposure (3 days) to iron activates a fast proliferative and phagocytic response in microglia cultured with astrocytes. Conversely, microglia chronically exposed to iron (30 days) show a senescent‐like phenotype characterized by flattened morphology, proliferation arrest, decreased phagocytosis, increased ß‐galactosidase activity, cell death resistance and enhanced EV production. Furthermore, the cells show increased activity of the NAD‐consuming enzyme CD38, decreased NAD content and downregulation of NAD‐dependent sirtuins. Interestingly, iron‐fed senescent microglia decrease NAD content and induce senescent traits in healthy microglia in non‐contact coculture but not in microglia in which NAD levels were boosted by pharmacological treatments. The impact of iron‐fed microglia on healthy microglia in vivo is under investigation.

Summary/Conclusion : Collectively these data advance our understanding of the microglial response to iron and identify iron‐loaded microglia as a powerful in vitro tool to study mechanisms governing microglial transition from the proliferative to the hypofunctional/senescent state typical of late‐stage DAM and to screen for molecules able to counteract microglia‐mediated paracrine senescence.

The assessment of large extracellular vesicle protein cargo following TGF‐beta induced apoptosis of mammary epithelial cells

Sarai A. Gomez Perez 1 , Daxime Genier 1 , Laura Montermini 2 , Janusz Rak 2,3 , Alicia Viloria Petit 1

1 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Canada; 2 McGill University Health Centre Research Institute, Glen Site, Canada; 3 Department of Paediatrics, Faculty of Medicine and Health Sciences, McGill University, Canada

Introduction : Extracellular vesicles (EVs) participate in cell‐to‐cell communication and may modulate tissue remodelling. TGF‐beta induced apoptosis plays a role in mammary gland involution, and we have shown that TGF‐beta promotes the release of apoptosis‐associated EVs (ApoEVs). Given that apoptotic bodies (a type of ApoEV) are generally large, here we analysed the protein cargo of large (> 200 nm) EVs released by mammary epithelial cells following TGF‐beta induced apoptosis to gain insights on the potential capacity of these EVs to modulate the phenotype of surrounding cells within the mammary gland.

Methods : Three variants of the murine mammary epithelial cell line NMuMG were employed: two apoptosis‐proficient and one apoptosis‐deficient. Cells were cultured in DMEM supplemented with 10% FBS, 10 mg/mL insulin and 500 mg/mL G418. Cells below passage 10 were grown to 90% confluency, starved for 2.5 h in DMEM containing 5% EV‐free FBS, and treated with or without 5 ng/mL of TGF‐beta1 for 24 h. The collected media was centrifuged at 400 × g for 20 min, and EVs were isolated by ultracentrifugation at 10,000 × g for 1 h at 4°C. The pellets were resuspended in PBS and characterized by NTA, Western blotting and TEM to demonstrate EV presence prior to lysing. Nine mg of protein were subjected to uHPLC MS/MS. Peptides were identified using Mascot 2.3, and spectral counting was conducted with Scaffold (v4.4.8). An ANOVA with FDR correction was performed to identify differentially expressed proteins ( p < 0.05). The data were further analysed and visualized in RStudio, and the PANTHER database was used for additional gene ontology (GO) analysis.

Results : A total of approximately 250 proteins were identified across the three cell variants. Comparative analysis between control and TGFb1 treatment revealed cargo associated with adipogenesis and tissue remodelling. GO analysis identified enrichment in biological processes related to cell junction integrity and metabolic pathways, confirming the connection between junction disruption and apoptosis we previously reported.

Summary/Conclusion : Our findings suggest that the protein cargo of large EVs from TGF‐beta1‐treated NMuMG cells is involved in the modulation of adipocyte phenotype and tissue remodelling. Additional replicates and biological studies on target cells are necessary to confirm our findings.

Overcome senescence through the reduction in sEV biogenesis

Sergio Lucio‐Gallego 1 , Rocío Mato‐Basalo 1 , Lama Berjawi 1 , Nerea Lago‐Baameiro 2 , María Pardo 2 , Mónica Carrera 3 , Concha Gil 4 , Lola Gutiérrez 4 , Jesús Mateos 5 , María C. Arufe 1 , Juan A. Fafián‐Laboura 1

1 Therapy Cellular & Medicine Regenerative Group, University of A Coruña (UDC), Interdisciplinary Center for Chemistry and Biology (CICA), INIBIC—University Hospital Complex A Coruña (CHUAC), A Coruña, Spain; 2 Obesidomics Group, Endocrinology area, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain; 3 Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain; 4 Proteomics Facility‐Complutense University and Scientific Park Foundation of Madrid, Madrid, Spain; 5 Clinic Pharmacology group (FarmaCHUS). Health Research Center of Santiago de Compostela (IDIS). University Hospital Complex of Santiago de Compostela (CHUS). Santiago de Compostela, Spain

Introduction : Cells secretome is composed of small extracellular vesicles (sEV) among other secreted molecules such as interleukins, cytokines or chemokines that can modulate the cellular microenvironment. Senescent cells, characterized by being less proliferative, acquire a specific secretome known as SASP (senescence associated secretory phenotype). This SASP leads the cellular microenvironment to a more pro‐inflammatory one and induces senescence in the neighbouring cells in a paracrine and autocrine way. With time, senescent cells accumulate in the tissues, triggering age‐related diseases. In this study, we focused on paracrine senescence transmission mediated by sEV. Our objective is to avoid the transmission of senescence by modulating the secretion of the sEV.

Methods : We reduced the levels of RAB27A, a protein implicated in sEV biogenesis, in human mesenchymal stem cells using CRISPR/Cas9 technology. Isolated sEV by ultracentrifugation from senescent and non‐senescent cells were used to treat control cells and RAB27A KO cells. Treated cells were analysed by proliferation and β‐galactosidase assays to assure the senescent phenotype. We also performed a shotgun proteomic study in the recipient cells, identifying interesting proteins.

Results : The reduction in RAB27A protein accomplished a reduction in the senescence phenotype of recipient cells. The proteomic analysis shows upregulated ORAI1 and COG4 and downregulated LAPTM4A, proteins associated with the endomembrane system, in RAB27A KO cells treated with senescent sEV.

Summary/Conclusion : We developed a model of paracrine senescent transmission in which RAB27A inhibition prevents the spreading of senescence. We further see by the proteomic study that proteins related to the endomembrane system, such as ORAI1, LAPM4A and COG4, could have a role in the process of paracrine senescence transmission and may be possible targets for the development of drugs against SASP and senescence.

Funding : Xunta de Galicia, grant number ED481D‐2021‐020, Ministerio de Ciencia e Innovación (RYC2021‐032567‐I), funded by MCIN/AEI/10.13039/501100011033 and from the European Union «NextGenerationEU»/PRTR», InTalent program from UDC‐Inditex for the research grant and Project PI23/01347, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. PI20/00497, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. Xunta de Galicia (ED431F 2023/30). Transference Project financed by FINIBIC (RESISFERRO and ENDOPROT, respectively).

Subtopic EV cell interaction 1

Differential uptake and trafficking of extracellular vesicles affected by oncogenic RAS and RAF

Presenter: Chul Won Seo

Soonchunhyang University, Asan, Chungcheongnam‐do, Republic of Korea

Introduction : Mutations in RAS and RAF play critical roles in the pathogenesis of various cancers, including colorectal and lung cancer, influencing their progression, diagnosis, and treatment strategies. Importantly, these mutations alter the release of extracellular vesicles (EVs), affecting their uptake efficiency, systemic circulation, and molecular heterogeneity. In this study, we applied nano‐flow cytometry, quantitative proteomics, high‐throughput uptake screening, and in vivo tracking to analyse EVs released by cancer cells with KRAS and BRAF mutations.

Methods : EVs were isolated by size exclusion chromatography from wild‐type (Caco2, H292, H1703), BRAF‐mutant (HT29, WiDr), and KRAS‐mutant (HCT116, LoVo, A549, H358) colorectal and lung cancer cells. EV uptake and their subcellular trafficking (endoplasmic reticulum, nucleus, lysosome) were measured using automated microscopy. The biodistribution of EVs in mice was measured using IVIS imaging. EV proteomes were analysed with an Orbitrap mass spectrometer and processed with MaxQuant. For nano‐flow cytometry, EVs were labelled with fluorescently tagged antibodies (CD147, CD44, CD63, CD9, CD81) to analyse their subtypes.

Results : High‐throughput uptake screening with fluorescent‐labelled EVs in A549 cells showed that HT29 and LoVo EVs had unique trafficking efficiency to the endoplasmic reticulum compared to other EVs. However, in HEK293T cells, these EVs were primarily trafficked to lysosomes, suggesting that EV uptake and cellular utilization may depend on both EV and cell types. Additionally, IVIS imaging showed that colorectal cancer HT29 EVs preferentially trafficked to the caecum and intestines, while lung cancer H292 EVs primarily targeted the lungs. Proteomic analysis revealed unique surface proteomes influenced by RAS and RAF mutations. Specifically, ECM‐receptor interaction, focal adhesion, and junction proteins were regulated by these mutations. Notably, surface proteins such as CSPG4, LSR, CD44, CD133, NRP‐1, and various integrins were increased in EVs with these oncogenic mutations.

Summary/Conclusion : In summary, this research on EV heterogeneity and preferential uptake advances our understanding of intercellular communication by EVs in the context of oncogenic transformation. By identifying targeting proteins on EVs and their specific trafficking to subcellular locations, this study would unlock new avenues for developing novel drug delivery platforms that enable the efficient transfer of therapeutic cargo.

Funding : RS‐2024‐00348103 and RS‐2023‐00219563.

Artificial intelligence‐supported platform for quantitative analysis of the intercellular communication network in multicellular tumour spheroids

Maria Harmati 1 , Akos Diosdi 1,2,3 , Ferenc Kovács 1,3 , András Kriston 1,3 , Timea Tóth 1 , Ede Migh 1 , Gabriella Dobra 1 , Timea Böröczky 1,4 , Matyas Bukva 1,5 , Krisztina Buzas 1,5 , Peter Horvath 1,3,6,7

1 Institute of Biochemistry, HUN‐REN Biological Research Centre, Szeged, Hungary; 2 Doctoral School of Biology, University of Szeged, Szeged, Hungary; 3 Single‐Cell Technologies Ltd., Szeged, Hungary; 4 Doctoral School of Interdisciplinary Medicine, University of Szeged, Szeged, Hungary; 5 Department of Immunology, University of Szeged, Szeged, Hungary; 6 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; 7 Institute of AI for Health, Helmholtz Zentrum München, Germany

Introduction : To better understand extracellular vesicle (EV)‐mediated cell‐cell communication, multicellular model systems that mimic the in vivo context are essential for live tracking of EVs at single‐cell resolution. Artificial intelligence (AI)‐driven image analysis revolutionizes the study of EV‐mediated communication, enabling high‐dimensional, quantitative mapping of vesicle transfer dynamics and addressing the limitations of conventional methods.

Methods : We established three distinct multicellular 3D tumour models by co‐culturing a physiologically relevant set of EV donor and recipient cells, including tumour cell lines (T‐47D ductal carcinoma, A375 melanoma, and MG‐63 osteosarcoma) alongside stromal cells (MRC‐5 fibroblasts, EA.hy926 endothelial cells), in U‐bottom plates. The vesicle crosstalk was tracked through in‐cell EV‐labelling with cytoplasm dyes and high‐throughput fluorescent light sheet microscopy at different time points (24, 48, and 96 h) with or without chemotherapeutic stress (0.6 µM doxorubicin). Automated image analysis was conducted using an image processing and machine learning platform called BIAS (Biological Image Analysis Software). Neural network‐based nuclei and cell segmentation were performed with Stardist 3D and U‐Net models, respectively; for EV detection an ImageNet model was used.

Results : Our integrative approach combining in situ EV tracking in multi‐cellular tumour models and AI‐based image analysis enabled the quantitative monitoring of EV crosstalk, revealing EV transfer dynamics between each cell type in both directions. Although the three tumour types differed in their spheroid structure, EV crosstalk activity, primary communication axes and drug‐induced effects as well, stromal cells generally showed a prominent role in the communication.

Summary/Conclusion : In this interdisciplinary study, we developed a platform for live tracking and automated analysis of EV crosstalk. The integration of AI opened a new dimension of analysis, revealing insights previously unattainable with traditional methods. This platform is well‐suited to predict drug effects, test EV‐blocking therapies, and support the development of high‐throughput personalized medicine platforms.

Funding : TKP2021‐EGA09, MEC_R 149801, Janos Bolyai Research Scholarship.

Pharmacokinetic analysis on the efficacy of intravenously administered exosomes

Presenter: Hyun‐Suk Jung

Yonsei University College of Medicine, Seoul, Republic of Korea

Introduction : Exosomes are small vesicles released by all human cells, playing a crucial role in intercellular signalling and containing cellular information. Given their potential in disease diagnosis and therapy, there is increasing interest in utilizing exosomes for therapeutic purposes. However, concerns remain about their practical use, particularly because exosome concentrations in in vitro experiments are much higher than those found in vivo. This discrepancy has led to scepticism about whether the efficacy observed in vitro can be replicated in human bodies. In this study, we perform a pharmacokinetic analysis to examine the distribution of intravenously administered exosomes and assess their potential for therapeutic applications in realistic in vivo conditions.

Methods : We calculated the distribution and dilution of intravenously administered exosomes across various organs. Based on these calculations, we estimated the intravenous dosage required for exosomes to exhibit efficacy in different organs, such as the liver, spleen, lungs, and kidneys.

Results : Modelling calculations indicated that exosomes administered intravenously were predominantly distributed to the liver, followed by the spleen, lungs, and kidneys, which is consistent with observed trends in in vivo experiments with mice. To deliver the same exosome concentration to liver cancer cells as in vitro, approximately 10 15 to 10 17 exosomes would need to be administered. This is significantly higher than the 10 12 exosomes typically present in the bloodstream.

Summary/Conclusion : Our study presents a pharmacokinetic model that helps understand the distribution of exosomes following intravenous administration. The results show that significantly higher exosome doses (10 15 to 10 17 ) are required in vivo to achieve the same efficacy observed in vitro. This highlights the need for exosomes with improved tropism and modified cargo to enhance therapeutic potential.

Funding : This work was supported by the National Research Foundation of Korea (NRF) and the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea government (Ministry of Science and ICT) (RS‐2024‐00432946), the NRF grant funded by the Korea government (MSIT) (No. 2021R1A2C3011254), and the Technology Development Program funded by the Ministry of SMEs and Startup s(MSS, Korea) (RS‐2024‐00506850).

Potential test of peripheral blood and umbilical cord blood‐derived dendritic cells with endogenic and synthetic specific antigen induction on T cell activation in nasopharyngeal carcinoma patients

Cynthia Retna Sartika 1,2 , Melisa Intan Barliana 2,3 , Rima Haifa 1

1 PT. Prodia StemCell Indonesia, Jakarta, Indonesia; 2 Faculty of Pharmacy, Department of Biological Pharmacy, Universitas Padjadjaran, West Java, Indonesia; 3 Center of Excellence for Pharmaceutical Care Innovation, Universitas Padjadjaran, West Java, Indonesia

Introduction : Nasopharyngeal carcinoma (NPC) is a type of cancer caused by the interaction of multiple factors, including chronic infection with Epstein‐Barr virus (EBV), environmental influences, and genetic predispositions, leading to abnormal growth of the nasopharyngeal epithelium. One alternative treatment for NPC involves dendritic cell (DC) vaccines, used as immunotherapy agents. DC can capture and present cancer‐related antigens to T lymphocytes, thereby initiating a targeted immune response against cancer cells, including NPC cells. This study aimed to evaluate the in vitro potential of DCs derived from Peripheral Blood (DC‐PB) of NPC patients compared to those derived from Umbilical Cord Blood (DC‐UCB) by inducing different types of antigens (endogenous and synthetic) to stimulate T lymphocyte activation in NPC patients.

Methods : This experimental study included three treatment groups: one group received DCs induced with endogenous antigens, another with synthetic antigens, and a control group. Each treatment was applied to DCs from peripheral blood (PB) and umbilical cord blood (UCB), with five repeats per group, totalling 30 samples. We assessed DCs' ability to activate T cells by measuring IL‐12 levels, T cell proliferation, and concentrations of TNF‐α, IFN‐γ, and Granzyme B. Correlations between IL‐12 and these activation markers were also analysed in NPC patient T cells.

Results : The results showed that DC‐UCB secreted significantly higher levels of IL‐12 than DC‐PB. In co‐cultures with NPC patient T cells, DC‐UCB induced significantly higher T cell proliferation and increased concentrations of TNF‐α, IFN‐γ, and Granzyme B compared to DC‐PB. A positive correlation was observed between IL‐12 levels and TNF‐α, IFN‐γ, and T cell proliferation, though no correlation was found between IL‐12 and Granzyme B concentrations.

Summary/Conclusion : This study shows that dendritic cells from umbilical cord blood (DC‐UCB) are more effective than those from peripheral blood (DC‐PB) in activating T cells in nasopharyngeal carcinoma (NPC) patients. DC‐UCB produced more IL‐12 and led to higher T cell activity, including increased levels of TNF‐α and IFN‐γ. These results suggest that DC‐UCB may be a promising option for improving immune responses in NPC patients, and further research could help develop this approach for treatment.

Development of a hybrid extracellular vesicles/liposome system for enhanced skin anti‐inflammation

Presenter: Hye Sun Park

Korea Basic Science Institute, Daejeon, Republic of Korea

Introduction : This research introduces a hybrid system combining extracellular vesicles (EVs) from Hydrangea macrophylla leaves (HML‐EVs) with liposomes loaded with terpinen‐4‐ol and azelamide monoethanolamine.

Methods : By leveraging vesicular membrane fusion, confirmed through two‐colour fluorescence resonance energy transfer (FRET), we successfully created a stable hybrid dispersion of HML‐EVs and liposomes, with a size range of 100–200 nm.

Results : The hybrid system demonstrated significant inhibition of apoptosis, nitric oxide, and tumour necrosis factor‐α production. Moreover, it suppressed the expression of key inflammatory mediators such as protease‐activated receptor‐2, interleukin‐6, c‐Jun, and thymic stromal lymphopoietin.

Summary/Conclusion : These results suggest that the hybrid EV/liposome system not only reduces skin irritation but also enhances anti‐inflammatory effects on the skin, presenting a promising approach for therapeutic applications.

Funding : This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI); funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HP23C0011); the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (KSH, 2020R1A2C2012011); and a grant (KSH, A423100) from the Korea Basic Science Institute.

Subtopic Stem cell‐derived EVs 1

Hoffa‐MSC‐derived EVs produced in 3D vertical wheel bioreactor culture more potently regenerate primary osteoarthritic chondrocytes than EVs from 2D culture

Alexander Otahal 1 , Karina Kramer 1 , Slavomira Gulová 1,2 , Markus Neubauer 1,3 , Zsombor Lacza 4,5 , Stefan Nehrer 1,3 , Andrea De Luna 1

1 Center for Regenerative Medicine, University for Continuing Education Krems, Krems, Austria; 2 Associated Tissue Bank, Faculty of Medicine, Pavol Jozef Safarik University and Luis Pasteur University Hospital, Kosice, Slovakia; 3 Universitätsklinikum Krems, Krems, Austria; 4 University of Physical Education, Department of Sport Physiology, Budapest, Hungary; 5 Semmelweis University, Inst. Clinical Experimental Research, Budapest, Hungary

Introduction : Adipose mesenchymal stromal cells (MSCs) are increasingly studied for regenerative therapies due to their ability to modulate inflammation and support tissue homeostasis. Recent findings suggest MSC‐derived extracellular vesicles (EVs) play a central role in therapeutic effects. The infrapatellar Hoffa fat pad is a promising MSC source for EVs. Conventional EV production in 2D cultures can limit EV yield and functional relevance due to its artificial conditions. Here, we developed a 3D dynamic culture approach using vertical wheel bioreactors, supplemented with different human blood products such as citrate‐anticoagulated platelet‐rich plasma or hyperacute serum to generate EVs in a cost‐efficient manner.

Methods : Hoffa MSCs were isolated from surgical waste from joint replacement surgeries, characterized by flow cytometry, and assessed for adipogenic, osteogenic, and chondrogenic differentiation. Cells were cultured on animal‐free microcarriers in 3D bioreactors and standard 2D flasks, both with blood products to simulate clinical treatment settings. EVs were isolated by ultrafiltration and characterized using nanoparticle tracking analysis in scatter and fluorescence mode and western blotting. EV miRNA cargo was analysed through RNA sequencing. Biological potency was assessed by evaluating gene expression changes in osteoarthritic chondrocytes via RT‐qPCR.

Results : Hoffa MSCs displayed expected stemness markers before and after EV production as well as tri‐lineage differentiation potential. Approximately 10,000 particles per cell were obtained in 3D culture versus 1000 in 2D. EV marker proteins were confirmed via western blot, and EV preparations were not contaminated with lipoproteins. miRNA profiles were consistent across treatments, with miR‐100‐5p most abundantly present in EVs. EVs from 3D culture significantly inhibited inflammatory and hypertrophic genes, while preserving extracellular matrix gene expression, exemplified by iNOS, type 10 and type 2 collagen, in contrast to EVs from 2D culture.

Summary/Conclusion : The 3D culture of Hoffa MSCs in bioreactors, combined with ultrafiltration, supports efficient EV production for potency assays and preclinical research. Dynamic culture conditions appear to enhance EV release and could prevent EV reuptake, while yielding high amounts of EVs. This method facilitates scalable EV production having substantially higher anti‐inflammatory and tissue homeostatic potency than conventional EV production approaches.

Funding : The study was funded by Gesellschaft für Forschungsförderung NÖ (GFF), grant number LSC18‐014.

Single‐cell analysis of L‐Myc‐expressing neural stem cells and their extracellular vesicles revealed distinct progenitor populations with neurogenic potential

Presenter: Patrick Pirrotte

TGEN, Phoenix, Arizona, USA

Introduction : Neural stem cell (NSC)‐based therapies offer significant potential for treating brain injuries by delivering neurotrophic factors and promoting endogenous brain repair. Despite promising evidence of the protective and restorative effects of NSCs and their extracellular vesicles (EVs), advancing these therapies to preclinical and clinical stages has been limited by inadequate molecular characterization of NSCs and their EV cargo. This study addresses this challenge by characterizing two human foetal NSC lines expressing the L‐MYC gene (LMNSC01 and LMNSC02) and their EVs through single‐cell RNA sequencing (scRNA‐seq) and proteomic analyses.

Methods : LMNSC01 and LMNSC02 cells were cultured and analysed for differentiation potential into neuronees and glial cells using scRNA‐seq. Key NSC markers (SOX2, nestin) and differentiation markers (doublecortin [DCX], vimentin) were evaluated. Single extracellular vesicle nanoscopy was utilized to measure the size, shape, and tetraspanin content (CD9, CD63, CD81) of EVs from each line. Additionally, protein and RNA concentrations, particle numbers, and batch‐to‐batch consistency were assessed. Global proteomics was conducted to identify biological pathways in EV cargo, and neuroprotective effects were tested in a methotrexate‐induced brain organoid toxicity model.

Results : scRNA‐seq revealed that both cell lines were homogeneous, expressing NSC and differentiation markers like DCX and vimentin. LMNSC01 was enriched in markers for astrocytes, excitatory neuronees, and glial cells, while LMNSC02 showed enrichment for inhibitory neuronees and oligodendrocyte progenitor cells (OPCs). LMNSC02 EVs had a higher tetraspanin content and larger size compared to LMNSC01. Proteomic analysis showed that LMNSC02 EVs were enriched in proteins involved in neuronee development and synaptic transmission, whereas LMNSC01 EVs were more linked to RNA processing. Both types of EVs demonstrated neuroprotective properties in the brain organoid model, suggesting potential in reducing chemotherapy‐induced brain damage.

Summary/Conclusion : This study highlights the unique therapeutic potential of LMNSC01 and LMNSC02 and emphasizes the importance of detailed molecular characterization for guiding NSC‐based therapies for neurological conditions.

Funding : This work was supported by National Institutes of Health grants P30CA033572, 1R01NS121037‐01 Kline/Gutova, and UG3/UH3 TR002878.

Adipose tissue‐derived mesenchymal stromal cells and their extracellular vesicles are functionally deficient in patients with systemic sclerosis

Presenter: Dalia Ibarissen

INSERM, Montpellier, France

Introduction : Systemic sclerosis (SSc) is a rare autoimmune disease characterized by 3 pathological processes: vasculopathy, fibrosis and dysregulation of the immune response. SSc is potentially a lethal disease with no curative treatment to date. On the other hand, we have previously demonstrated that adipose tissue‐derived mesenchymal stromal cells (ASCs) as well as their derived extracellular vesicles (EVs) can slow the progression of the pathology in the HOCl‐induced SSc murine model. However, regarding clinical application, the use of autologous ASCs remains a topic of ongoing debate. Here, we compared the functional properties of ASCs from healthy donors (h‐ASCs) and scleroderma patients (SSc‐ASCs), focusing on their anti‐fibrotic and immunosuppressive effects. We also characterized the EVs released by these cells to better understand potential differences in their therapeutic potential.

Methods : ASCs and their derived EVs were isolated from healthy donors and from diffuse SSc patients and then amplified. Their anti‐fibrotic effect was assessed in a TGF‐β1 induced model where the ASCs were cocultured with fibroblasts stimulated with 5 ng/mL of TGF‐β1 for 24 h. The myofibroblastic phenotype was assessed via RT‐qPCR. Their immunomodulatory effect was determined in a mixed lymphocyte reaction for 96 h. The size distribution of EV was evaluated by nanoparticle tracking analysis (NTA), expression of EV markers by flow nanocytometry and western blot, and the total amount of proteins and RNA using the Qubit technology. Proteomics analysis has been done on hEVs and SScEVs.

Results : Both ASCs decrease the expression of pro‐fibrotic markers (αSMA, COL1A1, COL3A1) as well as increase anti‐fibrotic markers (MMP1, MMP3), but only SSc‐ASCs seem to have a pro‐apoptotic effect on stimulated fibroblasts. MLR results showed that h‐ASCs inhibit PBMC proliferation in a dose‐dependent way, thus underlying their immunomodulatory effects. However, with SSc‐ASCs, the degree of inhibition was markedly reduced. Similar results were obtained with EVs from the respective ASCs. Proteomic analysis showed that 28 proteins are differentially expressed between h‐EVs and SSc‐EVs, with 14 were upregulated in h‐ASCs.

Summary/Conclusion : SSc‐ASCs and their EVs partially lose the properties of functional h‐ASCs and EVs. This underlines the interest of using allogenic ASCs and EVs for therapeutic applications in SSc.

Setting a protocol to incorporate mesenchymal stromal cell extracellular vesicles in platelet‐derived fibrin gel for cartilage repair

Enrico Ragni 1 , Paola De Luca 1 , Simona Landoni 1 , Michela Taiana 1 , Giuseppe Talò 2 , Matteo Moretti 2,3,4,5 , Laura de Girolamo 1

1 IRCCS Ospedale Galeazzi—Sant'Ambrogio, Labouratorio di Biotecnologie Applicate all'Ortopedia, Italy; 2 IRCCS Ospedale Galeazzi—Sant'Ambrogio, Cell and Tissue Engineering Laboratory, Italy; 3 Ente Ospedaliero Cantonale, Labouratories for Translational Research, Switzerland; 4 Ente Ospedaliero Cantonale, Service of Orthopedics and Traumatology, Switzerland; 5 Università della Svizzera Italiana, Faculty of Biomedical Sciences, Switzerland

Introduction : Therapies for cartilage regeneration may lead to incomplete or fibrous tissue formation. Fibrocartilage is poorly performant, and over time joint degeneration progresses, eventually requiring prosthesis. Mesenchymal stromal cells (MSCs) release extracellular vesicles (EVs) able to stimulate naïve cartilage that are studied in clinical trials. The pitfall of EV‐based interventions into the joint cavity is dispersion. This leads to low concentration at the lesion site, resulting in reductions of efficacy and inflation of production costs to increase EVs dose. Biological scaffolds embedding MSC‐EVs are a promising option to concentrate and release MSC‐EVs at the lesion site. This work aimed at producing an enhanced platelet‐derived fibrin gel, already used in clinical practice for cartilage repair, loaded with MSC‐EVs.

Methods : Ethics Committee approval and informed consent were obtained. MSCs were isolated from waste adipose tissue, and at passage 1 after serum starvation, EVs were collected by differential centrifugation (100,000 × g , 3 h, 4°C). For microscopy, conditioned medium was 10 µM CFSE supplemented (1 h, 37°C). Particles were characterized by NTA, TEM, WB (positive markers: CD9/81, TSG101, Flotillin; negative markers: Calnexin), FC (lineage markers: CD73/90; EVs markers: CD9/63/81), ELISA, and miRNA‐arrays. MSC‐EVs were added to platelet rich plasma, and fibrin gel was obtained with CaCl 2 . MSC‐EVs’ embedment efficiency, release and incorporation in osteoarthritis (OA)‐patients’ chondrocytes and cartilage explants were monitored by SEM, FC, microfluidic‐based confocal approaches and real‐time quantitative multimodal nonlinear optics imaging. The therapeutic effect of highly‐concentrated gel‐released MSC‐EVs was tested in OA chondrocytes by qRT‐PCR.

Results : A high‐efficiency incorporation protocol (> 70% input EVs) was defined, relying on a one‐step modification of standard procedures used in clinical practice for platelet‐gel preparation for cartilage lesions. MSC‐EVs were released for up to 4 weeks and taken up by OA‐chondrocytes and cartilage explants. The release from MSC‐EVs‐loaded platelet gel had synergistic anti‐inflammatory/matrix remodelling effects on chondrocytes with respect to pure MSC‐EVs or released factors from unloaded gels.

Summary/Conclusion : A platelet gel loaded at high concentration with MSC‐EVs is an enhanced and easy‐to‐translate tool to foster chondrocyte homeostasis, a key requisite for cartilage repair.

Funding : This project (PNRR‐MCNT2‐2023‐12377836) has been funded by the European Union—Next Generation EU—NRRP M6C2 ‐ Investment 2.1 Enhancement and strengthening of biomedical research in the NHS.

Analytical comparison of MSC‐EV quality attributes across tissue sources and production platforms

Stephen Lenzini, Madeline Cramer, Elie Zakhem, Jon A. Rowley

RoosterBio, Inc., USA

Introduction : Scalable MSC‐EV manufacturing processes are required to meet lot sizes for clinical applications. Understanding differences among MSC tissue types and donors and establishing consistent production processes are critical to overcoming scalability challenges. We used analytical tools to understand MSC‐EV quality attributes from different tissue sources when using a consistent media system across multiple production platforms.

Methods : Human MSCs from 3 bone marrow (BM) donors, 2 umbilical cord (UC) donors and 1 adipose tissue (AD) donor (RoosterBio sourced) were expanded in RoosterNourish‐XF in different scalable production platforms (stirred‐tank, vertical‐wheel, spinner flask and 2D T‐flask). EVs were collected in the defined media RoosterCollect‐EV for 2 to 5 days. Particle number and size were evaluated by NTA. Total RNA content was measured via Bioanalyzer. EV marker expression was evaluated by capillary western blot, and CD73 activity was measured as a potency indicator. Fold changes in various miRNA targets were measured by qPCR.

Results : Cells in all 3D bioreactor platforms produced significantly more particles than in 2D (4–5‐fold greater). Across 3D conditions, particle count increased from Day 2 to 5, with BM and UC donors demonstrating comparable maximum particle production (∼1E10/mL) while AD had less particle production (∼5E9/mL). Differences in particle size were observed with median sizes for UC 173 nm, BM 158 nm, and AD 143 nm. For all groups, protein content increased, from Day 2 to 5 as particle count increased showing a strong correlation. Total RNA content decreased from Day 2 to 5 across all 3D platforms, while RNA content remained stable in 2D. EV identity markers (CD63, CD81, CD9) and MSC‐EV identity marker CD73 were confirmed. CD73 expression and activity were demonstrated in all conditions, but the overall magnitude and trend over collection days varied between donors.

Summary/Conclusion : Overall, this comprehensive study evaluated MSC‐EV quality attributes among multiple scalable production platforms using different donors from different tissue sources in a standardized cell source and media system. While EV quality attributes were consistent for each donor across production platforms, some quality attributes (particle count and CD73 activity) were donor dependent. This work helps establish a foundation for selecting clinical manufacturing processes for EV production.

The role of miRNA‐enriched extracellular vesicles from induced MSCs in modulating inflammation and redox balance in an in vitro osteoarthritis model

Presenter: Matilde Balbi

Department of Experimental Medicine, University of Genoa, Genoa, Italy

Introduction : Osteoarthritis (OA) is a chronic disease characterized by joint inflammation. Since there is no effective cure, new approaches are under investigation and mesenchymal stromal cells (MSC) are considered a promising therapy. As MSC have different limitations, we use MSC differentiated from induced pluripotent stem cells (iMSCs), which could reduce heterogeneity and allow longer cell expansion. Due to the risks of a cell‐based therapy, iMSC‐derived extracellular vesicles (iMSC‐EVs) could represent a promising cell‐free approach for OA. This study aimed to investigate and characterize iMSC‐EVs, evaluating their anti‐inflammatory and redox potential on an in vitro OA model.

Methods : Three different iMSC lines were differentiated from the iPSC line and maintained in culture for a long passage time in serum free medium; the surface phenotype and the onset of cellular senescence were evaluated. EVs isolated from iMSC by ultracentrifugation, were characterized by protein quantification, BCA, NTA and unconventional flow cytometry analysis. Human primary chondrocyte culture (HAC) primed with IL‐1α to mimic chronic inflammation was exposed to iMSC‐EVs. The anti‐inflammatory effect was evaluated by RT‐PCR evaluating the expression of COX‐2, IL‐6 and IL‐8. Enzymatic activity was evaluated to study redox potential by luminometric assay. iMSC‐EVs miRNAs were extracted, and small RNA quality and size were assessed by Agilent and expression of EV‐miRNAs was investigated by microarray using the SurePrint Human miRNA platform.

Results : The results obtained showed for the first time the anti‐inflammatory effect of iMSC‐EVs treatment on HAC primed with IL‐1α, significantly reducing COX‐2, IL‐6 and IL‐8 expression. Moreover, iMSC‐EVs treatment significantly decreases oxidative damage and ROS accumulation and restores the antioxidant defences induced by IL‐1α. Primary chondrocytes were transfected with the identified predictive miRNAs miR‐17‐5p, miR‐20a‐5p, miR‐21‐5p, miR‐29a‐3p, miR‐29b‐3p and miR‐29c‐3p, and the obtained data partially confirmed the anti‐inflammatory and antioxidant effects on the OA model in vitro.

Summary/Conclusion : In conclusion, iMSC‐derived EVs and miRNAs cargo could represent a promising alternative therapeutic strategy for osteoarthritis, ascribed to the ability to reduce inflammation, oxidative damage and ROS accumulation and to restore the antioxidant defences.

Funding : This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671.

Investigation of LINE‐1, ORF1 and FTO in mesenchymal stem cell derived exosomes supporting oocyte maturation in vitro

DamLa Akogulları Celik 1 , H. Seda Vatansever 1,2

1 Manisa Celal Bayar University, Manisa, Turkey; 2 Near East University, Nicosia, Turkey

Introduction : Oocyte maturation is a complex process that occurs with paracrine and autocrine effects secreted from granulosa cells in its microenvironment. The most important agents that can replace granulosa cells in vitro are mesenchymal stem cells. In co‐culture studies with oocytes from different species, mesenchymal stem cells were used because they are autologous and do not cause ethical concerns, and their secretions have been shown to support oocyte maturation. However, in recent studies, mesenchymal stem cell‐derived exosomes have been focused on in terms of ease of obtaining, low immunogenicity, reduced tumourigenicity potential and low ethical factors. The findings of our current project caused differences in m6A RNA methylation in groups that added exosomes to the oocyte culture medium, suggesting that the exosome contents secreted by mesenchymal stem cell‐derived exosomes when co‐cultured with immature oocytes were different from those in normal culture medium. In this project, it was aimed to determine the presence of LINE‐1, ORF1 and FTO associated with oocyte maturation in mesenchymal stem cell‐derived exosomes.

Methods : This study was approved by the Manisa Celal Bayar University, Experimental Animal Ethics Committee (77.637.435). Culture medium of human umbilical cord and adipogenic mesenchymal stem cells which their confluency is approximately 70%–80%. The exosomes were isolated using the miRCURY Exosome Isolation Kit (Exiqon 300102) according to the manufacturer's protocol. Exosomal RNA isolation was provided with a total exosomal RNA isolation kit, and LINE‐1, ORF1 and FTO gene analyses were performed by RT‐PCR. In addition, LINE‐1, ORF1 and FTO protein analyses were detected by western blotting.

Results : In human umblical cord mesenchymal stem cells exosome, the expression of three proteins were higher than adipogenic mesenchymal stem cells.

Summary/Conclusion : In conclusion, exosome content may differ from different cells; therefore, using exosomes in research and clinical studies will be evaluated.

Funding : This study was funded by Manisa Celal Bayar University Research Unit (project number: 2021‐082) and TUSEB, Turkey (project number: 32954).

Induced mesenchymal stromal cells for therapeutic extracellular vesicle production

Alisa Jokela, Kerttu Airavaara, Julia Monola, Chris S. Pridgeon, Riina Harjumäki

University of Helsinki, Helsinki, Finland

Introduction : Mesenchymal stromal cell (MSC)‐derived extracellular vesicles (MSC‐EVs) hold great potential for regenerative medicine. However, the quality of tissue‐derived MSC‐EVs varies with donor characteristics, and MSC cultures often face challenges, for example, in early senescence and reduced therapeutic potency. To obtain an inexhaustible source for MSC‐EVs with consistent quality, induced pluripotent stem cells (iPSCs) could be used to produce induced MSCs (iMSCs). However, a standardized protocol for a large‐scale iMSC production has not yet been established. This study aims to optimize the iMSC differentiation protocol for therapeutic EV production and explore if tissue‐derived MSC‐EVs could enhance the differentiation. We hypothesize that this optimized protocol could be used in future tissue regeneration applications.

Methods : Three protocols, modified from previous studies, were explored using pathway inhibitor‐based approaches. The methods include direct differentiation and differentiation via trophoblast (TB) or neural crest cell (NCC) checkpoints. After optimization, tissue‐derived MSC‐EVs were tested at different time points. All EVs in this study were isolated by differential centrifugation and size‐exclusion chromatography. The characteristics of iMSCs and EVs were analysed using qPCR, proteomics, and nanoparticle tracking analysis.

Results : Differentiation via NCCs produced iMSCs with a typical MSC gene expression profile (CD73+, CD105+, CD90+), robust proliferation, and high cell yield. Tissue‐derived MSC‐EVs did not significantly enhance differentiation processes but showed a trend of increased gene expression profiles under some conditions. Further analysis of the resultant iMSC‐EVs is ongoing.

Summary/Conclusion : Based on the preliminary data, induction of iMSCs through NCCs appears to offer the most promising protocol for generating iMSCs with sufficient cell yield for EV production. This protocol will be further optimized to produce iMSCs with the most potential EVs for future tissue regeneration applications.

Funding : Research Council of Finland: GeneCellNano flagship project [#337430] and Finnish Cultural Foundation [#00220283].

Bone marrow and placenta mesenchymal stem cell‐derived extracellular vesicles increase ex vivo expansion of haematopoietic stem cells

Presenter: Kunye Kwak

Korea University College of Medicine, Seoul, Republic of Korea

Introduction : Mesenchymal stem cells (MSCs) are known to help proliferate haematopoietic stem cells (HSCs) in the bone marrow (BM). Our study aims to compare the potential of EVs derived from bone marrow and preterm and full‐term placental MSCs to enhance ex vivo proliferation of HSCs.

Methods : MSCs from preterm placenta, full‐term placenta and bone marrow were cultured in xeno‐free media for up to 5 passages. The cell culture medium was concentrated with a molecular weight cut‐off of 100 kDa. EVs were isolated using size‐exclusion chromatography. EV concentration and size were measured by nanoparticle tracking analysis (NTA). Western blotting identified EV markers. Morphology was confirmed by transmission electron microscopy (TEM). Human BM HSCs were cultured at 1 × 10 5 cells/mL for 10 days with or without the isolated MSC‐EVs at a concentration of 2.5 × 10 4 particles/cell. The culture conditions were classified into four groups: the control group, the preterm placental MSC‐EV group, the full‐term placental MSC‐EV group, and the BM MSC‐EV group. On Day 10 of expansion, total nucleated cell (TNC) number and viability were assessed. Flow cytometry was performed to evaluate surface markers. Colony forming unit (CFU) assay was performed to assess differentiation.

Results : Isolated EVs showed an average size of 153 nm on NTA. On western blot, EVs were positive for CD63 and TSG101. TEM revealed a round‐shaped morphology with diameters approximately 150 nm. Overall characterization of EVs did not show significant difference between MSC types. Co‐culture of human BM HSCs with MSC‐EVs showed a significant increase in TNCs in every group compared with the control group. Flow cytometry showed that the proportion of CD34+ cells was maintained by each group, demonstrating preserved stemness. The CFU assay showed enhanced differentiation ability in each EV‐treated group, which was significantly highest in the full‐term placental MSC‐EV group.

Summary/Conclusion : Co‐culture of bone marrow and placenta MSC‐derived EVs enhanced ex vivo proliferation of HSCs. It is notable that not only BM MSC‐EVs, but also full‐term placental MSC‐EVs enhance proliferation of BM HSCs with greater differentiation potential. Since obtaining BM MSCs involves an invasive procedure, the use of placental MSCs could be advantageous due to the easier procurement process.

Identification of a specific marker panel for human mesenchymal stem cell‐derived small extracellular vesicles

Presenter: Lei Luo

Shanghai Jiao Tong University, Shanghai, People's Republic of China

Introduction : Human mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEVs) have demonstrated significant immunomodulatory and pro‐regenerative potential in tissue repair and disease treatment. However, the lack of specific markers to define and qualify human MSC‐sEVs presents a major challenge for their clinical application.

Methods : We analysed proteomic datasets of MSC‐sEVs from three different cell sources to identify potential markers. Several surface antigens commonly found on MSCs were selected as candidate markers due to their high abundance in MSC‐sEVs. We isolated MSC‐sEVs from the three cell sources using ultracentrifugation, density gradient ultracentrifugation and size exclusion chromatography, and then stained them with fluorescein‐conjugated antibodies. Following the removal of unbound antibodies, the MSC‐sEVs were analysed by NanoFCM at single‐vesicle resolution. High‐resolution microscopy, as an orthogonal method, visually confirmed the presence of candidate markers in MSC‐sEVs. We also analysed six non‐MSC‐sEVs to assess the specificity of these markers.

Results : The results showed that the positive rates of CD13, CD29, and CD90 exceeded 60% across sEVs derived from all three MSC sources, whereas CD44, CD73, CD105, CD146, and CD166 generally exhibited lower positive rates. High‐resolution microscopy, as an orthogonal method, visually confirmed the strong presence of CD13, CD29, and CD90 in MSC‐sEVs. Moreover, none of the six non‐MSC‐sEVs showed concurrent positive rates for CD13, CD29, and CD90 exceeding 40%. This finding suggests that the marker panel comprising CD13, CD29, and CD90 can specifically distinguish MSC‐sEVs from non‐MSC‐sEVs.

Summary/Conclusion : In conclusion, we have identified a specific and quantifiable marker panel (CD13, CD29, and CD90) for MSC‐sEV characterization. This panel will facilitate the development of quantifiable, reproducible, and standardized assays for defining MSC‐sEV preparations, ultimately accelerating the clinical translation of MSC‐sEVs.

Funding : This work was supported by the National Natural Science Foundation of China (grant numbers 82072550 and 31971274), the Science and Technology Commission of Shanghai Municipality (grant number 21DZ2201300), the State Key Laboratory of Drug Regulatory Science (grant number 2024SKLDRS0208) and the Interdisciplinary Program of Shanghai Jiao Tong University (grant number: ZH2018ZDA20).

Bone marrow mesenchymal stromal cell‐derived extracellular vesicles for bone regeneration

Evangelia Bochti 1 , Monica Tsimbouri 1 , Biswajoy Bagchi 2 , Xin Li 2 , Matthew Dalby 1 , Catherine Berry 1

Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, USA 1 ; Medical Research Scotland, Sphere Bio, Cambridge, UK 2

Introduction : Bone tissue is the second most transplanted tissue worldwide, with over 4 million annual transplants. Bone marrow‐derived stromal cells (BMSCs) are used in bone tissue engineering due to their osteoblastic lineage. The beneficial effects of BMSCs are potentially mediated through extracellular vesicles (EVs). In collabouration with Sphere Fluidics Limited, this project aims to manufacture a novel BMSC‐EV microenvironment, encapsulating osteogenic‐primed EVs within hydrogel beads. The osteogenesis‐inducing beads could be used for localised, cell‐free bone regeneration and repair therapies.

Methods : BMSCs were osteogenically primed for 28 days using two distinct approaches: chemical induction and mechanical induction, via nanoscale vibrational displacements. Osteogenic differentiation of BMSCs was verified via protein analysis and histological staining. BMSC‐derived EVs were successfully isolated and characterised by size and concentration. The bioactive cargo of osteogenic EVs’ is undergoing analysis at cytokine, metabolomic, proteomic and RNA levels. Osteogenic EVs were encapsulated in GelMA and alginate hydrogels and will soon be encapsulated with naive BMSCs in 3D hydrogel beads using Sphere Fluidics’ microfluidic technology. The viability of naive BMSCs within the hydrogel beads along with the osteogenic potency of the EVs will be assessed using Alamar Blue (viability assay) and at a gene level (qRT‐PCR) for early osteogenic markers (RUNX2 and Osterix).

Results : Both chemical and mechanical inductions induced osteogenesis in BMSCs. EV isolation was verified, and EVs were consistently sized (60–250 nm). Differences in EV cargo are noted in the cytokine profile, with ongoing studies at a metabolomic, proteomic and RNA level between the osteogenic induction methods. Osteogenic EVs can be encapsulated within GelMA and alginate hydrogels, and their osteogenic potency in naïve BMSCs is to be investigated.

Summary/Conclusion : BMSC analysis indicated osteogenesis. Ongoing characterisation of BMSC‐EVs aims to determine any differences in osteogenic‐derived EVs cargo, both compared to the negative control and between the induction approaches. Encapsulation of naive BMSCs with osteogenic EVs in hydrogel beads will indicate the ability of the BMSCs to uptake EVs in a 3D environment, emphasising the osteogenic potency of the EVs’ bioactive cargo on naive BMSCs.

Funding : This PhD research project is funded by Medical Research Scotland (MRS) and Sphere Fluidics Limited, studentship ref. number: PHD‐50388‐2021.

Exosome produced by automation‐assisted derivation of mesenchymal stem cells from human induced pluripotent stem cells possess immunomodulatory potentials

Presenter: Chia‐Ning C. Shen

Genomics Research Center, Academia Sinica, Taipei, Taiwan (Republic of China)

Introduction : Exosomes derived from mesenchymal stem cells (MSCs) possess a wide range of biological functions like these cells by contributing to tissue repair. For example, exosomes released from MSCs can be used to treat by reducing inflammation and modulating extracellular matrix. Therefore, several preclinical and clinical studies have recently demonstrated the potential of MSC‐derived exosomes to be applied to treating osteoarthritis. However, limited production of exosomes has hampered their development for clinical translation. Here, a programmable all‐in‐one Panasonic automated culture machine was utilized to derive MSCs and exosomes from human induced pluripotent stem cells.

Methods : Mesenchymal stem cells (iMSCs) were generated by a stepwise differentiation protocol utilizing a programmable all‐in‐one Panasonic automated culture system. Exosomes were isolated from concentrated conditioned medium and followed by size exclusion chromatography. iMSC and exosomes were characterized by immunofluorescence staining and single‐cell RNA sequencing analysis using the BD Rhapsody system. The purity of exosomes was assessed by nanoparticle tracking analysis (NTA).

Results : Initial characterization revealed MSCs derived from automatic process possess multipotency. To further uncover the cellular homogeneity in MSCs derived from automated processes, FACS and single‐cell analysis using the BD Rhapsody system were performed. Automatic process derived MSCs were positive for CD29, CD44, CD73, CD90, CD105, and HLA‐ABC and negative for CD45 and HLA‐DR. To investigate whether RNA abundance is correlated between parental iMSCs and their exosomes, mRNA and small RNA sequencing were further conducted. The results suggest that exosomal RNAs indeed recapitulate the RNA abundance of their parental iMSCs. To validate whether exosomes derived from iMSCs can mediate their immune modulation activity, exosomes were further isolated from concentrated conditioned medium of iMSCs followed by size exclusion chromatography. These purified exosomes assessed by NTA and characterized by TEM were positive for CD9, CD63 and CD81. We validated exosomes isolated from iMSCs exerted similar effects on NK cells. We found exosomes derived from iMSCs can prime NK cells to produce increased levels of IFN‐γ in response to IL‐12 and IL‐18, which is just like their parent iMSCs.

Summary/Conclusion : The findings of the current study elucidated the possibility of production of exosomes with superior immunomodulatory properties from iMSCs generated from iPSCs utilizing automated production methods.

Non‐coding RNA and proteomic profiling of iPSC‐derived extracellular vesicles: new frontiers in cell‐free nanomedicine for regenerative therapy

Presenter: Noemi Elia

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy

Introduction : Extracellular vesicles (EV) are nano‐sized particles released by cells, carrying biomolecules like proteins, RNA, and lipids that impact physiological and pathological processes. While EV‐based regenerative therapies present advantages over cell‐based methods, their clinical application is hindered by challenges in isolating EV from primary cell lines such as mesenchymal stromal cells (MSC). To overcome these limitations, our study focuses on EV derived from human‐induced pluripotent stem cells (hiPSC) generated from clinical‐grade cord blood MSC, offering a renewable EV source.

Methods : We characterized hiPSC‐derived EV (hiPSC‐EV) using nanoparticle tracking analysis, western blotting, flow cytometry, and electron microscopy per MISEV2023 guidelines. Proteomic profiling employed high‐resolution mass spectrometry, while non‐coding RNA (ncRNA) analysis used a high‐throughput PCR array and circular RNA microarray to assess the miRNA and circRNA content within hiPSC‐EV.

Results : Proteomic analysis identified functional proteins related to growth, homeostasis, and immune modulation, with consistent profiles across three hiPSC‐EV batches. The ncRNA analysis revealed conserved expression of 147 miRNAs and a distinct circRNA profile, suggesting involvement in hypoxic, ischaemic, and apoptotic pathways. Comparative analysis highlighted key differences between hiPSC‐EV and MSC‐EV, suggesting superior regenerative potential.

Summary/Conclusion : Our findings reveal that hiPSC‐EVs possess a distinct proteomic and RNA profile that supports their role in tissue repair and regeneration. This study emphasizes hiPSC‐EV as promising candidates for cell‐free nanomedicine applications in regenerative medicine.

Funding : Supported by ERP‐2020‐EuronanoMed3‐NANO4STROKE, Project no. ERP‐2020‐23680649, and by the “Bando PNRR Missione 6 ‐ Componente 2 investimento 2.1 valorizzazione e potenziamento della ricerca biomedica del SSN” no. PNRR‐POC‐2022 12375642, CUP C43C22001430006, I73C22000550006, H73C22001630006, funded by the European Union—Next Generation EU—NRRP M6C2 ‐ Investment 2.1 Enhancement and strengthening of biomedical research in the NHS.

Physiology and pathology

Subtopic Cell reprogramming 1

EVs as mediators of indoor air pollutant‐driven inflammatory systemic effects

Presenter: Chiara Macchi

University of Milan, Milan, Italy

Introduction : According to the WHO, the entire global population is exposed to air pollution levels higher than recommended for health preservation. Ambient air pollution is a major health risk, leading to respiratory and cardiovascular mortality. The primary air pollutants are particulate matter (PM), nitric oxide (NO 2 ) and total volatile organic compounds (tVOC). Pathophysiologic mechanisms through which air pollution triggers cardiovascular events include the activation of oxidative stress/inflammation and the release of extracellular vesicles (EVs), which contribute to the local and systemic effects driven by air pollutants.

Methods : EVs were isolated from healthy volunteers following MISEV2023 guidelines. After plasma ultracentrifugation (110,000 × g for 90 min), they were purified from lipoproteins by SEC and characterized using NTA, flow cytometry analysis and TEM to confirm their integrity. Mitochondrial activity was assessed by mitostress test (Seahorse). Indoor pollutants (PM2.5, NO2, and tVOC) were monitored by the AirAssure Monitor for 7 days.

Results : In 17 healthy volunteers from the Lombardy region (Italy), increased exposure to indoor air pollutants increased the release of the inflammatory cytokine TNF‐α, as well as that of EVs derived from monocytes (CD14), macrophages (CD206), T and B lymphocytes (CD8, CD19, respectively) and neutrophils (CD66b). To explore potential molecular mechanisms linking exposure to air pollutants and systemic inflammation, A459 human lung cells were treated with PM (< 4 µm) for 24 h, which resulted in a higher release of EVs compared to untreated A459. EV isolated from PM‐exposed A459 cells (EVPM) were able to increase the release of proinflammatory cytokines (IL1α, IL1β and IL6) from primary cultures of PBMCs and reduce their mitochondrial functionality. When injected into zebrafish, EVPM raised the expression of proinflammatory cytokines IL8 and IL1β.

Summary/Conclusion : Air pollutants favour a proinflammatory state through EVs by reducing the mitochondrial activity and boosting the release of proinflammatory cytokines in PBMCs.

Funding : This work was supported by piano di sostegno alla ricerca 2023 (LINEA 2 AZIONE A), University of Milan.

Engineering extracellular vesicle protein composition for targeted angiogenesis

Ingrid Petersen, Vanessa Franco Carvalho Dartora, Alyssa Panitch

Georgia Institute of Technology, Atlanta, Georgia, USA

Introduction : Wound healing is a multi‐stage process involving haemostasis, inflammation, proliferation and remodelling. Within the proliferation stage, angiogenesis is a crucial step that can be induced through angiogenic factors in response to injury. Our lab has previously studied the application of a synthetic glycan therapeutic to stimulate angiogenesis. We showed that the pro‐angiogenic response was not solely the result of endothelial cell (EC) recruitment and direct cellular function but was additionally mediated by the released secretome. Further investigation highlighted the critical role of extracellular vesicles (EVs). Our goal involves engineering EV protein composition to enhance EV impact on angiogenic outcomes, with the expectation that modifying cellular expression levels of target proteins known to be trafficked to EVs will control the therapeutic potential of EVs as a targeted angiogenic treatment.

Methods : Cell culture media was collected from endothelial progenitor cells (EPCs) subjected to transfection of siRNA or plasmid expression vector to modulate levels of target protein (neuropilin‐1, β3 integrin, or thrombospondin‐4). EVs were isolated by ultracentrifugation, and the size and concentration were measured using an IZON Exoid. EVs were visualized using electron microscopy, and surface markers were verified using flow cytometry. Angiogenesis was assessed through a tube formation assay where EPCs were cultured on Matrigel. Connected network structures were quantified using ImageJ's angiogenesis analyser plugin. Cellular and EV target protein levels were verified using an enzyme‐linked immunosorbent assay (ELISA).

Results : Proteomic analysis identified a subset of proteins that were enriched in the EVs of EPCs that stimulated angiogenesis, suggesting protein candidates that promote angiogenesis and are naturally selectively loaded into EVs. Based on these findings, we selected proteins of interest to further investigate how the cellular level of target protein influences EV composition and the corresponding impact of EV treatment on angiogenesis. Preliminary data suggests that silencing of these proteins reduces angiogenesis of EPC‐derived EVs. Ongoing work is investigating the effects of overexpression of these target proteins on EV loading and angiogenesis.

Summary/Conclusion : This approach enabled the investigation of how modifying cellular expression levels of target proteins could enhance the therapeutic potential of EVs as a delivery tool to enhance angiogenesis.

Extracellular RNA analysis of neuronal cell models as surrogate non‐invasive markers for differentiation assessment

Patricia Hernández‐López 1 , Elisabeth Rackles 1 , Ana Talamillo 1 , Juan Manuel Falcon‐Pérez 1,2,3

1 Exosomes Laboratory, CIC bioGUNE‐BRTA, Derio, Spain; 2 Metabolomics Platform, CIC bioGUNE‐BRTA, Derio, Spain; 3 Ikerbasque, Basque Foundation for Science, Bilbao, Spain

Introduction : In vitro cellular models for human neurological diseases are important due to the lack of consistent, reproducible, homogenous and viable neuronal models of human origin. In neurobiology, the major types of human cell culture models include neuroblastoma cell lines such as SH‐SY5Y and reprogramming of induced pluripotent stem cells (iPSCs), among others. The SH‐SY5Y cell line is a thrice‐cloned subline of the bone marrow‐derived cell line SK‐N‐SH, which has catecholaminergic neuronal properties. It can undergo in vitro differentiation to develop a phenotype resembling dopaminergic human neuronees, with neuritic processes and neuronee‐specific enzymes and receptors (Kovalevich et al. 2013; Shipley et al. 2016; Kalinovskii et al. 2022). Moreover, iPSCs are a cell line derived from human reprogrammed fibroblasts. In this case, differentiation of those cells using small molecule inhibitors and growth factors is used to generate basal forebrain‐like cholinergic neuronees (Sanz‐Muñoz et al. 2020). So, we have studied by quantitative real‐time PCR (qPCR) the extracellular RNA of SH‐SY5Y and iPSCs as a readout for the status of the cell differentiation to neuronees.

Methods : SH‐SY5Y cells were differentiated into neuronal phenotype using a sequential treatment with retinoic acid (RA) and brain‐derived neurotrophic factor (BDNF) for 9 days. iPSCs were differentiated using the previously published protocol described by Sanz‐Muñoz et al. (2020) for 62 days. RNA from conditioned media (extracellular) and from the cells (intracellular) of a gene panel including markers of pluripotency, neural progenitors and mature neuronees were analysed by qPCR in order to be compared at different time of cell differentiation.

Results : In general, the expression of the mature neuronal genes CACNA1B (Calcium Voltage‐Gated Channel Subunit Alpha 1 B) and TUBB3 (Tubulin Beta 3 Class III) is reproducible both intra‐ and extracellularly across different time points of differentiation.

Summary/Conclusion : Extracellular RNA analysis of the described human neuronal cell models could be reliable read out for the assessment of differentiation status in a non‐invasive manner.

Funding : Alzheimer's Association Grant (Ref. AARG‐NTF‐22‐968911).

Brain pericyte‐derived small extracellular vesicles as modulators of blood‐brain barrier endothelial cells differentiation and functions

Océane Dusailly 1 , Camille Menaceur 1 , Paulo Marcelo 2 , Marie Naudot 2 , Fabien Gosselet 1 , Laurence Fenart 1 and Julien Saint‐Pol 1

1 Université Artois, UR 2465, Blood‐Brain Barrier Laboratory (LBHE), Lens, France; 2 Plateforme d'Imagerie Cellulaire et Analyses des Protéines ICAP, FR CNRS 3085 ICP, Université de Picardie Jules Verne, Amiens, France

Introduction : The induction and maintenance of the Blood‐Brain Barrier (BBB) features exhibited by brain microvessel endothelial cells (ECs) are induced by neighbour cells and particularly brain pericytes (BPs). Most studies highlighted the role of secreted factors such as cytokines and growth factors in the BBB barriergenesis steps throughout embryogenesis and the first postnatal days, but none of them focused on BP‐derived extracellular vesicles (EVs), which are known to carry epigenetic and regulatory compounds able to modulate the cell fate and/or differentiation of receipt cells. Moreover, our previous mass spectrometry (MS) data on BPs showed an induction of proteins involved in EV biogenesis by BBB ECs. Using a syngeneic human in vitro BBB model, we aimed to characterize the role of BP‐derived small EVs (BP‐sEV) in BBB ECs differentiation.

Methods : BP‐sEV were isolated by UC and SEC and used at different concentrations on endothelial cells for 4 days (2× 2 days), and the BBB main features were checked by immunofluorescence, permeability assays, TEER measurements and functional assays. BP‐sEV have been characterized according to the MISEV 2023 recommendations by TEM, SEM, mass spectrometry approaches and TRPS for size and zeta potential.

Results : We first observed that BP‐sEV are internalized by BBB ECs from their basolateral side and therefore able to be referred to as a cell‐cell communication vector between BPs and BBB ECs. We also demonstrated that BP‐sEV promotes a decrease in EC permeability for sodium fluorescein and an increase of trans‐endothelial electric resistance (TEER) in a dose‐response manner that is linked to a differentiation of naïve ECs into BBB ECs. MS proteomics data from BP‐sEV‐treated ECs showed a decreased expression of non‐differentiated ECs markers such as PLVAP. miRNA‐seq experiments led on BP‐sEV also provided some candidate miRNAs explaining such ECs differentiation.

Summary/Conclusion : These results highlighted the role and importance played by BP‐sEV in the BBB barriergenesis and opened some outcomes concerning the pathological impact of such BP‐sEV on BBB main features in neurodegenerative disorders.

Funding : This work is funded by the French Research National Agency (ANR‐21‐CE14‐0002).

Metabolic reprogramming of T cells by glioblastoma‐derived extracellular vesicles induces glycolysis

Vikas Kumar 1 , Oksana Musatova 2 , Yury Rubtsov 2 are co‐senior authors.

1 Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia; 2 Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia

Introduction : Extracellular vesicles (EVs) produced by glioblastoma (GBM) cells have been shown to be a crucial participant of the tumour microenvironment, in particular, by negatively affecting the function and metabolism of immune cells. Although GBM EVs have been previously reported to activate immune checkpoint molecules such as PD‐L1 in T cells, the contribution of GBM EVs in metabolic reprogramming of lymphocytes is not fully understood. Here, we wondered how GBM‐derived EVs impact the T‐cell metabolism, with emphasis on glycolysis activation.

Methods : RNA sequencing was performed on T cells after treatment with GBM‐derived EVs. Differential gene expression analysis was conducted to determine the metabolic pathways that were upregulated and, in particular, glycolytic cascades. To further validate activation of the pathway, processes. Gene set enrichment analysis (GSEA) was used. The use of EVs in this study was ethically approved with regard to the experiments that require the use of human blood. Additional metabolic assessments are planned for the next steps of this research, including glucose uptake and lactate secretion.

Results : RNA sequencing revealed significant upregulation of glycolytic genes such as hexokinase 2 (HK2), pyruvate kinase M (PKM), and lactate dehydrogenase A (LDHA) that were upregulated in T cells incubated with GBM‐derived EVs. Further, GSEA analysis revalidated and supported, among other pathways, glycolysis and pathways associated with it. The metabolic characterization of T cells treated by EVs reveals an enhanced rate of glucose uptake and lactate production, consistent with previous RNA evidence of glycolytic activation.

Summary/Conclusion : This study demonstrates that GBM‐derived EVs lead to metabolic reprogramming in T cells through the activation of glycolysis. These data reveal a new function of GBM EVs in modulating T‐cell metabolism with relevance to the development of immune‐targeted approaches in GBM. Further validation of these findings through metabolic assays will be completed prior to the presentation.

8CLC‐derived exosomes mediate regulatory T‐cell retinoic acid metabolism through CRABP1 to alleviate psoriasis

Ying Fu 1,2 , Qiang Guo 2,3 , Yunge Wang 2,3 , Mengwei Fu 2,4 , Yixin Yan 2 , Xuan Dong 2

1 Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China; 2 BGI Research, Hangzhou, China; 3 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China; 4 College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China

Introduction : Clinical management of psoriasis is often complicated by recurrence and challenges in maintaining long‐term efficacy. While exosomes derived from pluripotent stem cells have shown promising long‐term therapeutic effects, they are hampered by issues such as senescence and unclear effector molecules and mechanisms.

Methods : In our previous research, we developed a system to induce pluripotent stem cells into 8C‐like cells (8CLCs), which exhibit characteristics similar to human 8‐cell embryos. These cells demonstrate totipotency and enhanced genomic stability.

Results : Co‐culturing T cells with exosomes derived from 8CLCs (8CLC‐Exo) significantly reduces Th17 differentiation while promoting Treg generation. Topical application of 8CLC‐Exo alleviates psoriatic skin lesions and enhances Treg infiltration in an IMQ‐induced mouse model. Through integrated analysis of exosomal proteomics data and RNA sequencing data from psoriasis patients, we hypothesize that 8CLC‐Exo facilitates Treg differentiation and modulates immune homeostasis in psoriasis via the cellular retinoic acid‐binding protein 1 (CRABP1)‐dependent retinoic acid metabolism pathway.

Summary/Conclusion : This project integrates disease models with multidimensional omics techniques to elucidate the precise molecular mechanisms and roles of 8CLC‐Exo in alleviating psoriasis, offering potential insights and innovative therapeutic strategies for its treatment.

Funding : This work was supported by the General Program of the National Natural Science Foundation of China (32400746).

Proteomic profiling of plasma‐derived small extracellular vesicles of pancreatic neuroendocrine tumour patients and healthy control subjects

Presenter: Surabhi A. S.

All India Institute of Medical Sciences, New Delhi, India

Introduction : Pancreatic neuroendocrine tumour (PanNET) is the most common (44%) gastroenteropancreatic neuroendocrine tumours in India and is mostly malignant, presenting in clinics at an advanced stage of disease with metastasis. The major loophole in the management of the disease is the unavailability of an efficient biomarker for early detection, targeted therapy, or prognosis. Thus, exploring the early diagnostic markers is the need of the h. Over the past decades, small extracellular vesicles (sEVs) have gained considerable attention as mediators of intercellular signalling and their potential role in the pathophysiology, diagnosis, and treatment of diverse cancers has been studied. There is a scarcity of research in the proteomic profiling of plasma derived sEVs in PanNETs. So, this study is focused on proteomic profiling of plasma‐derived sEVs in PanNET patients (PanNETs) and healthy control subjects (HCs).

Methods : Five millilitres of blood was collected from 30 subjects (20 PanNETs and 10 age‐matched HCs) after getting clearance from the ethical committee AIIMS, New Delhi, India (IECPG‐514/30.06.2022). Plasma‐derived sEVs were isolated through chemical precipitation and characterised by transmission electron microscopy (TEM) and nano particle tracking analyser (NTA). Validation was done by Western blot using tumour suppressor gene (TSG101), flottilin as an exosomal marker, calnexin as a negative marker, glutamic acid decarboxylase (GAD65) as an islet cell marker and β actin as an internal control. The proteomic profiling of plasma‐derived sEVs in 9 PanNETs and 3 healthy control subjects (HCs) was done using liquid chromatography‐mass spectrometry (LC‐MS).

Results : Overall, 2.57% and 32.11% of proteins were uniquely identified in PanNET and HCs, respectively while 65.32% were commonly expressed in PanNETs and HCs. Furthermore, 37 proteins exhibiting significant differential expression between PanNET and controls were identified with downstream analysis. Four proteins namely filamin‐A (FLNA), integrin subunit alpha 2B (ITGA2B), fibronectin1(FN1), and apolipoprotein E (APOE) were selected by pathway analysis. Validation by western blot showed dysregulation of ITGA2B and FN1 proteins in PanNETs as compared to HCs.

Summary/Conclusion : The study for the first time shed light on the comparison of the proteomic profile of sEVs cargo of PanNETs and HCs. Identified novel biomarkers and their validation in a large number of samples may enhance early‐stage detection and prognosis.

Neuroblastoma extracellular vesicles mediate blood‐brain barrier permeability and carry tight junctions’ protein cargo

Tal Manko 1 , Ishai Luz 1 , Meshi Zorsky 2 , Gad Vatine 2 , Tomer Cooks 1

1 Faculty of Health Sciences, The Shraga Segal Department of Microbiology, Immunology & Genetics, Ben‐Gurion University, Beer Sheva, Israel; 2 The Department of Physiology and Cell Biology, Ben‐Gurion University, Beer Sheva, Israel

Introduction : Extracellular vesicles (EVs) play an important role in cell‐to‐cell communication, particularly when released by cancer cells, allowing them to interact with their tumour microenvironment. Neuroblastoma (NB) is a cancerous solid tumour commonly arising in paediatric children across the sympathetic nervous system, which causes multisite neuronal damage to children and might even lead to death. NB is considered an enigma with spontaneous remission, relapses, and primary tumour sites; despite extensive research of NB, there are still many gaps in our knowledge regarding its tumourigenesis, metastasis, and pathology.

Methods : In this research, we sought to explore the effects of EVs shed by NB cells (NS20 and N2A, cell lines representing late and early onsets, respectively) upon endothelial cells (bEND3 cell line) of the blood‐brain barrier (BBB), employing a novel in vitro BBB model to study their interactions. Measuring their stability and permeability via Trans Epithelial Electrical Resistance (TEER) measurements and FITC penetrance. In addition, we also re‐educated NSC34 (non‐cancerous motor‐neuronees cells) via co‐culture NB cells or EVs and measured their ability to affect BBB permeability.

Results : Our results indicate that NB EVs and educated NSC34 cells significantly impact bEND3 integrity. Mass spectrometry analysis of EVs from N2A EVs unravelled several proteins that affect tight junctions’ assembly, suggesting the first reported evidence of BBB manipulations via NB cancer cells. Higher levels of tight junctions’ proteins were found in the late‐stage NB EVs (NS20), including key proteins such as occludin, claudin and ZO‐2, matching with our TEER measurements showing higher degree of destabilization of the BBB.

Summary/Conclusion : This study presents characterization of NB EVs and shows their capacity to promote disassembly of tight junctions, leading to destabilization of the BBB. Our results also suggest that EVs from NB sources can contain proteins involved in tight junction biology, leading to a new frontier of EV biomarkers and potentially enabling full intact tight junction formations that can interact with target sites.

Subtopic Tissue stress and remodelling

Small extracellular vesicle release by corneal epithelial cells may serve as an early indicator of corneal dysfunction in dry eye disease

Belinda J. Hernandez 1 and Danielle M. Robertson 1

1 Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Introduction : Dry eye disease (DED) is a multifactorial disease that damages the ocular surface epithelium. DED is characterized by tear film instability, tear hyperosmolarity, inflammation, and neurosensory abnormalities. Autophagy has been shown to be increased in DED. Insulin‐like growth factor binding protein‐3 (IGFPB‐3) inhibits autophagy in the cornea. IGFBP‐3 is also downregulated in cells exposed to hyperosmolar stress. The relationship between corneal epithelial cell‐derived small extracellular vesicles (sEVs), hyperosmolar stress and IGFBP‐3 is unknown. We hypothesize that both knockout of IGFBP3 and hyperosmolar stress increase sEV release, and that release of these sEVs underlies the early stages of DED.

Methods : Confluent telomerase‐immortalized human corneal epithelial (hTCEpi) cells were grown on T‐175 flasks and treated with 680 mOsm NaCl for 48 hs to induce hyperosmolar stress. A CRISPR‐edited IGFBP3 knockout cell line and control cell line were generated using hTCEpi cells. Cells were cultured in T‐175 flasks for five days. sEVs were isolated using differential centrifugation followed by iodixanol density gradient flotation. sEV yield was assessed by NanoSight tracking analysis. Purity and protein content of sEV preparations were analysed by immunoblotting for sEV markers and markers for known contaminants. The EV Track detailing methodology is EV Track ID: 230370.

Results : Hyperosmolar stress induced by NaCl and loss of IGFBP3 in corneal epithelial cells both revealed changes in the endosomal pathway. In IGFBP‐3 knockout cells, there was a 3‐fold increase in sEV markers, including syntenin‐1, TSG101, and CD9, by immunoblotting. We also detected a 3‐fold increase of sEV release from corneal epithelial cells under hyperosmolar stress compared to control cells.

Summary/Conclusion : We show for the first time that both hyperosmolar stress and knockout of IGFBP3 in corneal epithelial cells alter the endosomal pathway. This alteration is a stimulus for the increased release of sEVs. These findings open a novel avenue for therapeutic interventions and early biomarkers for cellular dysfunction in DED.

Funding : NEI grants EY029258 (DMR), EY024546 (DMR), EY033505 (DMR), core grant for vision research EY030413 , a Challenge Grant from Research to Prevent Blindness, and the Shirley G. and Norman Alweis Fund for Vision (DMR).

Sex differences in specialized lipid mediators in EVs and intra‐, and extracellular compartments of human conjunctival goblet cells during allergic conjunctivitis

Presenter: Changrim Lee

Harvard Medical School, Boston, Massachusetts, USA

Introduction : Specialized pro‐resolving mediators (SPMs) and proinflammatory mediators are endogenous small molecules playing an essential role in health and disease. Biosynthesis and secretion of mediators from polymorphonuclear leukocytes are well understood, but those from epithelial cells still need to be studied.

Methods : Using histamine‐mediated allergic inflammation in ex vivo cultures of human conjunctival goblet cells (HCjGCs) as a disease model of allergic conjunctivitis, we performed ELISA‐based quantification on seven representative lipid mediators (prostaglandin E2 (PGE2), leukotriene B4 (LTB4), lipoxin A4 (LXA4), resolvin E1 (RvE1), resolvin D1 (RvD1), maresin 1 (MaR1), and protectin D1 (PD1)) in three types of samples (extracellular vesicles (EVs), EV‐free media, and cell lysates) collected under three conditions (homeostasis, mild, and severe allergic inflammation). Then the results were compared between and within male HCjGCs (‘males’) and females.

Results : Females biosynthesized and secreted higher levels of mediators than males in all three types of samples in all three conditions. Females had more than 50% of the proinflammatory mediators (PGE2 and LTB4) and SPMs (LXA4, RvE1, RvD1, MaR1, and PD1) in the extracellular space. This was also the case for SPMs in males; however, more than 50% of the proinflammatory mediators were retained in cells of males. In all samples, PD1 level was notably higher than the sum of the other four SPMs (LXA4, RvE1, RvD1, and MaR1).

Summary/Conclusion : We conclude that the profile of intra‐ and extra‐cellular lipid mediators of HCjGCs is sex dependent. Lower levels of synthesis and secretion of lipid mediators in males than females during homeostasis and allergic conditions may be linked to the male predisposition to severe types of allergic conjunctivitis.

Funding : This work is supported by National Institutes of Health (NIH) R01EY029789, EY029789 , T32EY007145, F32EY035529, P30EY003790; Korean‐American Scientists and Engineers Association (KSEA) Young Investigator Grant (C.L.).

Overexpression of CD73 reduces extracellular vesicle (EV)‐sequestered cyclooxygenase 2 (COX‐2) levels in activated J744A1 macrophages

Presenter: Ravi Shankar Akundi

South Asian University, New Delhi, India

Introduction : Inflammation is the first line of the body's defence system, mediated by macrophages, which produce prostaglandin E2 (PGE2) in addition to cytokines and chemokines. Failure of resolution of inflammation leads to chronic inflammatory conditions such as rheumatoid arthritis, neurodegeneration, and/or cancer. We have previously identified extracellular ATP (eATP) as a synergistic enhancer of cyclooxygenase 2 (COX‐2)‐mediated inflammation in macrophages and found that activation of purinergic P2 receptors enhanced the synthesis of PGE2. Ectonucleotidases such as CD39 and CD73 sequentially dephosphorylate ATP to ADP and AMP and further to adenosine, respectively. Removal of ATP by ectonucleotidases, therefore, is an essential step for the resolution of inflammation. In addition, adenosine acts as an anti‐inflammatory molecule by binding to P1 receptors. In the case of tumours, we have shown that P2 receptor‐mediated increased expression of COX‐2 causes tumour progression and metastasis. Extracellular vesicles (EVs) released by activated macrophages or tumour cells are a potential mechanism through which pro‐inflammatory mediators such as COX‐2, cytokines, and PGE2 are transported to distant sites, thereby spreading metastasis in the case of cancer cells. In this work, we check whether ectonucleotidases reduce COX‐2 expression in macrophages and affect the packaging of EVs released by these cells.

Methods : In this project, we cloned and overexpressed CD73 in J774A1 macrophages to isolate EVs. EVs were isolated and characterized using different conditioned media followed by differential ultracentrifugation and using various protein markers. The isolated EVs were used to stimulate naïve J744A1 macrophages to check for COX‐2 expression.

Results : We show that overexpression of CD73, the rate‐limiting ectonucleotidase, in murine macrophages shows a reduction in inflammation and PGE2 release. Furthermore, we also show that EVs released from CD73‐transfected cells have reduced sequestration of COX‐2 mRNA and its expression.

Summary/Conclusion : Modulation of inflammation through engineered EVs (EVs released from CD73 overexpressing cells) could be a potential alternative to non‐steroidal anti‐inflammatory drugs (NSAIDs) to combat chronic inflammatory conditions, autoimmune disorders, and neurodegeneration.

Bacterial stimulation alters lipid composition of macrophage‐derived extracellular vesicles: Implications for immune modulation

Júlia Orlovská 1,2 , Kristýna Turková 1,3 , Miriam Sandanusová 1,2 , Štěpán Strnad 4 , Vladimír Vrkoslav 4 , Berka Vratislav 5 , Lukáš Kubala 1,2,3 , Gabriela Ambrožová 1

1 Institute of Biophysics of the CAS, Brno, Czechia; 2 Faculty of Science, Department of Experimental Biology, Masaryk University, Brno, Czechia; 3 International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia; 4 Institute of Organic Chemistry and Biochemistry of the CAS, Brno, Czechia; 5 Contipro a.s., Dolní Dobrouč, Czechia

Introduction : Macrophages (Mfs) are key innate immune cells involved in pathogen defence, inflammation, and tissue repair. Their extracellular vesicles (Mf‐EVs) play important roles in various diseases, making them significant therapeutic targets. We focus on the effects of dysregulated microbiota on host cells during intestinal inflammation, where immune cells play a crucial role. While many studies have explored the protein composition of Mf‐EVs, little attention has been given to their lipid profiles. This study investigates the differences in the lipid composition of Mf‐EVs derived from quiescent Mfs compared to those treated with Lacticaseibacillus rhamnosus stimuli.

Methods : Mf‐EVs were isolated by ultracentrifugation from quiescent RAW 264.7 Mfs and those treated for 24 h with bacterial lysate (BL) from Lacticaseibacillus rhamnosus CCM7091. Characterization followed MISEV 2023 guidelines (cryo‐EM, NTA, typical EV markers), while lipid profiles of both Mf‐EVs and their maternal cells were determined using LC‐MS lipidomic analysis (Orbitrap IQ‐X Tribrid Mass Spectrometer connected to the Vanquish UHPLC system) and bioinformatics analyses.

Results : The immunomodulatory effect of BL on RAW 264.7 Mfs was confirmed by increased pro‐inflammatory marker production. Cryo‐EM and NTA confirmed that Mf‐EVs isolated via ultracentrifugation, purified with a sucrose cushion, possessed a bilayer membrane and fell within the size range of small EVs. Typical EV markers (TSG101, CD63, CD81) were detected, while ApoE, a negative EV marker, was absent. Fewer particles were observed in BL‐treated cells compared to control Mfs. Lipidomic analysis revealed significant differences in the lipid composition between BL‐EVs and those from untreated cells, as well as between their respective maternal cells. Unique lipid features were identified, suggesting functional relevance.

Summary/Conclusion : Our study highlights a crucial gap in current research, focusing on the lipid composition of Mf‐EVs under inflammatory conditions. Bacterial stimulation of Mfs alters the lipid profile of both maternal cells and their EVs, indicating that the activation state of Mfs may influence lipid distribution within EVs. The functional implications of these lipid changes warrant further investigation.

Funding : Supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104)—Funded by the European Union—Next Generation EU.

Extracellular vesicles and their miRNA cargo in response to physical exercise: potential involvement in oxidative stress regulation—Insights from the HEBE Study

Eva Dariol, Rachele Matsagani, Mirjam Hoxha, Laura Dioni, Simona Iodice, Davide Biganzoli, Federica Rota, Valentina Bollati, Elia Biganzoli

Elia Biganzoli on behalf of the HEBE Consortium, University of Milan, Milan, Italy Eva Dariol and Rachele Matsagani are co‐first authors .

Introduction : Physical exercise (PE) is crucial for maintaining health across all ages, with regular PE playing a key role in preventing non‐communicable diseases. Particularly, moderate‐intensity PE has been shown to reduce inflammation and oxidative stress, two highly interconnected processes. Both acute and chronic PE stimulate tissues and organs to release extracellular vesicles (EVs), which allow cell‐to‐cell long‐distance communication, contributing to whole‐body homeostasis. EVs carry bioactive molecules, including microRNAs (miRNAs), which are post‐transcriptional regulators of gene expression influenced by external factors like PE and promising biomarkers of effect. This study aims to investigate the role of EVs and their miRNA content as potential mediators of the relationship between PE, oxidative stress, and inflammation.

Methods : After signing informed consent, 91 participants from the HEBE project (Award number: PSRL621MCLER_01) were enrolled. Clinical parameters were assessed, and blood samples were collected for circulating marker measurements before (T0) and after (T1) a 6‐month personalized PE intervention. Plasma‐derived EVs were isolated by ultracentrifugation and characterized for size distribution and concentration using nanoparticle tracking analysis (NTA). After EV‐miRNAs extraction, the miRNome (755 miRNAs) was profiled using OpenArray technology. EV size distribution and total concentration were compared between T0 and T1. Differentially expressed miRNAs between T0 and T1 were identified through statistical analysis and evaluated by gene ontology (GO) enrichment analysis.

Results : No significant difference in EV size distribution was observed between T0 and T1. Following the PE intervention, five EV‐associated miRNAs were upregulated (hsa‐miR‐483‐5p, hsa‐miR‐181a, hsa‐miR‐376c, hsa‐miR‐376a, hsa‐miR‐222), while two were downregulated (hsa‐miR‐98, hsa‐miR‐664). Enrichment analysis revealed a significant involvement of upregulated EV‐miRNAs in key cellular pathways, including apoptosis and oxidative stress response.

Summary/Conclusion : Our findings suggest that EVs and their miRNA content play a role in the regulation of key biological pathways, including the cellular response to oxidative stress, in response to PE. This study provides new insights into the molecular mechanisms underlying the beneficial effects of PE, highlighting the potential role of EV‐miRNAs as mediators of PE‐induced adaptations. Further research is needed to fully elucidate their functional impact.

Funding : Abstract funding: HEBE project (Award number: PSRL621MCLER_01). First co‐authors PhD scholarship funding: MAMELI project (Grant agreement ID: 101086988).

Subtopic Organ systems 1

Unlocking cancer secrets: how short‐chain fatty acids drive extracellular vesicle production in colorectal cancer

Daniela Macedo 1,2,3 , Teresa Lage 1 , Lara Pierantoni 1 , Sara Abalde‐Cela 1 , Lorena Diéguez 1 , Ana Preto 2,3 , Carlos Honrado 1

1 International Iberian Nanotechnology Laboratory (INL), Braga, Portugal; 2 Centre of Molecular and Environmental Biology (CBMA), University of Minho, Braga, Portugal; 3 Institute of Science and Innovation for Bio‐Sustainability (IB‐S), University of Minho, Guimarães, Portugal

Introduction : Colorectal cancer (CRC) exhibits high incidence and mortality rates, often due to late‐stage detection and limited treatment options. Changes in gut microbiota, chiefly an imbalance in short‐chain fatty acids (SCFAs), have been linked to CRC development, since SCFAs induce cell cycle arrest, lysosomal membrane permeabilization, and apoptosis. Concurrently, extracellular vesicles (EVs) are emerging as promising non‐invasive biomarkers for cancer diagnosis, prognosis, and monitoring. However, while SCFAs influence CRC cells, their effects on EV production and phenotype remain unknown.

Methods : EV isolation methods, that is, ultracentrifugation (UC), size‐exclusion chromatography (SEC), ultrafiltration and an EV aggregation agent (ExoGAG), were compared. Particle size and concentration (NTA), co‐precipitated protein content (BCA), and double‐stranded DNA (dsDNA) levels (Qubit) were assessed. SCFAs were administered at IC50 concentrations, in typical normobiosis proportions (60% acetate, 25% propionate, 15% butyrate), to CRC cells to evaluate the impact on EV production and phenotype. Particle size, EV yield, protein content, dsDNA levels and integrity, and EV‐DNA analysis for microsatellite instability (MSI) using droplet digital PCR were assessed post‐treatment.

Results : A 16 h UC yielded the highest particle counts but resulted in greater protein contamination compared to SEC and the optimized two‐step UC protocol. SCFA treatment increased EV production by ∼2× and significantly elevated EV‐DNA, while maintaining co‐precipitated protein levels. Notably, MSI status was assessed for the first time on CRC‐derived EVs and was shown to be detectable in EV‐DNA from all cell lines, reflecting the parent cells’ profiles, even with low dsDNA levels and after SCFA treatment.

Summary/Conclusion : An optimized EV isolation protocol was developed, yielding high counts with minimal contamination. SCFA treatment significantly enhanced EV production and dsDNA content without altering co‐precipitated protein levels. Moreover, for the first time, MSI was detected in EV‐DNA, highlighting the potential of EVs as non‐invasive biomarkers for CRC, supporting their role in diagnostic and therapeutic applications.

Funding : Health From Portugal (C630926586‐00465198), through the NextGenerationEU Fund.

A short form of multidrug resistance‐associated protein 1 (MRP1) in plasma sEVs is a potential indicator of pancreatic ductal adenocarcinoma

Presenter: Kritisha Bhandari

University of Oklahoma, Oklahoma, USA

Introduction : Multidrug resistance‐associated protein 1 (MRP1) is an ATP‐dependent transmembrane efflux pump. MRP1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and other malignancies. However, its diagnostic value in cancer has not been explored. Cancer‐derived sEVs are attractive candidates for the development of noninvasive biomarkers. The present study examined MRP1 expression in plasma sEVs derived from PDAC and colon cancer patients.

Methods : The human plasma samples were obtained from the NCI‐sponsored Cooperative Human Tissue Network and Stephenson Cancer Center. The human pancreatic cancer cell lines PANC‐1, MIA PaCa‐2 and BxPC‐3, and the pancreatic ductal cell line HPNE were examined for MRP1 expression. sEVs were isolated using double filtration followed by the polymer precipitation method. Ultracentrifugation and size‐restricted columns were also used for plasma sEV isolation. Western blotting was performed to evaluate the expression level of MRP1, and proteomics and bioinformatics were applied to confirm the detection of MRP1 in plasma sEVs.

Results : MRP1 was overexpressed in PDAC cells and detected in sEVs derived from PDAC cells but absent in sEVs from HPNE cells. A short form of the MRP1 protein, representing the region between the QCRL‐1 and C‐terminus domains of the protein, was identified in plasma sEVs, and its expression level was significantly elevated in plasma sEVs derived from patients with early‐ and late‐stage PDAC, compared with matched healthy subjects ( n = 34 for each group). The level of the short form of MRP1 was also elevated in plasma sEVs derived from patients with colon cancer ( n = 44) but not in those derived from patients with breast cancer ( n = 15). The levels of the short form of MRP1 in plasma sEVs were not related to the chemotherapy or radiotherapy status of the patients.

Summary/Conclusion : A short form of MRP1 is identified in plasma sEVs and its expression level is significantly elevated in patients with early stage and late stage PDAC, irrespective of their treatment status. These findings reveal a novel circulating biomarker for PDAC with potential for early detection.

Funding : Congressionally Directed Medical Research Programs (W81XWH‐22‐1‐0317), Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P30GM145423), and the Presbyterian Health Foundation.

Host factor‐induced bacterial extracellular vesicles promote horizontal gene transfer in vibrio cholerae

Presenter: Dominik Fleischhacker

University of Graz, Graz, Austria

Introduction : Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. As a facultative pathogen, V. cholerae ’s lifecycle is marked by environmental persistence in aquatic reservoirs as well as colonization of the human intestinal tract. Recent research suggests that in the presence of antimicrobial agents, such as mitomycin C, a new type of vesicle is being formed, which relies on the cell death of a sub‐population. My research focuses on finding natural inducers for this type of vesicle and their physiological role.

Methods : Protein quantification (Bradford assay) Nucleic acid quantification (SYTO 9 Green‐Fluorescent Nucleic Acid Stain) Lipopolysaccharide (LPS) quantification (FM 4–64 assay) Size and nanoparticle amount measurement (Zetasizer Nano ZS90 and NanoSight NS300) Transmission electron microscopy Qualitative and quantitative proteomic analyses (LC‐MS), DNA sequencing and analyses (Illumina). Horizontal gene transfer experiment in vitro and in vivo (murine model).

Results : Our results show that V. cholerae produces explosive‐type BEVs in the presence of host‐derived antimicrobial stressors, that is, bile. State‐of‐the‐art BEV content characterization, morphological analyses, and OMIC‐based analyses identified several qualitative and quantitative differences between blebbing‐type and explosive‐type BEVs. Most importantly, the explosive‐type BEVs contained substantial amounts of cytoplasmic proteins and nucleic acids. The incubation of explosive BEVs derived from a tetracycline‐resistant V. cholerae isolate with a tetracycline‐sensitive wild‐type strain resulted in vesicle‐mediated HGT of the antibiotic resistance cassette in vitro and in vivo. Downstream experiments using diverse mutants of the natural competence system identified periplasmic, inner membrane and cytoplasmic components to be essential for vesicle‐mediated HGT, while outer membrane or exterior parts are dispensable. Our data suggest the possibility of vesicle‐mediated HGT during intestinal colonization facilitating the spread of virulence factors or antimicrobial resistance genes.

Summary/Conclusion : By applying the antimicrobial substances MMC and bile, vesicles containing significantly higher amounts of protein biomass and nucleic acids are being formed. These vesicles also have a higher degree of complexity and more closely resemble the WCL compared with the condition without additional stressors. These vesicles are also able to promote HGT of a chromosomal element in a ComEA‐dependent but PilA‐independent pathway both in vitro and in vivo (murine model).

Funding : FWF

Mechanism of MSC‐derived EVs to cross biological barriers and target specific tumoural cell types

Presenter: Marc Liébana

CIC bioGUNE, Derio, Spain

Introduction : Nowadays more personalized and efficient cancer treatment approaches are pursued. Nanoencapsulation systems allow decreasing drug dosages and better control of the dose‐response. Extracellular vesicles (EVs) are membranous vesicles released by virtually all cells and represent a plausible biocompatible option for drug encapsulation. Mesenchymal stem cells (MSCs) represent a promising EV source due to their low immunogenicity and tumour homing ability, among other properties. Artificial systems are used with this aim, but the immunocompatibility and specificity problems that they usually exhibit lead to the exploration of alternative systems as MSC‐EVs. Another challenge for nanotherapeutic agents is to reach their targets, needing to trespass physiological barriers to deliver their effect in most of the cases. The objective of this work is the study of the use of EVs as pharmacological nanovehicles in cancer. For this purpose, biological barriers have been evaluated due to their relevance in the clinical context, and different cell lines, including representative cells from specific malignancies, have been tested for their capacity to capture MSC‐EVs.

Methods : UC‐MSCs were used as EV producer cells. DiO labelling of MSCs was assessed by fluorescence microscopy and flow cytometry at periodic timepoints. EV isolation was conducted by differential ultracentrifugation. MSC‐EVs potential to cross biological barriers was evaluated in transwell co‐culture models. EVs effect on the biological barrier was assessed in terms of cytotoxicity (LDH assay) and barrier permeability (LY). Finally, MSC‐EV capture by malignant and healthy cell lines was assessed via fluorescence microscopy and flow cytometry.

Results : MSCs can be labelled with the lipophilic dye Vybrant DiO for multiple days, and their derived EVs retain this dye. These EVs can be tracked across a transwell model, and they can pass through biological barriers with no significant effect on the barriers themselves. Moreover, MSC‐derived EVs display homing properties towards recipient cell lines.

Summary/Conclusion : DiO‐EVs derived from MSCs could pass biological barriers and become differentially captured by distinct cell lines, thus offering a promising opportunity for their use as anticancer drug‐containing nano‐vehicles.

Funding : Spanish Ministry of Universities—FPU21/05332 MCIN & ERDF/FEDER—PID2021‐125104OB‐I00 EU's Horizon Twinning programme ‐ 101079264 (EVCA) EU's Horizon Staying Healthy Programme ‐ 101095679 (halt‐RONIN)

Urinary exosomes as non‐invasive window to glomerular and tubular cells and their communication

Presenter: Sindhu Thiagarajan

Universitätsklinikum Erlangen, Erlangen, Germany

Introduction : Although many renal diseases originate in the glomerular unit, subsequent damage of tubular cells may lead to alleviation of the disease condition. Exosomes are a type of extracellular vesicle, which play a major role in intercellular communication in many diseases. All cells secrete exosomes which carry nucleic acids, proteins, lipids and metabolites to recipient cells. Our previous studies showed the role of microRNAs in intraglomerular communication as well as their disease‐specific detection in the urine. However, little is known if such communication can be mediated by exosomes and if they also can serve as non‐invasive biomarkers.

Methods : Characterization of exosomes from the glomerular and tubular cell lines and patients’ urine was performed via nanoparticle tracking analysis to identify size and concentration, electron microscopic imaging to show intact morphology and appropriate size of the exosomes, as well as FACS staining and western blotting to detect common exosome markers. Proteome profiling and protease activity assay of exosomes from cell culture supernatant and respective ‘mother cells’ were used to characterize internal and external exosome cargo. Cell type‐specific markers in FACS sorting of exosomes were used to identify the cellular origin of urinary exosomes.

Results : Cell culture and urinary‐derived extracellular vesicles showed typical exosome size, morphology and marker expression (CD63, CD9, CD81). Proteomics of cell culture‐derived exosomes and mother cells allowed distinct protein cargo characterization of the exosomes. Protease activity could be detected on the exosome surface derived from glomerular cells. Glomerular cell‐derived exosomes could be detected and isolated via FACS sorting from the urine of patients with different glomerular diseases, giving a non‐invasive material for further characterization as a potential biomarker.

Summary/Conclusion : Podocyte‐derived exosomes might deliver cargo to tubular cells on their way to being secreted with the urine, where they might serve as non‐invasive snapshots of their cell of origin. Size‐wise exosomes should not be able to pass the glomerular basement membrane in vivo. However, the presence of proteases on exosomes might enable the passage of endothelial cell or even systemic exosomes through the GBM to reach podocytes or even tubular cells.

Funding : DFG, TRR374.

Extracellular vesicles in the vitreous as local mediators in proliferative diabetic retinopathy

Melanie E. SchwämmLe 1,2 , Paula Liang 1 , Stefaniya Boneva 1 , Yana Tarakanchikova 3 , Hansjürgen Agostini 1 , Günther Schlunck 1 , Irina Nazarenko 4 , Felicitas Bucher 1

1 Eye Center, Faculty of Medicine, University Medical Center Freiburg, Freiburg, Germany; 2 Faculty of Biology, University of Freiburg, Freiburg, German; 3 CapCo Bio GmbH, Germany. 4 Institute for Infection Prevention and Control, Faculty of Medicine, University Medical Center Freiburg, Freiburg, Germany

Introduction : Diabetic retinopathy (DR), a severe retinal disorder, is one of the most common causes of blindness in the Western world. Consequently, it is crucial to gain a profound understanding of the disease‐related cellular and molecular alterations within the retina. Due to the retina's intimate relationship with the vitreous humour, retinal cells secrete molecules into the vitreous, creating a unique microenvironment. As mediators of cell‐to‐cell communication, extracellular vesicles (EVs) have become a central focus of investigation. This project aimed to compare the EV profiles in vitreous samples from patients with and without proliferative DR (PDR) to enhance our understanding of DR pathophysiology.

Methods : At the beginning of vitrectomies, undiluted human vitreous and blood samples were collected from three different patient groups: (1) PDR with vitreous haemorrhage, (2) PDR without vitreous haemorrhage, (3) control (macular pucker), all with ethical approval by the Ethics Committee Freiburg (21/1296). Subsequently, large and small EVs were isolated by a combination of tangential flow filtration and size exclusion chromatography. Particle characterization was performed through NTA, MicroBCA and TEM. To identify the different EV subpopulations, EV‐specific biomarker concentrations (e.g., CD9, CD63 and CD81) were evaluated using MSD assays. Furthermore, functional aspects of vitreal EVs were assessed by EV uptake experiments in vitro using HRMVECs (human retinal vascular endothelial cells).

Results : NTA, TEM and EV‐specific biomarker analyses revealed significantly higher levels of small EVs in the vitreous of PDR patients compared to the control group, for which measurements frequently fell below detection thresholds (PDR with vitreous haemorrhage: n = 11–16, PDR: n = 7, control: n = 12–16). Particle and EV‐specific biomarker concentrations were comparable in diabetic patients with and without vitreous haemorrhage. Interestingly, no group‐specific differences in EV marker expression were found in corresponding blood samples. Vitreal EVs were internalized by HRMVECs in vitro within 30 min of exposure.

Summary/Conclusion : The selective elevation of EVs in the vitreous of PDR patients as well as their uptake by retinal endothelial cells underlines their potential impact in the complex pathophysiology of DR. Further studies exploring the molecular cargo and functional implications of vitreal EVs will be essential for a comprehensive understanding of their contribution to disease development.

Funding : MES, FB: Else‐Kröner Fresenius Stiftung

The gut‐retina axis: how extracellular vesicles influence diabetic retinopathy progression

Luana Macedo 1 , Beatriz Felgueiras 1 , Tatiana Burrinha 1 , Federico Herrera 2 , Maria Paula Macedo 1 , Sandra Tenreiro 1* , Rita Machado de Oliveira 1*

1 iNOVA4Health, NOVA Medical School Research, Universidade NOVA de Lisboa, Lisboa, Portugal; 2 Biosystems & Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal Sandra Tenreiro and Rita Machado de Oliveira contributed equally to this study

Introduction : Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of vision impairment. Current therapies targeting late‐stage DR are often ineffective. DR causes blood‐retinal barrier breakdown, disrupting retinal permeability and communication between the retina and other organs. Diabetes affects multiple organs, and understanding their complex communication is key to preventing complications like DR. Extracellular vesicles (EVs) are crucial mediators of inter‐organ communication and diabetes progression. Yet, their impact on the gut‐retina axis in DR is unclear. Retinal organoids (ROs), derived from stem cells, replicate the cellular structure of the human retina, providing a valuable in vitro model. This study aims to explore the molecular link between peripheral metabolic organs and DR progression, with gut EVs as key mediators.

Methods : The EVs released from ROs at 100, 150, and 180 days of differentiation were characterized by nanoparticle tracking analysis (NTA) and western blotting. The effect of gut‐derived EVs (GDEVs) on ROs was also evaluated. Additionally, diet‐induced obese and prediabetic C57BL/6J male mice were used to isolate GDEVs. ROs at 120 days were incubated with normal chow (NCD) and high‐fat diet (HFD) labelled GDEVs for 24 h to assess internalization using immunofluorescence.

Results : ROs release EVs throughout all differentiation stages tested, with the highest concentration observed at 100 days. All NTA profiles display a peak at approximately 100 nm, indicative of the presence of smaller EVs. Additionally, labelled GDEVs from both NCD and HFD conditions are internalized by ROs 24 h after exposure, and they can penetrate its different layers.

Summary/Conclusion : Our data show that ROs not only release but also absorb EVs from peripheral sources, specifically the gut. This in vitro model presents a promising tool to explore EV‐mediated mechanisms in DR. Future studies include the analysis of cargo of EVs from ROs that model DR and studying the effects of GDEVs from a diet‐induced prediabetic mouse model on healthy and DR ROs.

Funding : Fundação para a Ciência e Tecnologia (FCT) / Ministério da Ciência, Tecnologia e Ensino Superior (2023.02154.BD), iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), Associated Laboratory LS4FUTURE (LA/P/0087/2020), 2022.05764.PTDC—INSULT. Funded by the European Union, EVCA Twining Project (Horizon GA No. 101079264).

Bioinformatics‐driven approach for profiling the kidney source of single urine extracellular vesicles

Chin‐Ya Sophie Chiang 1,2 , Uta Erdbruegger 3 , Samantha Upson 3 , Fabian Braun 4,5,6 , Benedetta Bussolati 7 , Cristina Grange 7 , Helen G. Healy 1,2,8 , Andrew J. Kassianos 1,2,8 , Monica Suet Ying Ng 1,2,8

1 Kidney Health Service, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia; 2 Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Queensland, Australia; 3 Division of Nephrology, University of Virginia Health System, Charlottesville, Virginia, USA; 4 3rd Department of Medicine, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 5 Hamburg Center for Kidney Health, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 6 Martin Zeitz Center for Rare Diseases, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 7 Department of Medical Sciences, University of Turin, Turin, Italy; 8 Faculty of Medicine, University of Queensland, Herston, Queensland, Australia

Introduction : The majority of kidney‐specific biomarkers identified in urine extracellular vesicles (uEVs) are derived from bulk multi‐omic (protein/RNA) analyses. However, only a limited number of these kidney‐specific markers will be present on the surface of uEVs and amenable to single uEV immunostaining. We present a bioinformatics‐driven approach to identify kidney‐specific surface markers on uEVs, followed by confirmation using single uEV flow cytometry.

Methods : A bioinformatics pipeline was established to generate a list of kidney‐specific proteins predicted to be on the uEV surface. uEV protein data was extracted from Vesiclepedia (http://www.microvesicles.org; version 5.1) and crosslinked with subcellular location data from the UniProt database. Functional enrichment analyses using the STRING database (https://string‐db.org) were used to identify kidney‐specific uEV proteins localised to plasma and/or intracellular membranes. The resultant list was crosschecked against single‐cell RNA sequencing data from the Kidney Precision Medicine Project (KPMP, doi: 10.48698/mgd0‐gz70) to identify the potential nephron source of proteins. Protein expression in the kidney was confirmed using confocal microscopy. Subsequent marker validation was performed in uEVs isolated by sequential centrifugation followed by size exclusion chromatography and examined as per MISEV2023 guidelines using (i) single uEV flow cytometry and (ii) cryogenic electron microscopy and EV marker positivity.

Results : 5062 uEV proteins were extracted from 95 experiments: 1463 proteins localised to membranes, 1642 proteins localised to the cytoplasm, 859 proteins localised to the nucleus, 714 proteins localised to intracellular organelles and 435 secreted proteins (some proteins identified in multiple compartments). 190 (∼13%) of the membrane proteins were identified as kidney‐specific. Confocal microscopy of kidney tissue confirmed nephron segment location of putative markers. Validation studies identified PODXL (podocytes), LRP2 (proximal tubule), SLC12A1 (loop of Henle) and AQP2 (collecting duct) positive uEVs at mean concentrations of 3.5 × 10 8 EVs/mL urine, 9.0 × 10 8 EVs/mL urine, 5.3 × 10 8 EVs/mL urine and 6.1 × 10 8 EVs/mL urine, respectively.

Summary/Conclusion : Existing bulk uEV proteomics and single‐cell RNA sequencing datasets can be leveraged to identify surface markers for selecting kidney uEVs. This bioinformatics pipeline provides novel diagnostic targets for improved characterization of discrete glomerular versus tubular uEVs for the clinical and scientific evaluation of kidney diseases.

Funding : Queensland Advancing Clinical Research Fellowship, Metro North Health Clinician Research Fellowship.

Paving the way toward a urine‐based extracellular‐like vesicle assay for early diagnosis of kidney disease

Presenter: Giacomo Vacca

Kinetic River Corp., Mountain View, USA

Introduction : Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder leading to the development of fluid‐filled cysts in the collecting duct of the kidney. ADPKD greatly affects the quality and length of life of patients and can lead to renal failure. Most recent estimates show that ADPKD affects approximately 417,000 people in the United States alone. Despite this level of prevalence, current diagnostic methods for ADPKD are invasive, lacking sensitivity, or prohibitively expensive.

Methods : Our preliminary work (Dr Ward's Laboratory) involved proteomic analysis on 2008 proteins expressed on extracellular‐like vesicles (ELVs) using label‐free mass spectroscopy on sucrose/D 2 O gradient‐purified urine‐derived samples from ADPKD patients (PKD1 mutation). Of these, four proteins were identified as candidate quantifiable biomarkers of disease: polycystin‐1 (PC1), polycystin‐2 (PC2), exosomal polycystin‐1 interacting protein (EPCIP), and a fibrocystin homolog (TMEM2). We developed a fluorescent antibody conjugate specific to one of these (PC1); we then used a flow cytometer optimized for nanoparticle detection (Kinetic River) to evaluate the effectiveness of this approach to rapidly detect and quantify the presence of PC1‐positive ELVs in urine. Samples from first‐void urine of 7 PKD patients and 7 normal individuals were prepared (2000 × g , 10 min), stained (PE anti‐PC1 from Dr Ward's Laboratory and membrane dye Aco‐490 from Acoerela), filtered using size‐exclusion centrifuge columns, and analysed on the nanoparticle analyser. Background (sheath fluid), unstained, membrane‐stain‐only, and antibody‐stain‐only controls were collected.

Results : ELVs were identified against background events using a combination of scattering and fluorescence gates. Membrane‐stained, PC1‐expressing urine ELV populations were clearly resolved against non‐expressing ones for sizes down to approximately 150 nm.

Summary/Conclusion : Our results demonstrate that a PC1‐based assay run on an ultrasensitive flow cytometer can detect PKD‐related ELVs in urine and differentiate between normal and ADPKD patients. We plan to build on this approach to incorporate additional ADPKD biomarkers to increase the fidelity of the diagnostic assay.

Funding : This work was made possible in part by US government support under award number 1R43GM155492‐01 from the NIH.

Influence of oral sucrosomal iron versus intravenous iron sucrose administration in dialysis patients on the interaction of monocyte subsets with platelet‐derived extracellular vesicles

Tanja Eichhorn 1 , Marwa Mostageer 1 , Camille Fournier 2 , Martin Wiesholzer 2 , Martin Ursli 2 , Birgit Pfaller 2 , Prisca Neulinger 3 , Viktoria Weber 1 , Ida Aringer 2

1 University for Continuing Education Krems, Krems an der Donau, Austria; 2 University Hospital St. Poelten, Karl Landsteiner University of Health Sciences, St. Pölten, Austria; 3 University Hospital St. Poelten, St. Pölten, Austria

Introduction : Chronic kidney disease (CKD) is associated with anaemia due to decreased renal production of erythropoietin, coupled with abnormal iron metabolism. Intravenous (iv) iron administration, alongside erythropoiesis‐stimulating agents, has become standard care for managing anaemia in CKD patients receiving haemodialysis (HD) as renal replacement therapy. However, the administration of iron (oral vs. iv) in HD patients is still controversially discussed. There is evidence that platelet‐derived extracellular vesicles (pEVs) interacting with monocytes are capable of inducing a shift towards inflammatory CD16+ monocytes. Therefore, we compared a new generation of oral sucrosomal iron with improved gastrointestinal absorption and bioavailability to iv iron sucrose, regarding the effect on pEV‐monocyte aggregate formation and the induction of inflammatory CD16+ monocyte subsets.

Methods : A total of 28 HD patients receiving iv iron and with stable iron status (ferritin ≥ 100 ng/mL and transferrin saturation 20%–50%) were included in this crossover study. After written informed consent, patients were randomized either in group A (oral sucrosomal iron, OLEOvital, Fresenius Kabi) or group B (iv iron, Venofer, CLS Vifor GmbH). After 24 weeks of treatment, the groups were switched (group A receiving iv iron and group B receiving oral iron). Healthy donors ( n = 8) served as controls. Before treatment started and after 12, 24, 36, and 48 weeks, blood samples were collected to characterize the relative abundance of monocyte subsets (classical, CM; intermediate, IM; non‐classical, NCM) according to their differential expression of CD14 and CD16. The interaction of pEVs (defined as Lactadherin+CD41+ events) with monocyte subsets, as well as monocyte ferroportin expression, was assessed by flow cytometry.

Results : The relative abundance of monocyte subsets differed between healthy individuals and haemodialysis patients, with a shift towards IM (CD14++CD16+) and NCM (CD14+CD16++) in the patient group. Aggregates of pEVs and monocytes were higher in haemodialysis patients compared to healthy donors, and pEVs preferentially interacted with IM. Ferroportin expression did not differ between HD patients and controls. The mode of iron supplementation (oral vs. iv) neither influenced monocyte subset distribution nor pEV‐monocyte aggregate formation, nor ferroportin expression.

Summary/Conclusion : We provide evidence that oral sucrosomal iron is non‐inferior to intravenous iron sucrose regarding the induction of inflammatory monocytes.

The protein signature of plasma extracellular vesicles in the ischaemic AKI model

Raquel Silva Neres dos Santos, Dylan Burger, Marcela Sorelli Carneiro Ramos

Cardiovascular Immunology Laboratory, CCNH, Federal University of ABC, Santo André, São Paulo, Brazil; Department of Cellular and Molecular Medicine and School of Pharmaceutical Sciences, University of Ottawa, Ottawa, Ontario, Canada; Cardiovascular Immunology Laboratory, CCNH, Federal University of ABC, Santo André, São Paulo, Brazil

Introduction : Encompassing different pathophysiological mechanisms, including inflammation and oxidative stress, acute kidney injury (AKI) is a global concern. Extracellular vesicles (EV) are also emerging as marker sources or mediators of AKI. Urinary EVs have gained much attention as diagnosis and prognosis biomarkers of kidney diseases. However, less is known about the circulating EV cargo in AKI. Our objective was to analyse the proteome of EV following ischaemic AKI.

Methods : The left renal artery of C57Bl/6j male mice (20–26 g and 6–8 weeks) was clamped for 60 min, followed by 1 day and 8 days of reperfusion (uOttawa Animal Facility Protocol: OHRIe3961‐A1). Control mice only had the LK exposed. EV were isolated from 200 µL of plasma by size exclusion chromatography followed by ultracentrifugation (100,000 × g , for 1 h 30 min at 4 C) and characterized by size (nm) and concentration (particles/mL) on the NTA, and protein concentration (µg/mL) by BCA. Ten micrograms of EV protein was analysed by label‐free LC‐MS/MS. Proteins were identified on MaxQuant and statistically analysed by ANOVA and t‐test on Perseus. Bioinformatics was executed on DAVID 6.8.

Results : The particle size (sham: 180.33 + 17.18; 1 day: 178.50 + 20.08; 8 days: 178.23 + 3.32), the number of particles/mL (sham: 4.66 + 1.24; 1 day: 6.40 + 2.47; 8 days: 6.26 + 1.46), and protein concentration (sham: 0.58 + 0.09; 1 day: 1.07 + 0.62; 8 days: 0.87 + 0.47) were not different among all groups. Two hundred and twenty‐seven proteins were identified, including 8 classic EV proteins, such as transferrin receptor‐1 (Trfc) and HSP70. Gene ontology analysis showed that the significant biological processes were haemostasis and complement activation (adjusted p values 1.16 × 10 −8 and 1.97 × 10 −8 , respectively). KEGG pathway analysis showed that complement and coagulation cascade are the main pathways in all experimental groups (adjusted p values 1.19 × 10 −29 , 3.19 × 10 −31 , and 7.10 × 10 −28 ). The statistical analysis demonstrated that the complement proteins C1q, Masp1, Masp2, Mbl2, C4b, Properdin and the complement receptors 1a/b and Trfc were upregulated in 1‐day and 8‐day groups compared to the control group, the inflammatory response protein CD5L was upregulated only in 8 days (FDR < 0.05).

Summary/Conclusion : These results demonstrated that renal ischaemia and reperfusion injury induced the release of complement‐enriched proteins associated with EVs. These findings might be potential biomarkers for the diagnosis of AKI.

Funding : FAPESP (2021/2022/00153‐0 and 2022/10142‐6) and Kidney Foundation (KHRG‐920575).

Hypertonicity affects the composition of microRNAs in extracellular vesicles derived from renal collecting duct cells

Kristina Enge 1 , Dennis Gerloff 2 , Bayram Edemir 1,3

1 Department of Internal Medicine IV, Martin‐Luther‐University Halle‐Wittenberg Halle, Halle, Sachsen‐Anhalt, Germany; 2 Department of Dermatology and Venereology, Martin‐Luther‐University Halle‐Wittenberg Halle, Halle, Sachsen‐Anhalt, Germany; 3 Faculty of Health‐School of Medicine, Department of Physiology and Pathophysiology, Center of Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany

Introduction : One of the key functions of the kidney is the generation of a concentrated urine. The osmotic gradient that is generated between the renal cortex and inner medulla is the driving force for water reabsorption in the renal collecting duct. The cells in the inner medulla are challenged with a hypertonic environment. The cells have developed mechanisms to adapt to this challenge by different mechanisms that include the induction of a specific gene expression pattern. In the recent study, we analysed how environmental osmolality affects the microRNA expression in extracellular vesicles (EV).

Methods : We used primary cultivated murine inner medullary collecting duct (IMCD) cells. The cells were either cultivated at 300 or 600 mosmol/kg, and the EVs were isolated from cell culture supernatants. Total RNA was isolated using the QIAGEN miRNeasy Kit from the EVs and the cells. The RNA was sent for analysis by the company Novogene. Differentially expressed miRNAs were identified by the DESeq2 algorithm.

Results : We detected 418 out of 944 microRNAs in the EV isolated from IMCD cell supernatant that was cultivated at 300 mosmol/kg and 468 out of 944 microRNAs in cells that were cultivated at 600 mosmol/kg. From these, 340 were detected in both groups. The analysis of differentially expressed microRNAs led to the identification of 112 differentially expressed microRNAs. The mmu‐miR‐9b‐5p showed the highest difference in expression in the 600 mosmol/kg group with a log2‐fold change of 6.6, and mmu‐miR‐134‐5p showed the highest expression level in the 300 mosmol/kg group with a log2‐fold change of 6.8. In the IMCD cells, we were able to detect 768 in the 600 and 771 in the 300 mosmol/kg group. A number of 652 microRNAs were common to both groups. A number of 94 showed differences in the expression level, with mmu‐miR‐1941‐5p showing the highest expression in the 600 and mmu‐miR‐199b‐5p showing the highest expression in the 300 mosmol/kg group.

Summary/Conclusion : Taken together, we could show for the first time that environmental hypertonicity affects the microRNA expression profile in cells and also the composition of micro‐RNAs in EVs is affected. Similar differences in vivo might influence renal or systemic processes.

The surface charge of urinary extracellular vesicles as a possible predictor of kidney disease in diabetes

Melanie Tepus Petrsoric, Maria Pia Savoca, Elisabetta AM Verderio

Nottingham Trent University, Nottingham, UK

Introduction : Urinary extracellular vesicles (uEVs) are an attractive source of biomarkers; however, their physical properties, such as the zeta potential (ZP), also known as surface charge, are less characterized features. As the ZP of a nanoparticle relates to its stability and tendency to aggregate/interact, changes in ZP could mirror the properties of the cells from which they originate and be a sign of a pathological change. We hypothesized that the ZP of uEVs varies in diabetic nephropathy (DN) and diabetes with no CKD, with the potential to stratify DN.

Methods : Cell‐free urines were sourced from Sheffield Kidney Institute and Patras University Hospital, Greece, with provided informed consent: DN ( n = 36), diabetics with no CKD ( n = 16), and healthy (18) cohorts. DN cohorts were stratified based on the rate of eGFR loss: < 2 mL/min/year in the stable stage and > 5 mL/min/year in the progressive stage. Differences in ZP, size distribution, and concentration of small and large uEVs in PBS were analysed by nanoparticle tracking analysis. UEVs were characterized for the presence (Alix, Flotillin‐2, LAMP1, HSP70) or absence (Tomm20) of proteins by immunoblotting, while their morphology was visualized by TEM.

Results : The ZP of large uEVs from the diabetic cohort was significantly less negative compared to the DN and healthy groups. This could be explained by the loss of negatively charged glycocalyx, a feature of diabetes, and may result in a loss of uEVs negative ZP in the diabetic status compared to healthy. As fibrosis progresses in DN, glycocalyx loss may be compensated by heparan sulphate deposition, a feature of tissue fibrosis; therefore, the negative charge of uEVs in DN progression may increase compared to non‐CKD diabetes. Within the DN cohort, although large uEVs from patients with stable conditions had less negative ZP than those from progressive patients, the difference did not reach significance ( p = 0.2). No correlation could be drawn between ZP and the concentration of uEVs.

Summary/Conclusion : In conclusion, we have shown that the ZP of the large uEVs could help differentiate the diabetic from the DN status; however, the approach was not powerful enough to distinguish between DN stability and progression, although a trend difference was seen.

Extracellular vesicles represent a glomerular exit route for autoantigens in membranous nephropathy

Presenter: Sarah Froembling

University Medical Center Hamburg‐Eppendorf, Hamburg, Germany

Introduction : Membranous nephropathy (MN) is an autoimmune disease affecting the kidney podocyte, with characteristic autoantibody formation towards the target foot process antigens PLA2R1 or THSD7A. Glomerular autoantibody/antigen aggregates are found in the urinary space. In recent years additional autoantibodies directed against the intracellular ubiquitin C‐terminal hydrolase L1 (UCH‐L1), predominantly recognizing an oxidatively modified form, have been discovered. It is unknown through which route these autoantibodies are formed. We hypothesize that the podocyte release of oxidatively modified UCH‐L1 through extracellular vesicles (EVs) in the course of MN plays a role.

Methods : Human cultured podocytes and BALB/c mice were exposed to THSD7A autoantibodies for the induction of experimental MN. MN patient urines were collected. Autoantibody/THSD7A and UCH‐L1 abundance and their modification and activity were determined by biochemical readouts in the patient and experimental MN samples. Experimental and patient urinary EV abundance was measured by image stream, and immunoblotting was used to determine the abundance and activity of UCH‐L1 and of proteolytic proteasome subunits within enriched EVs.

Results : Podocyte EV release was increased in cell culture as well as in mice following autoantibody exposure compared to control conditions. Within the released EVs, THSD7A MN antibodies as well as the antigen were present. The EV‐enriched THSD7A antigen was processed. Further, UCH‐L1 protein present in experimental as well as MN patient EVs was structurally altered.

Summary/Conclusion : Extracellular vesicles represent a glomerular exit route for structurally altered antigens. Especially the release of altered UCH‐L1 might be a mechanism for further autoantibody formation.

Urinary extracellular vesicles as non‐invasive biomarkers for early detection of kidney transplant injury

Maja Vodušek 1,2 , Ivana Sedej 1 , Maja Štalekar 3 , Katja Goričar 2 , Marija Holcar 2 , Nika Kojc 4 , Polona Kogovšek 3 , Nika Breznik 2 , Vita Dolžan 2 , Katarina Trebušak Podkrajšek 2,5 , Metka Lenassi 2 , Miha Arnol 1

1 Department of Nephrology, University Medical Centre Ljubljana, Ljubljana, Slovenia; 2 Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 3 Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia; 4 Faculty of Medicine, Institute of Pathology, University of Ljubljana, Ljubljana, Slovenia; 5 Clinical Institute for Special Laboratory Diagnostics, University Children's Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia

Introduction : Monitoring kidney graft function is critical for long‐term survival and improved patient outcomes. Although biopsy remains the gold standard for assessing graft injury, it is invasive and may not fully reflect kidney status. Therefore, reliable, non‐invasive methods are increasingly needed. This study aimed to evaluate whether urinary extracellular vesicles (uEVs) and uEV‐bound DNA (uEV‐DNA) can detect and predict graft injury in surveillance kidney biopsy 1 year after transplantation (Tx).

Methods : In this prospective longitudinal study, 49 adult kidney transplant patients were followed at 1, 3, 6, and 12 months after Tx, with blood and second‐morning urine samples collected at each visit. Surveillance biopsies performed approximately one year after Tx allowed categorization of patients into those with normal histology (NH, n = 35) and those with histologic or molecular indicators of kidney injury (KI, n = 14) based on Banff histology and the molecular microscope diagnostic system (MMDx). uEVs were isolated by size‐exclusion chromatography (SEC) and analysed for their characteristics and DNA parameters by nanoparticle tracking analysis (NTA), genotyping, and digital droplet PCR.

Results : Six months after Tx, the KI group showed increased uEV‐DNA levels compared to the NH group ( p = 0.0220). Using a non‐linear mixed‐effects model, kidney function and uEV‐DNA levels were followed over the first year after Tx. While there were no statistically significant differences between the groups overall, uEV‐DNA levels in the KI group initially decreased until day 147 (approximately 5 months after Tx) ( p = 0.0106) before increasing again in both groups. After 6 months, a multivariate model that included uEV‐DNA, serum creatinine, and log‐eGFR successfully predicted graft injury. The model achieved an AUC of 0.84, a sensitivity of 58%, a specificity of 97%, and a positive and negative predictive value of 88% and 86%, respectively ( p = 0.006).

Summary/Conclusion : The integration of the novel, non‐invasive biomarker uEV‐DNA into a multivariate model together with established clinical parameters enables effective prediction of kidney graft injury 6 months after transplantation and provides insights into the condition of the graft after 1 year. This approach supports non‐invasive monitoring and early detection and has the potential to improve long‐term outcomes for transplant patients.

Funding : Slovenian Research and Innovation Agency (ARIS): Grant J3‐50117.

Shiga toxin in extracellular vesicles is more potent than toxin alone in a mouse model of kidney disease

Markus Wendler, Alexandra Gerogianni, Ida Arvidsson, and Diana Karpman

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden

Introduction : Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is a pathogen that causes severe gastroenteritis and haemolytic uremic syndrome characterized by non‐immune haemolytic anaemia, thrombocytopenia, and acute kidney injury. EHEC is non‐invasive and releases virulence factors in the gut, including Shiga toxin, that are crucial for strain pathogenicity. Our group has shown that blood cells release EVs containing the toxin, which thereby circulates and reaches the kidney, where the toxin is endocytosed. The aim of this study was to investigate if Shiga toxin‐positive EVs exhibit altered potency compared to free toxin.

Methods : HeLa cells were stimulated with Shiga toxin 2 (10 ng/mL) or PBS in 1% Exo‐free foetal bovine serum for 24 h to induce vesicle secretion. EVs were isolated by size exclusion using a 450 nm syringe driven filter unit (PES membrane). The EV suspension was purified with a centrifugal filter at 100 kDa to remove free proteins. EVs were characterized by immunoblotting and nanoparticle tracking. EVs were 100–450 nm (median 140 nm), and were identified by positivity for CD63, TSG101, and Alix. Cytotoxicity of the pure toxin and within vesicles was confirmed by performing a Vero cell assay. BalbC mice were injected iv with Shiga toxin 2 at 126 ng/kg, the same toxin concentration in EVs, EVs alone or PBS.

Results : All mice treated with toxin‐positive EVs ( n = 9) developed clinical signs of disease by day 3, whereas 4 mice treated with free toxin ( n = 6) developed signs of disease by day 6 and the remaining 2 survived until Day 7. Mice treated with EVs alone ( n = 6) or PBS ( n = 5) remained unaffected. Urea levels were highest in mice challenged with toxin‐positive EVs, indicating kidney injury. Kidney pathology and fibrinogen staining are being performed as well as specific staining for vesicles and toxin in target organs.

Summary/Conclusion : Shiga toxin 2‐positive EVs are more potent than free Shiga toxin 2 when delivered iv in a mouse model of EHEC virulence. The results suggest that bacterial toxin packaged within vesicles is capable of reaching target organs, possibly by evading a host response.

Understanding impact of proteinuria on uEV excretion in patients with diabetic kidney disease over the course of a day

Presenter: Samantha Upson

University of Virginia Health System, Virginia, USA

Introduction : Over 500 million people worldwide suffer from diabetes, which is the leading cause of end‐stage kidney disease. Early diagnosis and treatment of Diabetic Kidney Disease (DKD) is essential for improving patient outcomes. Urine can be used as a noninvasive biopsy for DKD. Urinary extracellular vesicles (uEVs) may mirror physiologic and pathologic processes in the kidney. The goal of this study is to examine changes in uEV excretion in proteinuric urine in patients with DKD.

Methods : Ten patients (5 M, 5 F) with DKD Stage 3 (GFR 30‐59 mL/min) collected every urine void separately over a 24‐h period. Five of the patients were microalbuminuric (30–300 mg over 24 h), and five were sub‐nephrotic range proteinuric (300–3000 mg over 24 h). A urine dipstick (Siemens Multistix 10SG) was done to assess pH, specific gravity, osmolarity, etc. The protein and creatinine content were analysed through a Bradford and Jaffe reaction assay. uEVs were isolated using differential centrifugation with a low ionic strength buffer to reduce uromodulin. uEV size and concentration were determined by Nanoparticle Tracking Analysis (PMX‐120, Zetaview). Daily patterns of uEV excretion were analysed using GraphPad Prism with a Friedman test.

Results : Each urine void was collected over the day, with a total of 75 voids processed into separate uEV‐enriched pellets. The patients’ average age was 73, for serum creatinine 1.8 mg/dL and for eGFR 34 ± 7.5 mL/min/1.73m 2 . For an individual void over the day, the max protein/creatinine ratio was 2675 and the min was 2.653 mg/g. The maximum uEV concentration was 1.13 × 10 9 and the minimum was 2.86 × 10 7 (particles/mL of urine). The maximum uEV median size was 232.4, and the minimum was 141.7 (nm). At this point there was no significant difference in uEV excretion or size between different voids.

Summary/Conclusion : A 24‐h collection accounted for variability of protein/creatinine concentrations compared to spot urine. uEV size and concentration varied over the day but were not statistically different. More samples need to be collected. The results will guide us to understand how proteinuria affects uEV collection and isolation. Future analysis will include the study of disease and site‐specific biomarkers in these urine samples.

Funding : ADA 7‐22‐ICTSPM‐19 NIH 5R25DK124918‐05.

Mechanical stress induces phenotypic alterations in extracellular vesicles derived from renal cell carcinoma tissues

Gaia Bianchi 1,2 , Elisa De Lorenzis 3 , Deborah Mattinzoli 4 , Giuseppe Castellano 1,4 , Giovanni Montini 1,2 , Emanuele Montanari 1,3 , Giancarlo Albo 1,3 , Benedetta Bussolati 5 , Federica Collino 1,2

1 Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy; 2 Laboratory of Translational Research in Paediatric Nephro‐Urology, Paediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; 3 Department of Urology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 Renal Research Laboratory, Department of Nephrology, Dialysis and Kidney Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 5 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy

Introduction : Renal cell carcinoma (RCC) accounts for 90% of all kidney malignancies, and nephrectomy represents the mainstay treatment. However, 20%–30% of RCC patients experience recurrence after surgery, with up to 10% reporting a higher risk for distant metastases. Tumour mechanical stresses can induce immune evasion and metastasis formation. Extracellular vesicles (EVs) are relevant players in the tumour microenvironment, with roles in angiogenesis, immune modulation, metastases, and drug resistance. Their composition changes upon external stimuli and stresses, including those occurring during surgical intervention for tumour exportation. Our study aims at defining the phenotypic changes occurring in EVs released by the tumour following a mechanical stress and their potential effects on the surrounding microenvironment during tumour progression.

Methods : Twenty‐five RCC patients with tumour grades 2, 3 and 4 (pT1a‐pT3a stage) were recruited after signing an informed consent. The cellular composition of tumour tissues was analysed through spatial proteomics by MICS (MACSima Imaging Cycling Staining), and the presence of EV‐like particles within the tissue was assessed by transmission electron microscopy (TEM). EVs were extracted from the tumour using an optimized enzymatic protocol followed by either simple dropping (matrix‐EVs) or application of a mechanical stress (compression and shears) to the tissue (stress matrix‐EVs). Particle number was determined by nanoparticle tracking analysis, and the expression of 37 surface proteins was analysed by flow cytometry using the MACSPlex kit. Sequencing of matrix‐ and stress matrix‐EVs extracted RNAs was performed.

Results : Application of a mechanical stress during the extraction procedure yielded an increased number of released particles. The three tetraspanins were equally expressed on the surface of EVs obtained through either isolation method. Stress matrix‐EVs presented an augmented expression of cellular markers characteristic of the tissue‐resident populations detected through MICS (i.e., immune‐related, inflammatory, and cell adhesion molecules). The epithelial CD326 was the only significantly decreased marker in the comparison with matrix‐EVs.

Summary/Conclusion : The application of mechanical stress affected the number of vesicles released from the tissues and the expression of immune‐ and inflammatory‐related markers on their surface. Stress conditions by changing the EVs phenotype can account for their possible role in tumour progression and immune escape.

Subtopic Metabolism 1

Extracellular vesicles from adiponectin knockout mice have an altered proteome and mediate direct adverse functional metabolic effects in target cells

Hyekyoung Sung 1 , Sungji Cho 1 , Khang Nguyen 1 , Yue Qu 1 , Kacey Prentice 2 and Gary Sweeney 1

1 York University, Toronto, Toronto; 2 University of Toronto, Toronto, Toronto Hyekyoung Sung and Sungji Cho are co‐first authors .

Introduction : Adiponectin is an important endogenous metabolic regulator, promoting insulin sensitivity and cardiometabolic health. While adiponectin was identified as an extracellular vesicle (EV) cargo and as a modulator of EV biogenesis, its role in shaping EV proteomes and their subsequent metabolic effects remains underexplored. Here, we first investigated how the genetic deletion of adiponectin in mice alters circulating EV proteome profiles and then examined the functional impact of EV on insulin sensitivity and glucose metabolism under high‐fat diet (HFD)‐induced metabolic stress.

Methods : Plasma EVs were isolated from wild‐type (WT) and adiponectin knockout (AdKO) mice ± HFD for 4 weeks. A glucose tolerance test (GTT) was performed after 3 weeks of HFD feeding. EVs were isolated by size exclusion chromatography, and characterization included analysis of EV number, size and protein cargo.

Results : Plasma EV size and number showed no significant differences between WT and AdKO mice; however, proteomic analysis of EVs revealed significant differences in the EV cargo between WT and AdKO mice. Pathway analyses indicated EVs from WT mice were enriched in the proteins associated with acute inflammatory responses compared to EVs from AdKO mice. In WT HFD‐fed mice, EV proteome enriched pathways related to glycolysis, the TCA cycle and the pentose phosphate pathway. Pathway analysis of proteome profiles in AdKO HFD‐fed mice EV identified a reduction in proteins related to catabolic processes, including glycolysis and gluconeogenesis, indicating impaired glucose handling. Using skeletal muscle cells expressing an Akt biosensor as a target, we found that the direct functional effect of EV purified from AdKO, but not WT, mouse plasma was to induce insulin resistance. Only AdKO EV reduced glucose‐stimulated insulin secretion in pancreatic beta cells.

Summary/Conclusion : Lack of adiponectin in mice significantly alters the circulating EV proteome, impacting the functional metabolic effects on recipient cells. In AdKO mice, these changes promote insulin resistance in skeletal muscle and impair insulin secretion in beta cells. These results highlight the potential of targeting adiponectin and its impact on EV composition in developing new therapeutic strategies for the treatment of cardiometabolic diseases.

Adipocyte‐derived extracellular vesicles accelerate obesity‐associated comorbidities

Presenter: Xilal Y. Rima

The Ohio State University, Columbus, Ohio, USA

Introduction : The prevalence of obesity is continuously rising, with half the global population predicted to be overweight or obese by 2035. Insulin resistance (IR), atherosclerosis, and metabolic‐dysfunction‐associated steatohepatitis (MASH) have paralleled the rising trend in obesity. The adipocyte, the dominant cell in adipose tissue (AT), performs innate and adaptive immune functions by releasing adipokines and contributing to antigen presentation. However, the mode by which the adipocyte affects distant organs remains unclear. Extracellular vesicles (EVs) are key mediators of interorgan communication, which harbour microRNAs (miRNAs). 65% of circulating EV‐encapsulated miRNAs are derived from the adipocyte. Therefore, we hypothesize that adipocyte‐derived EVs (AdEVs) affect obesity‐associated comorbidities.

Methods : AdEVs were collected from the visceral AT (VAT) of lean and obese mice and lean and obese bariatric patients who voluntarily participated under informed consent. AdEVs were characterized using transmission electron microscopy, tunable resistive pulse sensing, western blot, and single‐EV phenotyping. Mice modelling the metabolic syndrome, middle‐aged Ldlr − / − mice given a western high‐fat diet, were retro‐orbitally injected with AdEVs from lean and obese mice. Insulin tolerance tests, en‐face aortae analyses, and histology were conducted to measure IR, atherosclerotic extent, and MASH. In vitro experiments were performed to elucidate human disease. Lastly, miRNA sequencing was executed to uncover candidates for disease progression.

Results : Mice administered AdEVs from obese mice had increased IR ( p < 0.05), accelerated atherosclerosis by ∼50% ( p < 0.0001), and higher MASH activity scores ( p < 0.01) than mice delivered AdEVs from lean mice and saline injections, which were comparable. Human AdEVs were internalized by macrophages and trafficked intracellularly into endosomal/lysosomal compartments. AdEVs from obese patients induced greater pro‐inflammatory responses in recipient macrophages ( p < 0.01) compared to AdEVs from lean patients and saline injections. AdEVs from lean and obese patients differentially packaged miRNAs that may have direct effects on the obesity‐associated comorbidities accelerated by AdEVs from obese AT.

Summary/Conclusion : Our results offer insight into the role of adipocyte interorgan signalling in the pathophysiology of IR, atherosclerosis, and MASH, which are major complications of obesity. Furthermore, we have identified therapeutic targets that may alleviate these diseases and benefit an increasing patient population.

MSTN mutant pig serum exosomes reduce fat deposition in obese mice by inducing browning of white fat

Presenter: Sitong Lyu

Yanbian University, Yanji, People's Republic of China

Introduction : Myostatin (MSTN) is an inhibitor of skeletal muscle growth and development. MSTN mutant pigs mainly exhibit a phenotype of increased muscle and decreased fat deposition, which has great potential in the treatment of obesity. Exosomes are nanovesicles with characteristics of donor cells and are important mediators of intercellular communication. It is worth investigating whether MSTN‐mutated porcine exosomes (MSTN‐Exos) have similar metabolic benefits on obesity.

Methods : In this study, obese mice were established by high‐fat diet (HFD). Serum exosomes were isolated from MSTN mutant pigs and injected into adipose tissue of mice. The effects of serum exosomes on adipose tissue morphology and biochemical indexes were observed. At the same time, the 3T3‐L1 cell line was used to treat adipose differentiation with MSTN‐Exos. The effect of MSTN‐Exos on adipose differentiation was evaluated by measuring cell differentiation degree and triglyceride content. In addition, we performed biological sequencing analysis of MSTN‐Exos to reveal its underlying molecular mechanism.

Results : The results showed that after local injection of MSTN‐Exos into adipose tissue, fat deposition was significantly reduced in HFD mice. Meanwhile, an obvious browning trend was observed in white adipose tissue, which was manifested by significantly increased expression of brown adipose‐related genes PGC‐1α and UCP1. In vitro experiments showed that MSTN‐Exos significantly decreased the adipose differentiation ability of 3T3‐L1 cells, which was manifested as decreased triglyceride accumulation and decreased expression of CEBP‐α and PPAR‐α genes related to adipose differentiation. Through biological sequencing of MSTN‐Exos, the mechanism of MSTN‐Exos promoting browning of white fat was analysed.

Summary/Conclusion : Our study shows that MSTN‐Exos significantly reduces fat deposition in HFD mice by promoting browning of white fat, which has the potential to promote fat metabolism and improve obesity. The detailed elucidation of the reaction molecules and their mechanisms of action will provide new insights into the fight against obesity and related diseases.

Metabolomic profiling of size‐exclusion chromatography‐isolated plasma‐derived extracellular vesicles from healthy donors

Estelle Maret 1,2 , Sylvain Le Gludic 3 , Caroline Samer 1,2 , Aurélien Thomas 3 , Youssef Daali 1,2

1 Division of Clinical Pharmacology and Toxicology, Geneva University Hospital, Geneva, Switzerland; 2 Department of Anaesthesiology, Pharmacology, Intensive Care and Emergency Medicine, Faculty of Medicine UNIGE, Geneva, Switzerland; 3 University Center of Legal Medicine, Unit of Forensic Toxicology and Chemistry, Lausanne, Switzerland

Introduction : In the past few years, plasma‐derived extracellular vesicles (EVs) have emerged as a promising source of biomarkers for various pathological conditions, particularly in cancer research (Witwer et al.). However, limited research has been conducted on EVs from healthy individuals, despite their potential value in clinical studies. This study aims to characterize the metabolome of EVs from healthy donors, addressing the challenge of lower EV secretion rates in non‐pathological states and exploring their potential as a source of biomarkers for future research in drug exposure prediction.

Methods : Six mL of whole blood from 42 healthy donors was processed within 2 h via 3 centrifugation steps. Plasmas from 6 donors were pooled together, reaching a final volume of 10 mL. EVs were isolated using size exclusion chromatography (QEV10, Izon), either from 10 mL plasma loading ( n = 3) or from 5 mL plasma loading in two separated columns ( n = 4). Particles were eluted with 5 mL PBS, and fractions were characterized for total protein concentration (Micro BCA), EVs (LAMP2), and lipoproteins (APOC3 and APOB100) using commercially available ELISA kits. Untargeted metabolomics analysis was performed using LC‐HRMS.

Results : Particles elution profiles shifted by one fraction when 10 mL plasma was loaded compared to 5 mL. The 5 mL loading showed significantly reduced APOC3 contamination in fraction 8 ( p = 0.002). APOB100 and APOC3 showed an elution ratio at 5%–7% in fractions 8–9 (5 mL) or 9–10 (10 mL), while LAMP2 showed 15%–21% elution. Untargeted metabolomics of these fractions revealed particle‐enriched features when compared to plasma. Fraction clustering was found to be independent of the volume loaded onto the column, suggesting a different metabolome across early and late fractions. Several features showed positive correlation with LAMP2, indicating EV‐associated metabolites. LAMP2‐correlated features will be further identified.

Summary/Conclusion : This research highlighted features potentially associated with or encapsulated within EVs, thus providing a methodological framework for exploring the subtle yet significant metabolic signatures carried by EVs which could be used for clinical studies involving healthy cohorts.

Funding : This research is funded by the Swiss National Science Foundation.

Tumour extracellular vesicles and particles (EVPs) induce aberrant lipid metabolism

Presenter: Yue Yao

The University of Hong Kong, Hong Kong, People's Republic of China

Introduction : Tumour extracellular vesicles and particles (EVPs) serve as vital cargos for intercellular communication within the tumour microenvironment, and can be widely involved in cell metabolism, but EVPs remain poorly understood on lipid metabolism. Chaperone‐mediated autophagy (CMA) is a type of autophagy that can target lipid droplets, and this study aims to explore the regulatory mechanism of EVPs involved in hepatic cell metabolism through chaperone‐mediated autophagy mediated lipolysis.

Methods : sEVs and exomeres derived from human cancer HLE and MHCCLM3 cell lines were collected. Mice were injected with sEVs or exomeres via the tail vein. The livers of mice educated with EVPs or PBS control were analysed by lipidomic mass spectrometry. The components of EVPs from HLE and MHCCLM3 cell lines were detected by protein mass spectrometry.

Results : We found a reduction in the number and size of hepatic lipid droplets following injection with both sEVs and exomeres. Lipidomics data derived from livers of PBS‐injected control and EVPs mice revealed that glycerophospholipid metabolism and fatty acid metabolism were significantly affected. The internalization of the EVPs in the recipient cells and EVPs‐mediated exogenous Hsc70 transfer into cytoplasm were examined by confocal microscopy and western blotting. Knockdown of Hsc70 resulted in a decrease in the lysosomal stability of LAMP‐2A, reduced CMA activity and lipid accumulation in hepatocytes.

Summary/Conclusion : These results indicate that the EVPs alter hepatic lipid metabolism in vivo and in vitro. EVPs transported Hsc70, leading to increased lipid droplet catabolism and promoting CMA‐mediated lipolysis in hepatic cells.

Funding : National Natural Science Foundation of China (grant numbers 82300921, 81872340, 82072626, and 81902431); Hong Kong Scholars Program (grant number XJ2020012 and 2020–036); Marshal Initiative Funding of Harbin Medical University (grant numbers HMUMIF‐22008); Natural Science Foundation of Heilongjiang Province (grant number LH2023H043)

Lysosomal GBA1 alterations affect extracellular vesicle size and frequency in Gaucher disease

Presenter: Reena Kartha

University of Minnesota, Minneapolis, Minnesota, USA

Introduction : Lysosomal diseases occur due to genetic defects in enzymes or cofactors required to hydrolyse cellular waste products. The ensuing lysosomal dysfunction leads to accumulation of toxic substrates within the cells, which can trigger a multitude of cellular anomalies, including oxidative stress, mitochondrial dysfunction, inflammation, and so forth. Gaucher disease (GD) is one such disease caused by defects in the glucocerebrosidase enzyme encoded by the GBA1 gene. Over 300 different mutations have been identified, each affecting the enzyme activity to varying extents. This leads to glycosphingolipid accumulation triggering multisystem symptoms including anaemia, thrombocytopenia, hepatosplenomegaly and bone issues. Notably, mutations in the GBA1 gene have emerged as a major risk factor for Parkinson's disease, although the exact mechanism underlying this is not clear. Lysosomes are crucial in extracellular vesicle (EV) biogenesis and can influence its cargo by participating in the regulation of cell sorting mechanisms. Alterations in lysosomal function, especially in the context of diseases, can therefore directly impact the characteristics of EVs, highlighting their potential as biomarkers for these diseases.

Methods : Patient‐derived GD fibroblasts harbouring either mild (N370S/84GG) or severe (L444P/S364T) variants were compared against a normal human fibroblast line (NHDF). These were characterized as mild or severe based on the percentage of glucocerebrosidase enzyme activity. EVs were isolated from three independent passages of each cell line using the ultracentrifugation method. The collected vesicles were analysed using the ZetaView next generation nanoparticle tracking analyser, to determine EV concentration and size distribution.

Results : We observed significant differences in EV sizes between normal and GD cells. Specifically, both GD cells showed lower frequency of EV between 100 and 200 nm size compared to normal cells. However, only milder GD cells showed higher concentrations of EVs above 200 nm. Notably, EV size did not vary between different passages of normal cell lines that served as a control.

Summary/Conclusion : Our initial findings indicate EV size and potentially EV cargo to be altered by lysosomal dysfunction and to be dependent on the severity of the defect. We are further investigating the EV biogenesis pathway genes in cells and examining the alterations in the EV cargo.

Funding : Department research funds.

Study of extracellular vesicles (EVs) biology in 3D‐culture hepatic models of liver disease

Presenter: Silvia López‐Sarrió

CIC bioGUNE, Derio, Spain

Introduction : Metabolic syndrome (MetS) is characterized by a cluster of conditions which increase the risk of developing metabolic‐dysfunction‐associated steatotic liver disease (MASLD). MASLD is marked by lipid accumulation in hepatocytes and can progress to cirrhosis. Extracellular vesicles (EVs) have gained attention as they transport lipids, proteins, and RNAs that affect liver inflammation and fat metabolism, with potential as biomarkers for MASLD. However, 2D hepatocyte cultures lose liver‐specific functions, limiting their relevance for liver studies. This work aims to establish a 3D hepatocyte culture model for studying EVs and lipid accumulation in MASLD.

Methods : Primary hepatocytes were isolated from murine models with albumin‐expressing GFP‐labelled hepatocytes and cultured as 3D spheroids. Spheroid formation was monitored via microscopy, hepatocyte‐specific gene expression was tested by qPCR, and viability was assessed using flow cytometry and histological analysis. EVs were isolated from culture media through ultracentrifugation, with characterization by western blotting, nanoparticle tracking analysis, and cryo‐electron microscopy, and analysed by flow cytometry. In addition, murine models of Rab7 and Rab11 knockout in hepatocytes under a high‐fat diet were used to investigate vesicular trafficking roles in lipid metabolism.

Results : Hepatocyte spheroids formed consistently across various cell densities, with a single spheroid per well. Compared to 2D cultures, 3D spheroids displayed gene expression patterns more similar to fresh hepatocytes. EVs isolated from 3D cultures contained characteristic markers (CD81, Flot1) and showed a size profile consistent with small and medium EVs. Time‐course tracking indicated a gradual reduction in EV concentration. In Rab7 knockout spheroids, lipid accumulation was reduced, as evidenced by perilipin2 (AP125) and BODIPY 493/503 staining and confocal microscopy, suggesting Rab7's role in lipid droplet formation.

Summary/Conclusion : Our 3D hepatocyte model reliably supports liver‐specific functionality, making it suitable for studying EV dynamics and lipid metabolism in MASLD. These findings highlight Rab7's role in lipid storage and provide a platform for further exploring EV‐mediated intercellular communication in liver pathology related to MetS. Future directions include exploring the role of Rab11 under these conditions and developing more complex liver models to investigate cell‐cell interactions relevant to human MASLD pathology.

Funding : Science and Innovation Spanish Ministry (Reference PRE2022‐103544), MCIN/AEI/10.13039/501100011033 and FSE, and Halt‐RONIN (No. 101095679).

Extracellular vesicles: novel biomarkers of endocrine disrupting chemical exposure in metabolic disfunction associated steatotic liver disease (MASLD)

Alison Rapin, Stéphanie Brisset, Valentine Brouard, Laura Morin, Dominique Lagadic‐Gossmann, Normand Podechard, Corinne Martin‐Chouly

University of Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)—UMR_S1085, Rennes, France

Introduction : Metabolic dysfunction‐associated steatotic liver diseases (MASLD), beginning with steatosis (lipid accumulation in hepatocytes) and potentially advancing to steatohepatitis, represent the most common chronic liver disease worldwide, affecting over a quarter of the global population. Environmental pollutants, particularly endocrine disruptors (EDs), are now acknowledged as risk factors. EDs are known to promote metabolic disorders such as type 2 diabetes and obesity (closely linked to MASLD) and may directly affect liver health. However, hepatic dysfunctions induced by EDs exposure in an MASLD context are not fully understood. Extracellular vesicles (EVs), which play roles in MASLD pathophysiology and may be modulated by EDs, are of interest here. We aim to investigate EVs biological roles in EDs impact on MASLD and explore their potential as biomarkers of exposure and effects due to their accessibility in biological fluids.

Methods : To examine steatosis development and EVs production, we utilized HepaRG cells with or without lipid supplementation to model normal and steatotic hepatocyte states, respectively. These cells were then exposed to DEHP (Di(2‐ethylhexyl) phthalate), our EDs of interest. Steatosis was quantified using AdipoRed assays, while EVs isolation, production and characterization were carried out via ultracentrifugation, NTA analysis and western blot analysis. To evaluate the impact of EVs on MASLD progression, primary human macrophages were exposed to HepaRG‐derived EVs. Markers of inflammation, indicative of progression to steatohepatitis, were measured in macrophages by ELISA and RT‐qPCR.

Results : Our initial findings indicate that DEHP exposure does not significantly influence lipid accumulation in HepaRG under either normal or steatotic conditions. However, DEHP markedly increases EVs production. Additionally, macrophages exposed to HepaRG‐derived EVs exhibit elevated secretion of pro‐inflammatory cytokines IL‐6 and TNFα, indicating a shift toward a pro‐inflammatory state.

Summary/Conclusion : These preliminary results show that EVs may contribute to MASLD by promoting liver inflammation. Further characterization of EVs content is needed to clarify underlying mechanisms and assess their potential as biomarkers within this context.

Funding : OBERON European project, under Grant Agreement no 825712; ENDOMIX European project, under Grant Agreement 101136566 Université de Rennes.

Effects of aerobic‐strength training on glucose metabolism, cognitive functions, and proteome of circulating extracellular vesicles in patients with Parkinson's disease

Marček Malenovská K. 1,2 , Alchus Laiferová N. 1 , Slobodová L 1 , Amiri A. 1 , Konečná M. 3 , Straka I. 2 , Kurdiová T. 1 , Mytiai O. 1,2 , Novotová M. 1 , Litvákova V. 1 , Pustka V. 4 , Klepochová R. 5 , Košutzká Z. 3 , Minár M. 3 , Blanco A. 6 , Krššák M. 5 , Mc Gee M. 7,6 , Zbyněk Zdráhal 4 , Valkovič P. 3 , Ukropec J. 1 , Ukropcová B . 1,2

1 Institute of Experimental Endocrinology and Center of Physical Activity, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia; 2 Faculty of Medicine Comenius University, 2nd Department of Neurology, University Hospital Bratislava, Bratislava, Slovakia; 3 Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia; 4 High‐Field MR Centre, Department of Biomedical Imaging and Image‐Guided Therapy, Medical University of Vienna, Vienna, Austria; 5 Proteomic Core Facility, Central European Institute of Technology, Brno, Czech Republic; 6 School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland; 7 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland

Introduction : Type 2 diabetes contributes to the progression of Parkinson's disease (PD), while exercise improves glucose metabolism, cognitive performance and clinical state in patients with PD. The aim of this work was to assess the effects of regular exercise on glucose metabolism, cognitive functions and dynamics of concentration and protein composition of circulating extracellular vesicles (EVs) in patients with mild to moderate PD.

Methods : Patients (M/F 14/7) aged 63.8 ± 8.7 years; H&Y score I‐III, BMI 27.7 ± 4.9 kg m −2 , completed a 4‐month supervised aerobic‐strength training (3 × 1 h/week). Clinical status was assessed by MDS‐UPDRS in ON/OFF states. Cognitive performance (ACE‐R, Memtrax, CogState), body composition (bioelectrical impedance; MRI), energy metabolism (indirect calorimetry), and insulin sensitivity (euglycemic hyperinsulinemic clamp) were assessed before and after the 4‐month intervention. EVs were analysed by flow cytometry (Beckman Coulter CytoFLEX‐S) and NTA (NanoSight NS300). Proteomic analysis (mass spectrometry) of EVs isolated from serum (SEC, qEV columns, Izon) before, immediately after and 60 min after acute aerobic exercise (40 min cycling, 60%–70% VO 2 max) was performed both before and after a 4‐month training intervention. EVs were characterized by TEM and immunoblotting.

Results : Four months of aerobic‐strength training improved body composition (reduced: BMI, p = 0.004; body fat, p = 0.08; visceral fat, p = 0.022), insulin sensitivity ( p = 0.04), muscle strength ( p < 0.05) and cognitive performance (psychomotor function, p = 0.04; visual learning, p = 0.06 and memory, p = 0.01). Clinical status, including motor and non‐motor symptoms of PD (MDS‐UPDRS, p < 0.05) was improved in both ON and OFF states. Four‐month training increased basal concentration of serum EVs (NTA; p = 0.001) and modulated the concentration of serum EVs during recovery 60 min after acute exercise (FACS; p = 0.001). Out of 2550 proteins, 82 were regulated by 4‐month training, with 42 proteins being upregulated and 40 downregulated ( p < 0.05). Regulation of specific EVs proteins by a 4‐month training was observed 60 min post‐acute exercise recovery.

Summary/Conclusion : Regular exercise improved clinical state, cognitive and motor functions and glucose metabolism in patients with mild to moderate Parkinson's disease. Both acute and regular exercise modulated the concentration and protein profile of serum‐derived extracellular vesicles, indicating their potential role in the exercise‐induced adaptive response and related health benefits.

Funding : APVV 20‐0466, VEGA‐2/0076/22, ADDIT‐CE Horizon Europe 101087124, FWF KLI1122 (Austrian Science Fund), APVV 23‐0604, VEGA‐2/0161/24.

Subtopic Organ systems 1

Stimulatory effects of a subpopulation of bovine milk extracellular vesicles with collagen‐binding properties in 2D and 3D models of osteogenesis

Presenter: Peng Wang

Radboud University Medical Center, Nijmegen, The Netherlands

Introduction : Bovine milk‐derived EVs (BMEVs) have gained interest in recent years for the treatment of bone loss. However, little is known about BMEVs homogeneity and the functionality of subpopulations. Emerging evidence indicates that EVs inherently interact with the meshwork of the extracellular matrix (ECM) through surface molecules. In this case, we utilized the collagen‐binding property of BMEVs for selection and to explore the effects of this ECM‐binding subpopulation in 2D and 3D models of osteogenesis.

Methods : BMEVs were isolated by ultracentrifugation and size exclusion chromatography, and their effects on osteogenesis were evaluated using osteogenic differentiation of human bone marrow stem cells (BMSCs) cultured on 2D plastic plates and 3D human bone organ culture models. Collagen‐binding of EVs was observed using IVIS optical imaging. The cargo protein content of BMEVs and collagen‐unbound BMEVs was determined through LC‐MS/MS proteomics and validated by western blotting. A subpopulation of BMEVs was obtained by immunomagnetic separation, and its effect on human BMSCs osteogenic differentiation on 2D collagen plates and 3D collagen hydrogel constructs was investigated through calcium assay and alizarin red staining.

Results : BMEVs increased type I collagen expression, alkaline phosphatase level and calcium mineralization of BMSCs and stimulated new bone formation in the defect site of human bone organ cultures as determined by fluorochromes calcein green and alizarin red. Approximately 5% of BMEVs adhered to the collagen plate, and those strongly promoted human BMSCs mineralization. However, the unbound BMEVs showed no enhancing effect on mineralization by human BMSCs when cultured on a 2D collagen plate or in 3D collagen hydrogel culture. Furthermore, removal of the collagen‐binding BMEVs led to a reduction of CD9 and Annexin V protein levels in BMEVs, and Annexin V‐depleted EVs diminished BMEV‐induced mineralization by human BMSCs.

Summary/Conclusion : Utilizing the ECM‐binding property of EVs enables selection of a subpopulation of BMEVs that is positive for annexin V and has robust stimulatory effects on osteogenesis.

The role of galectins in extracellular vesicle‐mediated tissue communication in osteoarthritis

Christine Strauss 1,2 , Juergen Alphonsus 1,3 , Silvia Hayer 2,4 , Sara Savic‐Ivanovic 1 , Maximilian Haertinger 5 , Herbert Kaltner 6 , Reinhard Windhager 3 , Stefan Toegel 1,2

1 Karl Chiari Lab for Orthopaedic Biology, Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Vienna, Austria; 2 Ludwig Boltzmann Institute for Arthritis and Rehabilitation, Vienna, Austria; 3 Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Austria; 4 Department of Internal Medicine III, Division of Rheumatology, Medical University of Vienna, Austria; 5 Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Vienna, Austria; 6 Chair of Biochemistry and Chemistry, LMU Munich, Munich, Germany

Abstract unavailable

Exosomes from myostatin‐mutant pig serum promote C2C12 cell differentiation and skeletal muscle growth

Presenter: Zhouyan Li

Yanbian University, Yanji, People's Republic of China

Introduction : Blood, a circulating fluid in vertebrates, contains exosomes that may influence the muscle microenvironment. Skeletal muscle, the largest organ in the body, secretes exosomes rich in muscle‐specific microRNAs into the bloodstream. Myostatin, a member of the transforming growth factor‐β superfamily, plays a critical role in regulating muscle growth. Mutations in the myostatin gene, whether natural or induced, can enhance muscle quality and produce a double‐muscle phenotype. Our previous research successfully generated myostatin‐mutant pigs.

Methods : In this study, we isolated exosomes from the serum of myostatin‐mutant pigs (MSTN Exo) using ultracentrifugation for in vitro and in vivo experiments to elucidate their effects on skeletal muscle growth. For the in vitro experiments, we added MSTN Exo to the C2C12 differentiation medium to evaluate cell differentiation, myotube formation, and the expression of myogenic markers. In vivo, we administered repeated MSTN Exo injections into the skeletal muscle of 8‐week‐old C57BL/6 mice. We assessed the effects 4 weeks post‐injection by measuring gene and protein expression related to muscle cross‐sectional area and myogenesis. The miRNA expression profiles of MSTN Exo were then determined using second‐generation sequencing.

Results : Our findings indicate that the addition of 25 µg of MSTN Exo to the C2C12 differentiation medium enhances myoblast differentiation and fusion index while increasing the expression of myogenin (myoG) and myosin heavy chain (MyHC) proteins and genes. In vivo studies demonstrated that after intramuscular injection, MSTN Exo is absorbed by muscle fibres, leading to increased muscle mass, greater single muscle fibre diameter, and enhanced muscle cross‐sectional area, along with increased expression of genes and proteins related to myogenesis. Furthermore, the miRNA expression profile in MSTN Exo revealed a high expression of miRNAs associated with myoblast differentiation.

Summary/Conclusion : Our findings from in vitro, in vivo, and bioinformatics analyses indicate that MSTN Exo plays a significant role in promoting skeletal muscle growth.

Exploring the effect of extracellular vesicles derived from human dental pulp stem cells in dexamethasone‐induced muscle atrophy

Seong Eun Kim, Hyekyoung Hannah You, Young Kim

Chonnam National University, Gwangju, Republic of Korea

Introduction : Extracellular vesicles (EVs) are nano‐sized functional molecules such as nucleic acids, proteins, and lipids, which reflect the characteristics of their parent cells. Dental stem cells are considered promising candidates for tissue regeneration due to their non‐invasive sources and similarities to the regeneration potential of mesenchymal stem cells. Muscle atrophy, characterized by a loss of skeletal muscle, can result from pathogenic conditions such as insulin resistance, ageing, and cancer. In this study, we aimed to explore the therapeutic potential of EVs derived from human dental pulp stem cells (hDPSC‐EVs) in muscle atrophy.

Methods : hDPSCs were obtained from extracted supernumerary teeth in healthy children. hDPSC‐EVs were isolated using the TFF system and the exosome isolation kit. The effects of hDPSC‐EVs on myogenesis were analysed by assessing proliferation, migration, and the expression of myogenic markers such as myogenin and MHC in C2C12 myoblast cells by WST‐1, scratch wound healing assay, and western blot. The therapeutic potential and related signalling pathway of hDPSC‐EVs were evaluated in dexamethasone (DEX)‐induced muscle atrophy in C2C12 myotubes by qRT‐PCR and western blot assay. In a mouse model of DEX‐induced muscle atrophy, the expression of atrogenes and histological analysis were evaluated. Additionally, proteomics analysis of hDPSC‐EVs was conducted to identify candidate proteins related to alleviating muscle atrophy.

Results : hDPSC‐EVs exhibited increased cell viability, wound closure, and expression of myogenin and MHC in C2C12 myoblasts. Treatment with hDPSC‐EVs significantly reduced the expression of atrogenes (Atrogin‐1 and MuRF‐1) via the mTOR/Akt/FoxO3 signalling pathway in DEX‐induced C2C12 myotubes. In a mouse model of DEX‐induced muscle atrophy, hDPSC‐EVs exhibited decreased expression of atrogenes and recovered muscle fibres in tissue analysis. In proteomics analysis of hDPSC‐EVs, IGFBP5, FTH1, and SEMA3A, which may contribute to the biological effects in muscle atrophy, were identified.

Summary/Conclusion : Our findings showed that hDPSC‐EVs promote myogenesis and alleviate DEX‐induced muscle atrophy via the mTOR/Akt/FoxO3 signalling pathway. These results suggest that hDPSC‐EVs could potentially serve as a new therapeutic strategy for treating muscle‐related disease and improving muscle health.

Plasma EVs‐derived complement proteins as biomarkers of sarcopenia progression and functional decline in the Korean Frailty and Aging Cohort Study

Ji Yeon Kim 1 , Tae‐Hwan Gil 1 , Do Hyun Han 2 , Sung Hye Kong 3 , Ok Hee Jeon 1

1 Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea; 2 Department of Transdisciplinary Medicine, Seoul National University Hospital, Seoul, Republic of Korea; 3 Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea

Introduction : With the rapid aeging of society, early identification of individuals at risk for sarcopenia is crucial. Among its diagnostic criteria—muscle mass, strength, and physical performance—muscle performance has emerged as an indicator of sarcopenia progression and functional decline. This study aimed to identify plasma extracellular vesicle (EV)‐derived protein biomarkers associated with sarcopenia progression, focusing on muscle performance. By analysing plasma‐derived EV proteomes, we sought to uncover biomarkers and molecular mechanisms linked to functional decline in sarcopenia.

Methods : Proteomic analysis was conducted on EVs isolated from plasma in the Korean Frailty and Ageing Cohort Study (KFACS), a prospective community‐based cohort of older adults aged 70–86 years. Ninety participants were included for both cross‐sectional and 2‐year longitudinal analyses of sarcopenia‐related parameters. EVs were isolated using density gradient ultracentrifugation, and their protein profiles were analysed via LC‐MS. Multivariate regression analyses were performed, adjusting for sex, age, BMI, and comorbidities. Participants were grouped based on 2‐year changes in sarcopenia‐related factors, following the Asian Working Group for Sarcopenia (AWGS) thresholds. Individuals who stayed robust were compared to those with declining muscle performance over 2 years and those non‐sarcopenic at baseline who progressed to sarcopenia.

Results : At baseline, the mean age of the participants was 78 years, and 47% were female. Longitudinal analyses revealed that complement activation pathways were significantly associated with muscle performance changes but not muscle mass or strength. Among the complement‐related proteins identified, C1QA, C2, C4B, and MBL2 exhibited distinct patterns. C4B levels were elevated in individuals with worsening muscle performance compared to those who remained robust (normalized protein abundance: 9.27 ± 0.71 vs. 8.70 ± 0.84, p = 0.034) and those progressing to sarcopenia (9.14 ± 0.63 vs. 8.69 ± 0.76, p = 0.002). C2 levels were higher in those with declining performance (6.05 ± 0.56 vs. 5.60 ± 0.24, p = 0.002), whereas C1QA was lower in individuals progressing to sarcopenia (6.54 ± 1.18 vs. 7.22 ± 0.93, p = 0.047), suggesting a protective role in muscle maintenance.

Summary/Conclusion : This study identifies plasma EV‐derived biomarkers and pathways linked to sarcopenia progression, highlighting complement activation in functional decline and supporting early diagnosis and intervention.

Denervated skeletal muscle‐derived extracellular vesicles: insights into molecular responses to nerve injury

Presenter: Maria Vittoria Giraudo

Umeå University, Umeå, Sweden

Introduction : Peripheral nerve injuries lead to muscle loss due to the disruption of neural input. If reinnervation does not occur, muscle fibres become atrophic, significantly hindering functional recovery. Our study aims to investigate extracellular vesicles (EVs) from denervated muscles to understand their role and molecular content in response to nerve injury.

Methods : Denervation of the rat gastrocnemius, tibialis, and soleus muscles was induced through sciatic nerve transection. Muscle samples were collected and weighed at 1 day and 2 weeks post‐injury. EVs were isolated from these tissues using enzymatic digestion and ultracentrifugation and characterized via western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Proteome profiling was also performed.

Results : Initial proteome profiling revealed distinct differences in the protein composition of EVs from control and denervated tissues, as well as between samples collected at 1 day and 2 weeks post‐injury. These findings highlight potential injury‐related changes in the protein content of EVs that may correlate with the tissue's state of denervation, with some protein changes potentially involved in atrophic reprogramming and axonal regeneration. Further studies are underway to explore the mRNA content and biological functionality of these EVs.

Summary/Conclusion : This study suggests that EVs derived from denervated muscle tissue could serve as biomarkers for the physiological state of muscles following nerve injury and may provide insights into new therapeutic approaches to promote muscle repair. Detailed analysis of their mRNA and functional profiles is expected to deepen our understanding of muscle responses to nerve injury and identify potential targets for treatment.

Subtopic Nervous system 1

Hypoxia‐upregulated membrane protein ATXN2 enhances the uptake of neuronal small extracellular vesicles to ameliorate ischaemic brain injury

Presenter: Yousheng Wu

The First Affiliated Hospital of Jinan University, Guangzhou, People's Republic of China

Introduction : Cellular hypoxia results in an elevated release of small extracellular vesicles (sEV), which can be more readily absorbed by surrounding cells. This process may lead to the dissemination of harmful substances and initiate the ischaemic cascade, a response associated with ischaemic stroke. Nonetheless, sEV derived from various cell types have different effects, and there is presently an absence of specific markers to identify sEV originating from hypoxic neuronees.

Methods : Isolating membrane proteins of SH‐sy5y‐derived sEV for proteomic analysis. Identifying sEV membrane proteins through Western blotting, immunoelectron microscopy, and ELISA. Extracting sEV from ischaemic brain tissue to verify the presence of membrane proteins. In vitro labelling of sEV is performed using PKH67 and CFSE to evaluate the efficiency of sEV uptake. Administering sEV from ischaemic brain tissue via stereotactic injection into mice prior to middle cerebral artery occlusion (MCAO), with or without monoclonal antibodies against membrane proteins, and evaluating behavioural and neurological outcomes in mice three days post‐surgery.

Results : Biotin successfully isolated the sEV membrane protein and identified the hypoxia‐upregulated membrane protein Ataxin2 (ATXN2). ATXN2 was found to be expressed in the sEV membrane protein fractions of hypoxic neuronees and ischaemic brain tissue, and it was present on the surface of the sEV membrane. Fluorescently labelled hypoxic sEVs were taken up by cells more readily than normoxic sEVs; however, the uptake of hypoxic sEVs was significantly reduced following the knockdown of ATXN2, and the use of an ATXN2 monoclonal antibody greatly diminished the uptake of hypoxic sEVs. Furthermore, the stereotactic injection of hypoxic neuronee‐derived sEVs into mice subjected to MCAO facilitated the recovery of neurological function. The outcome for MCAO mice receiving sEVs derived from infarcted brain tissue was more favourable than that of MCAO mice treated with a combination of infarcted brain tissue sEVs and the ATXN2 monoclonal antibody at the three‐day mark.

Summary/Conclusion : Hypoxic conditions lead to an elevated expression of the ATXN2 membrane protein in neuronal sEV and improve the uptake of these vesicles.

Astrocyte‐enriched extracellular vesicles as mediators of synaptic endurance after stroke

Hind Haj Ahmad 1 , Santra Brenna 1 , Ali Biabani 2 , Jelena Navolic 3 , Yvonne Pechmann 4 , Frank Heisler 4 , Julia Neumann 3 , Matthias Kneussel 4 , Tim Magnus 1 , Mathias Gelderblom 1 , Berta Puig 1

1 Neurology Department, Experimental Research in Stroke and Inflammation, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 2 Section for Mass Spectrometry and Proteomics, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 3 Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 4 Institute for Molecular Genetics, Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany

Introduction : Following an ischaemic stroke, brain cells directly supplied by the affected vessel (the core of the stroke) die within min. However, the neuronees surrounding this area (the penumbra) remain metabolically active yet enter a state of electric silence. Due to the energetic demands of synaptic activity, this temporary suppression of synaptic function may act as an adaptive response to sustain cell viability. Depending on the signals these neuronees receive in the hours and days that follow the ischaemic insult, they may either survive or undergo cell death. Current therapies are focused on rapid reperfusion, but a lot of effort is put into understanding how to rescue these neuronees to improve neurological recovery. We have previously shown that 24 h after ischaemia/reperfusion, the relative proportion of astrocyte‐enriched EVs (AEVs) within the pool of brain‐derived EVs (bdEVs) was increased in the affected hemisphere in a mouse stroke model (transient middle cerebral artery occlusion, tMCAO), and in the present study we aimed to analyse the content of AEVs and investigate their functional implications.

Methods : We isolated both small and medium/large AEVs using antibody‐based magnetic sorting and performed mass spectrometry analysis on samples from both stroke and sham‐operated mice at 24 h and 7 days after stroke. Additionally, we performed functional studies using calcium imaging by incubating AEVs with primary neuronal cultures subjected to oxygen‐glucose‐deprivation (OGD) as an in vitro model of stroke.

Results : Our results show that one day after stroke, proteins related to synaptic formation, integrity, plasticity and activity were upregulated. At 7 days after stroke, while some proteins related to synaptic processes remained upregulated, proteins related to metabolic pathways showed increased expression. Functional studies point to a role of AEVs in synaptic preservation and neuronal survival after ischaemic insult.

Summary/Conclusion : In this study, we have developed a protocol suitable for directly studying AEVs from the brain and providing new insights into the role of AEVs in neuroprotection, laying the groundwork for identifying potential therapeutic targets.

Funding : Werner Otto Stiftung, Deutsche Forschungsgemeinschaft (DFG), and Hermann und Lilly Schilling Stiftung für Medizinsiche Forschung.

Investigating the role of exercise linked‐irisin and extracellular vesicles in Alzheimer's disease

Presenter: Tayná Rody Souza Ferreira

Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Introduction : Exercise is known to prevent and improve Alzheimer's disease (AD). Physical exercise upregulates irisin, a myokine derived from fibronectin type III domain‐containing protein 5 (FNDC5) previously shown to mediate the beneficial actions of exercise on memory in AD mouse models. Exercise causes an overall increase in plasma extracellular vesicles (EVs) and modulates the levels of different myokines in EVs. Interestingly, EVs have been reported to modulate some of the physiological roles of FNDC5/irisin in peripheral tissues. However, the involvement of EV‐associated FNDC5/irisin in the brain has not been investigated. Herein, we investigated EVs as a therapeutic approach. We hypothesize that exercise modulates irisin production in EVs and might elicit a beneficial effect on cognition.

Methods : Three‐month‐old and 14‐month‐old C57BL/6 mice, AP/PS1 transgenic mice, and FDNC5 knockout mice were obtained from the Federal University of Rio de Janeiro (Rio de Janeiro, Brazil) facility and Queen's University (Kingston, Canada) facility. Plasma EVs were isolated using ExoQuick‐ULTRA. Mice underwent an exercise protocol consisting of 60 min of swimming sessions, 5 days per week for 5 weeks. Mice were intravenously injected with plasma EVs of exercised mice and mice that overexpress FNDC5/irisin. Memory was assessed using behavioural tests: the Radial Arm Water Maze and the Novel Object Recognition Test. We determined the levels of EV‐associated FNDC5/Irisin using ELISA. FNDC5 and BDNF expression were evaluated using PCR.

Results : Our results show that FNDC5/irisin is increased in plasma EVs of exercised mice or in mice that overexpress FNDC5. We report that the intravenous treatment with EVs obtained from exercised wild‐type mice, but not from exercised FNDC5 knockout mice, elevates the expression of brain‐derived neurotrophic factor (BDNF) in the cortex and rescues memory in an AD mouse model. In addition, EVs obtained from mice overexpressing FNDC5/irisin rescue memory in a transgenic AD mouse model.

Summary/Conclusion : Results indicate that EVs may be necessary for FNDC5/irisin‐related crosstalk between the periphery and brain, providing insight into the role of EVs as carriers of FNDC5/irisin during exercise. In summary, our results provide a basis for developing a therapeutic approach designed to enrich EVs with FNDC5/irisin to treat AD patients.

Visualizing proteopathic Tau seeds within Alzheimer's disease brain‐derived extracellular vesicles by single molecule localization microscopy

Dalya Gulseren 1,2 , Christian G. Specht 2 , Mehdi Kabani 1

1 Université Paris‐Saclay, CEA, CNRS, Labouratoire des Maladies Neurodégénératives (UMR 9199), Fontenay‐aux‐Roses, France; 2 Université Paris‐Saclay, INSERM, Maladies et Hormones du Système Nerveux (U1195), Le KremLin‐Bicêtre, France Christian Specht and Mehdi Kabani are co‐senior authors .

Introduction : Misfolding and aggregation of tau into highly ordered amyloid filaments is characteristic of Alzheimer's disease (AD) and other tauopathies. Tau lesions progressively invade the brain by a prion‐like mechanism of self‐replication and spreading of diffusible tau species or ‘seeds.’ Extracellular vesicles (EVs) are major vehicles for tau dissemination, but the ultrastructural and molecular properties of Tau‐containing EVs are scarcely known. Here, we aim at characterizing tau‐containing EVs isolated from AD brains at single‐vesicle resolution using single‐molecule localization microscopy (SMLM).

Methods : Post‐mortem human brain tissue samples (parietal and frontal cortex) from AD (Braak V/VI) and non‐demented age‐matched control subjects were obtained from the NeuroCEB brain bank, in accordance with French bioethics laws. After gentle dissociation of the tissues with collagenase, EVs were isolated by ultrafiltration and size‐exclusion chromatography and characterized by immunoblotting and negative staining transmission electron microscopy (TEM). For SMLM, EVs were selectively captured on multichannel coverslips, and then fixed, permeabilized and labelled with specific antibodies against EV marker proteins and their pathogenic cargo.

Results : We were able to visualize pathological tau seeds within brain‐derived EVs from AD patients. The simultaneous detection of thousands of immobilized EVs per recording generates comprehensive datasets for categorizing and quantifying tau‐containing EV subpopulations.

Summary/Conclusion : Due to its high spatial precision and outstanding sensitivity, SMLM is ideally suited to provide both detailed ultrastructural information as well as quantitative molecular information about the association of Tau with EVs. We have thus far succeeded in detecting tau in AD brain‐derived EVs. We will now apply this methodology for a more systematic analysis of the ultrastructure and molecular composition of EVs in neurodegenerative diseases.

Funding : This study was funded by grants from France Alzheimer (M.K.) and the Graduate School of Life Science and Health of University Paris‐Saclay (M.K., C.S.).

Regenerative potential of mesenchymal stem cell‐derived extracellular vesicles loaded with PTEN small interfering RNA for facial nerve injury

Presenter: Ayelet Lotan

Technion—Israel Institute of Technology, Haifa, Israel

Abstract unavailable

Intercellular communication through Schwann cell‐derived extracellular vesicles in peripheral neuropathy

Hui Su Jeong, Ji Eun Lee

SungKyunKwan University, Seoul, Republic of Korea

Introduction : Chronic inflammation impairs the repair ability in the peripheral nervous system, including the neuromuscular junction (NMJ). Chronic inflammation inhibits Wallerian degeneration/regeneration of presynaptic structures and leads to NMJ disorders. Although Schwann cells (SCs) have been suggested as a key player in neuronee regeneration, their precise mechanism responsive to inflammation is poorly understood. In our study, we investigated the role of SCs in the NMJ and muscle regeneration in a state of inflammation.

Methods : SCs in transverse abdominal and lumbrical muscles of young (4m) and old mice (22‐30m) were visualized via immunohistochemistry using NMJ and cilia markers. Cardiotoxin and LPS were used to induce tissue regeneration and inflammation. S16 SC lines were cultured for extracellular vesicle (EV) isolation that was characterized using nanoparticle trafficking analysis, western blotting, and proteomics analysis. Comparison of EVs from inflamed and non‐inflamed SCs revealed differences in size, number, and protein content. We treated EVs at damaged C2C12 myoblast cells to know their positive function in muscle regeneration.

Results : NMJ defects accompanied by abnormal SC were observed in both aged and inflammation‐induced mice, suggesting SC involvement in NMJ abnormalities. While normal SCs exhibited dynamic changes after injury to regeneration, inflammation‐hyperinduced SCs lost this activity, indicating impaired molecular functions in the regenerative environment. Given NMJ's unique multicellular composition, we hypothesized that SCs would communicate through mediators, and we developed our experiments based on the hypothesis that these mediators were EVs. We performed a proteomic analysis of culture media from SCs and identified a cluster of proteins involved in both neuronee development and muscle growth factor pathways, including those regulating early myoblast differentiation. To validate the functional role of these proteins, we treated myoblasts with SC‐derived EVs and demonstrated their ability to accelerate myoblast differentiation following muscle injury.

Summary/Conclusion : These findings reveal a novel mechanism whereby SCs regulate NMJ maintenance and muscle regeneration through EVs containing specific proteins, providing insight into potential therapeutic strategies for age‐ and inflammation‐related NMJ dysfunction.

Microvesicles carrying EV71 virions cross the blood‐brain barrier through endocytic pathway to induce brain injury

Presenter: Xiaoyan Tian

Nanjing University, Nanjing, People's Republic of China

Introduction : Enterovirus 71 (EV71) is a major etiologic pathogen for hand‐foot‐and‐mouth disease (HFMD) in young children. Severe cases of EV71 infection could lead to neurological complications and even death, while the mechanism inducing neurological complications remains poorly understood.

Methods : Microvesicles (MVs) were isolated from HeLa cells infected or not infected with EV71 by differential centrifugation. The morphology and size of the separated MVs were examined by electron microscopy and nanoparticle tracking analysis (NTA). We investigated whether MVs contained viral proteins and/or virions, and a monolayer BBB model was established to detect the ability of MVs to cross BBB in vivo. EV71 or MVsEV71 was injected into mice via the intraperitoneal (ip) route, and the brains were prepared for subsequent analysis by H&E staining and QPCR assay. Subsequently, we investigated whether MVs contained damaged mitochondria using co‐immunoprecipitation assay and transmission electron microscopy (TEM).

Results : We found that MVs could carry EV71 virions and mediate infection of target cells at a higher efficiency. MVs containing virions (MVsEV71) could cross the BBB with greater efficiency than EV71 alone both in vivo and in vitro. Meanwhile, MVsEV71 infection of animals induced more severe inflammatory infiltration in the brain compared to animals infected by EV71. Moreover, we elucidated that MVs crossed the BBB via disrupting tight junctions (TJs) and non‐clathrin‐dependent endocytosis pathways. Damaged mitochondria, as the ‘cargo’ of MVs, also facilitated MVsEV71 crossing the BBB. MVsEV71 crossing the BBB further induced mitochondrial damage and activated the NOX4‐derived ROS pathway in U251 cells.

Summary/Conclusion : Our findings suggested that MVs transported EV71 virions across the BBB, while damaged mitochondria facilitated this process. MVsEV71 induced mitochondrial damage and activated the NOX4‐derived ROS pathway in U251 cells, leading to further brain injury.

Funding : This study was supported by the National Natural Science Foundation of China.

Raman Spectroscopy analysis of alpha synuclein in salivary extracellular vesicles for the early diagnosis of Parkinson disease

Alice Gualerzi 1 , Rita Martino 1 , Luana Forleo 1,2 , Davide Moscato 3 , Riccardo Conte 3 , Silvia Picciolini 1 , Martina Gerli 1 , Valentina Mangolini 1,4 , Francesca Rodà 1,5 , Aurora Mangolini 1 , Stefano Faggiano 1 , Michele Ceotto 3 , Marzia Bedoni 1

1 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milan, Italy; 2 Medical and Surgical Pathophysiology and Transplantation Department, University of Milan, Italy; 3 Department of Chemistry, University of Milan, Milan, Italy; 4 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; 5 Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy

Introduction : Parkinson's disease (PD) is a neurodegenerative disorder lacking a robust biomarker for early diagnosis and monitoring of its progression. Recent studies suggest that extracellular vesicles (EVs) are crucial in the aggregation and propagation of α‐synuclein (α‐syn), a protein mainly present as oligomers and fibrils in people with PD (pwPD) and as a monomeric conformation in healthy controls (HC). For this reason, this protein is studied as a possible biomarker for early PD diagnosis. From computational simulations, Raman Spectroscopy (RS) can discriminate α‐syn conformation in healthy and pathological conditions, showing changes in its peak in the spectral range between 1600 and 1700 cm −1 . The aim of this study is the development of an analytical method based on RS to detect pathological α‐syn on salivary EVs in pwPD, compared to HC.

Methods : After signing written informed consent, 30 pwPD and 30 HC were recruited at Fondazione Don Carlo Gnocchi. Saliva was collected through a cotton swab. After concentration of saliva by ultrafiltration, EVs were isolated from saliva combing size exclusion chromatography (qEV, Izon) and ultracentrifugation (100,000 × g for 70 min). For RS acquisition, 5 µL of EVs were deposited and air‐dried on calcium fluoride slide. The measurement was performed using a 532 nm laser in the spectral ranges 600–1800 cm −1 and 2600–3200 cm −1 . Simulations were performed using the Tinker suite of codes and the AMOEBABIO18 Force Field.

Results : EVs were successfully isolated from the saliva of all subjects. Nanoparticle tracking analysis and dot blot showed no significant difference between pwPD and HC in size distribution and protein markers, respectively. RS analysis of salivary EVs showed differences in multiple spectral ranges attributable to multiple macromolecules (proteins, lipids and nucleic acids). Despite remarkable biological variability among pwPD, a significant change in the amide I RS peak attributable to aggregated forms of α‐syn was observed.

Summary/Conclusion : RS analysis on salivary EVs proved to be a promising method for the detection of pathological α‐syn in pwPD, showing the potentiality to be transferred to clinical settings to improve the diagnosis and monitoring of PD.

Funding : This research was funded by ERC‐2022‐POC2, HORIZON‐ERC‐POC and Fondo di Beneficenza di Intesa Sanpaolo.

Neurone‐targeted nanoparticle‐EV complex for specific brain delivery: a platform for early diagnosis and treatment of neurodegenerative disease

Jisook Moon 1 , Jae Hyun Park 1 , Chul‐Woo Lim 1 , Jinwoo Hawng 1 , Soo‐Hyeon Nam 1 , Tae Hee Han 1

1 Department of Biotechnology, CHA University, South Korea Jisook Moon and Jae Hyun Park contributed equally to this study. Jisook Moon is the corresponding author .

Introduction : Early diagnosis and effective treatment of neurodegenerative diseases remain challenging due to the difficulty of capturing brain‐specific molecules in the blood. Amyloid precursor‐like protein 1 (APLP1), a membrane protein highly expressed in the brain, has emerged as a promising biomarker for isolating brain‐derived extracellular vesicles (EVs). However, differentiating APLP1 from homologous proteins like APLP2 and APP770 and developing targeted delivery systems have posed significant obstacles.

Methods : This study addresses these challenges by designing APLP1‐specific peptide sequences that bind to its extracellular domain, enabling selective brain targeting. We developed a nanoparticle‐EV complex by integrating these peptides, creating a robust platform for precise delivery to neural tissues and brain regions. By analysing structural differences within the APLP family, we optimized two peptide sequences that specifically recognize APLP1. These sequences were conjugated onto EVs to enhance targeting efficacy, while the integration of nanoparticles allows for advanced applications such as controlled delivery and imaging.

Results : The resulting Nanoparticle‐EV complex demonstrated high selectivity and efficiency in binding to APLP1‐expressing areas, validating its use in brain‐targeted delivery. Importantly, APLP1+ EVs isolated from human plasma were enriched with neuronal markers and neurodegenerative disease‐related proteins, including those implicated in Alzheimer's and Parkinson's diseases. Small RNA profiles further confirmed brain‐specific signatures, with putative miRNA targets highly expressed in brain tissues.

Summary/Conclusion : This innovative approach, which combines APLP1‐targeted peptides with nanoparticles and EVs, establishes a foundational platform for the early diagnosis and treatment of neurodegenerative diseases. It provides an efficient and precise system for brain‐targeted delivery, offering significant advancements in neurotherapeutics and diagnostics.

Funding : This work was supported by the Korea government (MSIT) (RS‐2024‐00462182) and a grant (C390000) from the Korea Basic Science Institute and the Korean Fund for Regenerative Medicine funded by the Ministry of Science and ICT and the Ministry of Health and Welfare (RS‐2022‐00070658, 22C0617L1‐12).

Tracking adipose‐derived stem cell exosomes applied in a mouse crush injury model: insights from fluorescent labelling and spatial transcriptomics—an experimental study

Presenter: Ching‐Hua Hsieh

Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (Republic of China)

Introduction : Peripheral nerve injuries often result in disability and functional loss, with current treatments showing limited efficacy. Adipose‐derived stem cell exosomes (ADSC‐exos) have shown promise in nerve regeneration, but their precise mechanisms remain unclear. This study aimed to investigate the effects and mechanisms of ADSC‐exos on crushed sciatic nerves using fluorescent labelling and spatial transcriptomics.

Methods : The study employed a mouse sciatic nerve crush model. ADSC‐exos were labelled using biorthogonal click chemistry with Cy5 fluorescent markers and applied locally to the crushed nerve. L4‐L6 spinal cords and proximal nerve segments were examined through immunohistochemistry. Spatial transcriptome analysis was performed to investigate gene expression changes and cell‐cell interactions. The study included exosome characterization, fluorescence tracking, and comprehensive analysis of cellular and molecular responses.

Results : Labelled exosomes were detected in spinal neuronees and proximal nerve segments post‐application. Spatial transcriptomics revealed significant upregulation of genes in neuronees and Schwann cells, while oligodendrocyte‐related genes were downregulated. Key pathways involved included PSAP, PTN, FGF, SPP1, SLITRK, VEGF, and GAS signalling. ADSC‐exo treatment enhanced cell‐cell interactions, particularly between Schwann cells and astrocytes. Gene ontology analysis indicated improvements in metabolic activity, cell communication, and structural support.

Summary/Conclusion : This study provides novel insights into the molecular mechanisms of ADSC‐exo‐mediated nerve regeneration. The comprehensive approach revealed complex interplay between multiple cell types and signalling pathways, suggesting that ADSC‐exos promote a regenerative environment through enhanced cellular communication and metabolic activity. These findings advance our understanding of nerve regeneration mechanisms and provide new perspectives for developing therapeutic strategies for peripheral nerve injuries.

Funding : This research was supported by the Chang Gung Memorial Hospital (grant numbers CMRPG8L0201, CMRPG8M0141, and CMRPG8M0921).

investigating the influence of IL‐17A signalling in astrocyte‐enriched extracellular vesicle cargo and its impact on neuronal recovery post‐stroke

Presenter: Hind Haj Ahmad

University Medical Center Hamburg‐Eppendorf, Hamburg, Germany

Introduction : Ischaemic stroke remains one of the leading causes of long‐term disability. It is characterized by the acute occlusion of brain‐supplying arteries, resulting in oxygen and glucose deprivation, which leads to significant neuronal damage and inflammation in the ischaemic brain. Among the key inflammatory cascades driving the early immune response, IL‐17A plays a critical role. While most IL‐17A signalling occurs in non‐haematopoietic cells like astrocytes, which express the IL‐17RA/IL‐17RC receptor complex, the short‐ and long‐term effects of IL‐17A signalling in astrocytes on neuronal regeneration remain poorly understood. Astrocytes, key players in neuroinflammation, communicate through extracellular vesicles (EVs), an essential process for both healthy brain function and pathological conditions such as stroke. In this study, our aim was to investigate the specific influence of IL‐17A on the cargo of astrocyte‐enriched EVs (AEVs) during post‐stroke recovery, a topic that has yet to be explored.

Methods : We utilized a genetically modified mouse model (Gfap‐Cre + / − × Il17rafl/fl) with a targeted deletion of the IL‐17 receptor A (IL‐17RA) in astrocytes. After inducing stroke in both Gfap‐Cre + / − × Il17rafl/fl and wild‐type littermate mice, we collected AEVs from ischaemic brain tissue using magnetic sorting and immunoprecipitation at 24 h (acute phase) and 7 days (recovery phase).

Results : Preliminary data from mass spectrometry reveal that IL‐17RA knockout in astrocytes post‐stroke leads to distinct time‐dependent changes in ADEV cargo. At day 1, ADEVs are enriched with protein related to immune activation and mitochondrial pathways, reflecting an early focus on inflammation and cellular metabolism. By day 7, ADEVs exhibit enhanced proteins in pathways related to synaptic structure, ion transport, and neuronal recovery, suggesting a delayed yet improved neuronal repair response in the absence of IL‐17A signalling.

Summary/Conclusion : Our findings reveal for the first time that IL‐17A influences the composition of ADEV cargo.

The transfer of sensory neuronee‐derived extracellular vesicles to resident macrophages in dorsal root ganglia maintains persistent inflammatory pain

Presenter: Silvia Oggero

King's College London, London, UK

Introduction : Persistent pain in rheumatoid arthritis is a clinical feature that can be modelled by the K/BxN serum transfer (ST) model of inflammatory arthritis. In naïve mice, two systemic injections of serum obtained from K/BxN arthritic mice result in paw swelling and pain hypersensitivity within 7 days. However, whilst paw swelling resolves by 25 days, hypersensitivity persists because nociceptive neuronee interaction with immune cells along the pain pathway sustains pain signalling. Here we delineate a crosstalk between sensory neurones and macrophages mediated by extracellular vesicles (EVs).

Methods : In vitro, cultured DRG neuronees (10,000 cells/well) were incubated with capsaicin (1 µM, 3 h) with(out) EV biogenesis inhibitor GW4896 (5 µM). EVs were isolated from neurone media by centrifugations at 4000 × g 15 min, 13,000 × g 2 min, and 100,000 × g 1 h at 4°C and analysed by imaging flow cytometry, nanoparticle tracking, and transmission electron microscopy. Neurone‐derived EVs were incubated for 3 h with bone marrow‐derived macrophages (BMDMs; 1 × 10 6 cells/well). Lipid content in EVs and BMDM culture media was assessed by ELISA. In vivo: GW4896 was injected intrathecally (100 pmol/5 µL) on days 7 and day 25 of K/BxN ST, and mechanical thresholds were assessed by von Frey filaments.

Results : Capsaicin activation of cultured sensory neuronees resulted in a 2‐fold increase of EV numbers compared to controls. Sensory neuronee‐derived EVs were smaller than 150 nm and expressed tetraspanins CD9 and CD63. Arachidonic acid (AA) content in capsaicin released EVs was 2‐fold higher than controls. Both EV numbers and AA levels remained at control values after GW4896 incubation prior to capsaicin. A single intrathecal injection of GW4869 at day 7 and day 25 K/BxN‐ST exerted anti‐nociceptive effects at 1 and 2 h after injection, suggesting that release of EVs within the DRG contributes to persistent hypersensitivity. In BMDMs fed with AA through exposure to neuronee‐derived‐EVs, 5‐lipoxygenase converted AA to LTB4, and the higher content of AA in capsaicin‐released EVs resulted in more LTB4 formation.

Summary/Conclusion : Under persistent inflammatory pain, activation of neuronee cell bodies results in releases of EVs that are engulfed by macrophages, where AA is converted to LTB4, which facilitates leukocyte trafficking and nociceptive signalling.

Funding : This study was supported by the Arthritis UK grant 21961.

A brain‐wide interneuronal network mediated by extracellular vesicle

Presenter: Cho Hyun Byun

Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

Abstract unavailable

Contribution of human and murine blood serum exosomes in depressive‐like behaviour: focus on protein content

Vásquez‐Pérez Jorge Manuel 1,2 , Flores‐Ramos Mónica 1 , Avendaño‐Estrada Arturo 2 , Ruiz‐May Eliel 3 , Ramos‐Godínez María del Pilar 4 , Ramírez‐Rodríguez Gerardo Bernabé 1

1 National Institute of Psychiatry Ramón de la Fuente Muñiz, Mexico City, Mexico; 2 National Autonomous University of Mexico, Mexico City, Mexico; 3 National Institute of Ecology, Mexico City, Mexico; 4 National Institute of Cancerology, Mexico City, Mexico

Introduction : Major depressive disorder (MDD) is one of the most common and disabling psychiatric disorders worldwide. It is classified as an affective disorder, as described in the DSM‐V. It is considered a multifactorial disorder, with influence on the central nervous system (CNS) and endocrine and immunological systems, impacting different neurochemical and inflammatory pathways through common signalling mechanisms such as metabolites, soluble factors, and even extracellular vesicles, such as exosomes.

Methods : Four groups of samples were included: healthy female controls ( n = 12), women diagnosed with depression ( n = 12), healthy female Balb/C control mice ( n = 10), and Balb/C mice under the mild chronic unpredictable mild stress (CUMS) protocol ( n = 10). EVs were isolated from human and murine blood serum using size exclusion chromatography (qEV1 70 nm, Izon Science). We characterized the exosomes by transmission electron microscopy, Exo‐Check Antibody Array (Neuro) Standard Kit and ExoELISA‐ULTRA CD63 assay (SBI, System Biosciences). We used human and murine (AAM‐CYT‐1000‐8, RayBiotech) arrays to analyse their protein content. Human CTRL and MDD exosomes were administered using robotic stereotactic surgery, and we performed a positron emission tomography (PET) functional analysis to identify CNS metabolic changes and depressive‐like behaviour associated with the exosomes that were administered.

Results : Three statistically different protein sets were identified ( p < 0.05): 37 proteins from human exosomes (CTRL vs. MDD), 18 proteins in murine exosomes (CTRL vs CUMS), and 12 proteins from the groups of human and murine exosomes. We used STRING and UniProt to identify protein‐protein interaction (PPI), and gene ontology functional bioinformatics analysis was performed using WEBGESTALT and the KEEG database. Signalling pathways associated with MDD and chronic stress pathophysiology were identified: cytokine‐cytokine receptor interaction, JAK‐STAT signalling, and chemokine signalling. In addition, the exosomes administered caused depressive‐like behaviours in the mice in the forced swim and tail suspension tests.

Summary/Conclusion : Our findings suggest the importance of exosomes in MDD, highlighting in part the validity and homology of murine models and their correlation with clinical observations, highlighting CUMS as an efficient model for the development and experimental modelling of subjects that replicate depressive‐like behavioural aspects with apparently similar signalling pathways.

Funding : S.I.C.‐2000, Fondo Sectorial de Salud‐CONACYT 262307; Ciencia de Frontera 2023, CONAHCYT, Mexico (Proyecto CF‐2023‐I‐2651).

Extracellular vesicles derived from HIV‐infected microglia induce neuroinflammation and apoptosis

Jun Yang 1 , Zhimin Feng 1 , Ge Jin 1,2

1 Case Western Reserve University, Cleveland, Ohio,vUSA; 2 The MetroHealth Research Institute, Cleveland, Ohio, USA

Introduction : Despite the significant reduction in mortality and morbidity related to HIV infection due to the effective combined antiretroviral therapy (cART), recent studies have shown that over 50% of HIV‐infected patients develop HIV‐associated neurocognitive disorder (HAND). The role of HIV‐associated extracellular vesicles (HIV+ EVs) in the development and exacerbation of HAND is not well understood. This study utilized an optimized brain environment model to investigate the potential involvement of HIV‐associated EVs in the development or worsening of HAND, as well as the underlying mechanisms.

Methods : EVs were isolated using a differential ultracentrifugation protocol from culture media of latently HIV‐infected T cells (J1.1) and microglial cells (HC69), as well as non‐HIV‐infected T cells (Jurkat) and microglial cells (C20). Cells were grown in media supplemented with EV‐depleted FBS before EV isolation. The ZetaView Twin system was used for EV quantification. Cerebral organoids were created using human induced progenitor stem cells (hiPSCs). The blood‐brain barrier (BBB) model was established with primary human brain microvascular endothelial cells, astrocytes, and pericytes. The immunopathogenic response was evaluated using immunofluorescence staining following cryo‐sections of cerebral organoids, BBB, and microglial cells.

Results : EVs isolated from HC69 microglial cell cultures and J1.1 T cell cultures contained HIV RNA, including the transactivation regulatory element (TAR), Tat, and Nef RNA. These HIV+ EVs were capable of entering various brain cell types, such as microglial cells, neuronee cells, astrocytes, and pericytes. They traversed the in vitro BBB model and reactivated latent HIV in HC69 cells, leading to TNF‐α and IL‐1β production, resulting in apoptosis in neuronees of the cerebral organoids within the BBB model.

Summary/Conclusion : The study demonstrates that HIV‐associated EVs induce inflammation and cell death within the central nervous system, contributing to the progression or exacerbation of HAND.

Funding : This study was supported by a startup fund from the MetroHealth Research Institute and R01CA264910 from NIH/NCI.

Gut microbiota‐derived extracellular vesicles influence alcohol intake preferences in rats

M. Díaz‐Ubilla 1 , H. Tobar‐Durán 2 , A. Figueroa‐Valdés 2 , M. E. Quintanilla 3,4,5 , E. Díaz 3 ; P. Morales 3,4,5 , P. Berríos‐Cárcamo 1 , D. Santapau 1 , J. Gallardo 1 , C. De Gregorio 1 , J. Ugalde 6 , C. Rojas 7,8 , A. González‐Madrid 1 , M. Ezquer 1 , Y. Israel 3,5 , F. Alcayaga‐Miranda 2,7 *, F. Ezquer 1,9 *

1 Center for Regenerative Medicine, Faculty of Medicine, Clínica Alemana‐Universidad del Desarrollo, Santiago, Chile; 2 Center of Interventional Medicine for Precision and Advanced Cellular Therapy (IMPACT), Santiago, Chile; 3 Molecular and Clinical Pharmacology Program, Institute of Biomedical Science, Faculty of Medicine, Universidad de Chile, Santiago, Chile; 4 Department of Neuroscience, Faculty of Medicine, Universidad de Chile, Santiago, Chile; 5 Faculty of Medicine, Specialized Center for Prevention of Substance Use and Treatment of Addictions (CESA), Universidad de Chile, Santiago, Chile; 6 Center for Bioinformatics and Integrative Biology, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile; 7 Centro de Investigación e Innovación Biomédica (CIIB), Facultad de Medicina, Universidad de Los Andes, Santiago, Chile; 8 Faculty of Dentistry, Universidad de Los Andes, Santiago, Chile; 9 Research Center for the Development of Novel Therapeutic Alternatives for Alcohol Use Disorders, Santiago, Chile

Introduction : Growing preclinical and clinical evidence suggests a link between gut microbiota and problematic alcohol consumption. Extracellular vesicles (EVs) are an important component in bacteria‐to‐host communication. However, their potential role in mediating addictive behaviour remains unexplored. This study aims to investigate the role of gut microbiota‐derived bacterial extracellular vesicles (bEVs) in promoting high alcohol consumption and their possible dependence on vagus nerve function.

Methods : EVs were isolated from three experimental groups: UChB rats (a high alcohol‐drinking rat strain) with chronic alcohol consumption, ethanol‐naïve UChB rats, and Wistar rats as a control group. bEVs were characterized following the MISEV2023 guidelines and were intraperitoneally administered for three consecutive days to Wistar rats. Voluntary alcohol intake was evaluated using a three‐bottle choice test. Animals were then euthanized, and tissue samples were collected for inflammation analysis. To assess the role of the vagus nerve in mediating the effects of bEVs, subdiaphragmatic bilateral vagotomy or sham surgery (control) was performed.

Results : Particles isolated from the three experimental groups exhibited high‐integrity vesicles, displaying different sizes, electron density profiles, and a variety of vesicle morphologies, suggesting a good representation of gut microbiota. Enrichment of bacteria‐derived vesicles was confirmed by the detection of bacterial markers lipoteichoic acid (LTA) and lipopolysaccharide (LPS). Both types of UChB‐bEVs increased voluntary alcohol consumption in Wistar rats by up to 10‐fold ( p < 0.0001), indicating that bEVs can effectively transmit drinking behaviour across different rat strains. No systemic, liver, or neuroinflammation was detected. Additionally, we demonstrated that the vagus nerve mediates the increase in alcohol consumption, as vagotomy completely abolished the effect of bEVs on ethanol intake (97.9% reduction compared to the sham group injected with bEVs, p < 0.01).

Summary/Conclusion : In conclusion, this is the first study proposing gut microbiota‐derived extracellular vesicles as a novel mechanism mediating high alcohol intake. Furthermore, we demonstrate that these isolated particles can transmit reinforcing/addictive behaviour between different rat strains in a vagus nerve‐dependent manner.

Exercise‐induced small extracellular vesicles: exploring miRNA's role in alleviating major depressive disorder

Reine Khoury 1,2 , Dariusz Żurawek 2 , Georgia Kruck 1,2 , Sierra Codeluppi 2 , Minh Nguyen 2 , Corina Nagy 2,3

1 Integrated Program in Neuroscience, McGill University, Montreal, Québec, Canada; 2 McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, Québec, Canada; 3 Department of Psychiatry, McGill University, Montreal, Québec, Canada

Introduction : Exercise is a crucial non‐pharmacological intervention for addressing mental health conditions like major depressive disorder (MDD), with known benefits for brain function, including enhanced plasticity and neuroprotection. Recent research highlights the brain‐muscle and brain‐bone axes, suggesting that exercise influences not only local adaptations but also systemic effects, including interneuronal communication. While the molecular mechanisms remain unclear, small extracellular vesicles (sEVs), released during exercise, are emerging as potential mediators of these effects. Given the role of miRNAs in regulating various diseases, including depression, this study explores the role of exercise‐induced sEVs and their miRNA content in mitigating MDD symptoms.

Methods : The first objective is to characterize changes in sEV size, concentration, and molecular content (miRNA and proteins) in different tissues following an aerobic exercise regimen in mice. We conducted a 2‐week study with young‐adult mice undergoing treadmill running to examine the release of sEVs from plasma, bone marrow, and skeletal muscle. sEVs were collected and analysed for size, quantity, and molecular cargo, particularly focusing on miRNAs. The second objective is to assess the antidepressant effects of exercise‐induced sEVs by examining their impact on stress‐susceptible mice after treadmill exercise and chronic stress exposure.

Results : The study revealed that exercise significantly increased the release of skeletal muscle‐derived sEVs in both male and female mice, with a transient spike that declined hours post‐exercise. These vesicles were consistent in size across groups. miRNA analysis showed that miR‐133b‐3p and miR‐199a‐5p were upregulated post‐exercise, while miR‐21a‐5p and miR‐200a‐3p were downregulated, among others. Notably, miR‐133b‐3p's upregulation points to its involvement in muscle adaptation, synaptic plasticity, neuronal communication, and cardiovascular function, emphasizing the broader physiological and neuroprotective benefits of exercise.

Summary/Conclusion : These findings suggest that exercise‐induced sEVs from peripheral tissues like skeletal muscle may have systemic effects, including influencing neuronal communication. The enrichment of synaptic‐related pathways in miR‐133b‐3p targets points to the role of muscle‐derived EVs in muscle‐nervous system cross‐talk, supporting evidence of exercise's therapeutic potential via tissue‐derived EVs.

Sex‐ and size‐specific protein cargo variations in extracellular vesicles from oligodendrocyte progenitor cells in response to hyperoxic stress

Donna Elizabeth Sunny 1 *, Elke Hammer 1 , Rabea Schlüter 2 , Stephan Michalik 1 , Uwe Völker 1 , Matthias Heckmann 1

1 University of Medicine Greifswald, Greifswald, Germany; 2 University of Greifswald, Greifswald, Germany

Introduction : Cerebral oxygenation differences in preterm infants due to exposure to high oxygen levels during intensive care and sex‐specific oxidative stress responses can disrupt oligodendrocyte maturation, affecting neuronal development and function differently in male and female brains. Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication and stress response in the brain. Here, we used a proteomic approach to identify and characterize the cargo composition of small (< 200 nm) and large (< 1000 nm) EVs released by primary, mouse‐derived oligodendrocyte progenitor cells (OPCs) of both male and female origins separately. We aim to pinpoint molecular alterations within this cargo, triggered by hyperoxic conditions, in a sex‐ and size‐specific manner.

Methods : Male‐ and female‐derived OPC spheroids, obtained from neurosphere cultures, were treated with 80% oxygen for 24 h, and EVs were isolated using the precipitation method. This was followed by size separation using the filtration method. The EVs were characterized to meet the Minimal Information for Studies of EVs 2023, including electron microscopy and western blot analysis. The EVs subsequently underwent an untargeted mass spectrometric analysis on an ESI‐LC‐MS/MS system followed by initial data analysis using Spectronaut (Biognosys) and subsequent quantification of differences by the SpectroPipeR pipeline.

Results : Our analysis identified distinct sex‐ and size‐specific protein signatures with only 17% and 22% overlap in large (lEVs) and small extracellular vesicles (sEVs) between males and females after hyperoxia. In response to hyperoxia, male‐derived lEVs showed a greater number of significantly altered proteins than female‐derived lEVs, whereas female‐derived sEVs exhibited more protein changes than those from males, suggesting enhanced stress response signalling in females via sEVs and a significant role for lEVs in male cells. Nanoparticle tracking analysis revealed a slight increase in sEV intensity in female samples post‐hyperoxia. Male lEVs showed an upregulation of mitochondrial proteins, including those involved in oxidative phosphorylation and ATP synthesis, while female sEVs showed an enrichment of proteasomal complex proteins, with ingenuity pathway analysis highlighting post‐translational protein phosphorylation pathways.

Summary/Conclusion : Our results suggest that intercellular stress response signalling is mediated differentially by male and female OPCs via size‐specific secretion of EVs, whereby large and small EVs carry different protein cargo.

Influence of air pollution exposure on the microRNA content of neuronal extracellular vesicles in subjects affected by major depressive disorder

Monti Paola 1 *, Borroni Elisa 1 , Matsagani Rachele 1 , Dariol Eva 1 , Dioni Laura 1 , Davide Biganzoli 1 , Iodice Simona 1 , Pesatori Angela Cecilia 1,2 , Bollati Valentina 1,2 , Buoli Massimiliano 3,4 , Carugno Michele 1,2

1 Department of Clinical Sciences and Community Health, EPIGET Lab, University of Milan, Milan, Italy; 2 Occupational Health Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 3 Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy; 4 Department of Neurosciences and Mental Health, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy

Abstract unavailable

Subtopic Organ systems 1

KRAS/LKB1 co‐mutated non‐small cell lung cancer reshapes fibroblasts toward an inflammatory‐like phenotype through extracellular vesicles

Presenter: Cristina Borzi

IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy

Introduction : KRAS/LKB1 co‐mutated non‐small cell lung cancers (KL NSCLCs) show a very aggressive clinical behaviour. They present a peculiar tumour microenvironment, with pro‐inflammatory features. The role of extracellular vesicles (EVs) in tumour‐stroma communication has been extensively demonstrated, as well as their contribution to inflammation, immunosuppression and tumour dissemination. Fibroblasts are the major component of the stroma. They are characterized by phenotypic and functional heterogeneity; however, the molecular mechanisms responsible for the modulation of their phenotype are largely unknown. Here, we investigated how EVs released by KL NSCLCs could affect fibroblast phenotype reshaping.

Methods : EVs were isolated by ultracentrifuge from conditioned medium of the KRASWT/LKB1WT H1299 NSCLC cell line (WT‐EVs) and the KRASG12C/LKB1DEL isogenic clone (KL‐EVs). EVs were characterized by NTA, TEM, and EVs‐associated markers (flow cytometry – FC, western blot). Normal lung fibroblasts (CCD‐19Lu) and immortalized cancer‐associated fibroblasts (CAF154hTERT) were used for functional studies. EVs uptake was evaluated by FC and confocal microscopy. Fibroblast phenotype was assessed by gene expression analysis and ELISA. Patient‐Derived Xenografts (PDXs) were established by subcutaneous injection of WT and KL NSCLC tumour samples in SCID mice. The cBioPortal open‐source tool was used for in silico analyses.

Results : KRAS/LKB1 co‐mutation significantly increased EVs secretion by H1299. However, KL‐EVs had lower protein content than WT‐EVs. EVs size distribution was not affected by the mutational status. In vitro data showed that uptake of EVs by fibroblasts is comparable between WT‐ and KL‐EVs, with 100% of EV+ CCD‐19Lu at 4 h of EVs exposure. We revealed that KL‐EVs are able to induce an inflammatory phenotype in normal and immortalized cancer associated fibroblast by upregulating the expression and secretion of IL‐6. KL‐EV long‐term exposure modulates other immune‐related genes, including CCL2, CXCL1, CXCL3, and IL‐8. The increase of IL‐6 expression level in KL compared to WT tumour samples was confirmed in vivo in PDXs and in silico analyses (CPTAC series).

Summary/Conclusion : KRAS/LKB1 co‐mutation impacts EVs release and phenotype. Our results support the hypothesis of an active role of EVs in switching fibroblasts toward an inflammatory‐like subtype in KL NSCLC. Further studies are needed to fully elucidate the molecular mechanisms behind this modulation.

Funding : Italian Association for Cancer Research (AIRC)

Pharmacological inhibition of acid sphingomyelinase activity reduces extracellular vesicle release from murine alveolar leukocytes during lipopolysaccharide‐induced acute lung injury

Xingzhi Cheng 1 , Jonathan Stephens 1 , Wolfgang Kuebler 2 , Masao Takata 1 , Sanooj Soni 1

1 Imperial College London, London, UK; 2 Charité—Universitätsmedizin Berlin, Berlin, Germany

Introduction : Leukocyte extracellular vesicles (EV) are vital mediators of intercellular communication within the alveolar space and have been implicated in the pathophysiology of acute lung injury (ALI). Previous work has demonstrated the importance of acid sphingomyelinase (ASM) in the formation of EVs and packageing of their molecular cargo. We hypothesise that inhibition of ASM will diminish pro‐inflammatory EV production, thereby ameliorating ALI.

Methods : C57BL/6J mice received intratracheal instillation of lipopolysaccharide (LPS) to induce ALI. Imipramine, a functional inhibitor of ASM activity (FIASMA), was intratracheally administered into some mice 24 h prior to the LPS challenge. Bronchoalveolar lavage fluid (BALF) was collected for analyses of leukocyte EVs and albumin levels, and the lungs were harvested to measure leukocyte infiltration and surface ASM expression in single‐cell suspension by flow cytometry. EVs were defined and analysed by flow cytometry using their size and surface markers, with CD45+CD11c+ events as alveolar macrophage (AM) EVs and CD11b+Ly6G+ events as neutrophil (N) EVs.

Results : LPS stimulation at 1 h caused increases in BALF AM‐EVs, accompanied by upregulation of ASM expression on AMs, and both were significantly reduced by pre‐treatment with imipramine (EVs: 1563 ± 318 vs. 1164 ± 201 EVs/µL, n = 8, p < 0.01. ASM: 13270 ± 3025 vs. 9935 ± 1502 MFI, n = 6, p < 0.05). LPS stimulation at 4 h caused substantive increases in N‐EV release as well as upregulated ASM expression on neutrophils, which were massively ablated with imipramine (EVs: 1639 ± 445 vs. 977 ± 379 EVs/µL, n = 7, p < 0.05. ASM: 1747 ± 431 vs. 1163 ± 417 MFI, n = 5, p < 0.05). Furthermore, imipramine attenuated the increase in BALF albumin levels post LPS stimulation (2.16 ± 0.374 vs. 1.67 ± 0.290‐fold change, n = 6, p < 0.05), suggesting ASM inhibition may have a protective effect against pulmonary permeability and oedema.

Summary/Conclusion : Our study demonstrates that ASM is crucial in leukocyte EV formation in vivo, and inhibition of ASM activity reduces consequent EV release and offers protection against LPS‐induced alveolar oedema and inflammation. These data provide valuable insights into the therapeutic potential of repurposing FIASMAs for prevention and treatment of ALI.

Funding : Friends of Hammersmith Hospital Charity.

The role of glycan modification in altering lung uptake of HBEC‐derived EVs for drug delivery

Kazuki Kawajiri 1,2 , Reika Kaneko 1 , Misato Yamamoto 1 , Shota Fujimoto 1,2 , Jun Araya 2 , Yu Fujita 1,2

1 Division of Next‐Generation Drug Development, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo, Japan; 2 Division of Respiratory Diseases, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan

Introduction : Extracellular vesicles (EVs) can serve as natural drug delivery carriers. To apply EVs as a drug delivery system for respiratory diseases, we need to find an efficient method to target EVs to the lungs. However, it remains unclear which type of EVs would be the most effective for drug delivery carriers. Unmodified EVs may have limited uptake into diseased target cells. In our recent study, EVs derived from human bronchial epithelial cells (HBEC) demonstrated therapeutic effects for idiopathic pulmonary fibrosis. Here, we focus on the potential of HBEC‐EVs as drug carriers for the lungs. In this study, we compare the lung uptake of HBEC‐EVs with that of other EV types. Additionally, we evaluate how de‐glycosylation alters the biodistribution of HBEC‐EVs.

Methods : HBEC, HEK293 and mesenchymal stem cells (MSCs) were cultured in serum‐free medium for 48 h, and EVs were purified from the culture supernatant by ultracentrifugation. These EVs were characterized by western blotting and nanoparticle tracking analysis. Surface glycan patterns of HBEC‐EVs were analysed using a lectin microarray system. EVs were treated with glycolytic enzymes. After co‐culturing cells with PKH67‐labelled EVs, EV uptake into the cells was evaluated by fluorescence microscopy. For in vivo analysis, EVs were fluorescently labelled with DiR, administered intratracheally to mice, and the fluorescence intensity of each organ was observed using an in vivo imaging system (IVIS).

Results : Lectin microarray analysis revealed the glycan profiles of HBEC‐EVs. HBEC‐EVs were significantly more taken up by HBECs than HEK293‐EVs and MSC‐EVs. In IVIS evaluation, HBEC‐EVs accumulated efficiently in lungs following intratracheal administration in mice. Removal of surface glycans on HBEC‐EVs altered their lung accumulation.

Summary/Conclusion : HBEC‐EVs showed higher incorporation into lung tissue compared to other EV types. Using EVs derived from cells of target organs may enhance the efficiency of drug carriers. Glycan modification of EVs could be an important strategy for optimizing their use as drug delivery carriers.

Evaluation of extracellular vesicles as non‐invasive early predictive markers for severe COVID‐19

Klara Eriksson 1 , Meera Ravi 1 , Swamy Adapa 2 , Oliver Schildgen 3 , Michaela Ludmilla Merten 3 , Ionita Ghiran 4 , Christian Karagiannidis 3 , Philippe Krebs 5 , Dieter Kressler 6 , Michael Stumpe 6 , Rays Jiang 2 , Pierre‐Yves Mantel 1,7 , Paola Martínez‐Murillo 7

1 Department of Oncology, Microbiology, and Immunology, University of Fribourg, Fribourg, Switzerland; 2 Department of Global Health, University of South Florida, Suite 404, Tampa, Florida, USA; 3 Lungenklinik—Lungenintensivstation Kliniken, Universität Witten/Herdecke, Cologne, Germany; 4 Division of Allergy and Infection, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA; 5 Institute of Pathology, Division of Experimental Pathology, University of Bern, Bern, Switzerland. 6 Department of Biology, University of Fribourg, Fribourg, Switzerland; 7 Christine Kühne—Center for Allergy Research and Education, Davos, Switzerland

Introduction : Identification of novel biomarkers to detect as early as possible severe cases of COVID‐19 is important. Because of cellular activation, extracellular vesicles (EVs) concentration is often increased during disease development and specific EV biomarkers can be found in biofluids. We investigated the EV proteome composition in the lungs (bronchoalveolar lavage, BAL) and matching plasma of severe COVID‐19 patients by using mass spectrometry‐based proteomics.

Methods : For this retrospective, double‐centre study, EVs were isolated from small amounts of human plasma (200 µL, n = 27) and BAL ( n = 117) by using size exclusion chromatography (SEC) and characterized by western blot (WB) and transmission electron microscopy (TEM). Proteins were extracted in a urea‐containing buffer and digested with trypsin and endoproteinase Lys‐C. Generated peptides were purified and analysed by LC‐MS/MS. MS files were analysed using MaxQuant and relative protein levels were determined by label‐free quantification. Ethical approval was obtained from the Bernese Cantonal Ethics Committee and the Ethics Committee of Witten/Herdecke University, and informed consent was signed by all participants or their legal representatives.

Results : After SEC, EVs were contained in fractions 1‐4 (F1‐4). WB showed that only F1‐4 were enriched with expression of the EV markers CD63, stomatin and TSG101, while albumin was only detected from F6‐12. EV preparations (F1‐4) showed typical EV structures (size and morphology) by TEM. Proteomics of EVs preparations isolated from BAL showed that COVID‐19 positive samples had a higher median of unique proteins detected (1518, CI 118‐2027). Twelve conventional EVs markers examined were found in all our BAL samples and were present in > 70% or > 85% of the samples negative and positive for COVID‐19, respectively. Overall characterization of EVs’ COVID‐19 lung proteome shows infection signatures, both in normalized data and overall protein abundance. Gene set enrichment analysis showed cellular pathways specifically involved in COVID‐19, including inflammation and tissue remodelling, as compared to other types of lung infections. Different machine learning models, random forests, neural networks, and lasso, can all predict COVID‐19 based on this proteome signature.

Summary/Conclusion : BAL EVs proteome showed that they are rich in common and new EV markers and identified a specific lung signature in COVID‐19 that includes inflammation and tissue remodelling.

Funding : University of Fribourg.

Is EV‐associated TG2 a marker of idiopathic pulmonary fibrosis in biofluids?

Presenter: Maria Pia Savoca

Nottingham Trent University, Nottingham, UK

Abstract unavailable

Polymicrobial bEVs elicit a hyperinflammatory response in cystic fibrosis bronchial epithelial cells

Lily Charpentier 1 , Roxanna Barnaby 1 , Fabrice Jean‐Pierre 2 , George A. O'Toole 1 , Douglas Taatjes 3 , Bruce A. Stanton 1

1 Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; 2 Département de Microbiologie, Université de Sherbrooke, Sherbrooke, Québec, Canada; 3 Department of Pathology and Laboratory Medicine, The University of Vermont, Burlington, Vermont, USA

Introduction : Despite highly effective modulator therapy (HEMT; ETI) that improves outcomes in people with cystic fibrosis (CF), HEMT does not eliminate antibiotic‐tolerant polymicrobial lung infections and inflammation, the major cause of morbidity and mortality in CF. Bacteria in the CF lung reside in mucus, and we have shown Pseudomonas aeruginosa communicates with human bronchial epithelial cells (HBEC) by secreting extracellular vesicles (bEVs) that induce inflammation. Since little is known about how polymicrobial bEVs, which are more representative of lung infections than a single genus, affect host‐cell immune responses, we developed a model containing four of the most common bacteria in CF to gain insight into how polymicrobial bEVs impact the immune response of HBEC.

Methods : bEVs secreted by P. aeruginosa , Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown together in artificial sputum medium (ASM) were isolated by ultracentrifugation and Optiprep density gradient. bEVs were characterized by NTA, 16S rRNA sequencing, flow cytometry, and transmission electron microscopy according to MISEV 2023 recommendations. Wild‐type (WT), CF, and CF+ETI HBEC ( n = 5 donors/group) were treated with processed, uninoculated ASM (processed control; PC) or polymicrobial bEVs for 6 h. Gene expression was assessed by RNA‐Seq and cytokine secretion by the Millipore 48‐plex cytokine array. CFTR Cl‐ secretion was measured in Ussing chambers.

Results : Characterization of bEVs revealed that all four bacteria secrete bEVs (∼30–100 nm) and induce a hyper‐inflammatory response by CF HBEC. CXCL3 secretion (which increases chemotaxis of monocytes and lymphocytes) is increased in CF HBEC compared to PC ( p = 0.02). TNFα secretion (which stimulates inflammation) also increases ( p = 0.001). bEVs increase inflammatory pathways, including TLR and MAPK signalling, in CF HBEC + ETI. bEVs reduce CFTR Cl‐ secretion by WT ( p = 0.002) and CF HBEC+ETI ( p < 0.0001).

Summary/Conclusion : Polymicrobial bEVs elicit a robust inflammatory response in HBEC and reduce Cl‐ secretion by CF HBEC + ETI, effects that are predicted to increase recruitment of immune cells to CF lungs, which cause lung damage, and reduce the ability of ETI to clear polymicrobial infections. Studies in progress are investigating approaches to mitigate the adverse effects of bEVs on HBEC.

Funding : CF Foundation (STANTO19R0) and NIH (P30‐ DK117469 , R01HL151385, T32AI007519, and F31HL172440).

Technologies and methods

Subtopic Analysis of EV cargo 1

Single‐pot extracellular vesicle isolation and multi‐omics workflow for ovarian cancer biomarker discovery

Presenter: Tyler T. Cooper

Queen's University, Kingston, Ontario, Canada

Introduction : High‐grade serous carcinoma (HGSC) is the most common and lethal form of epithelial ovarian cancer, largely due to the lack of effective biomarkers for early detection. Ovarian cancer is often diagnosed at an advanced stage when treatment options are limited, and prognosis is poor. Current biomarkers, such as CA‐125, are insufficient for early‐stage detection, as they lack the sensitivity and specificity to identify the disease at its onset. Therefore, there is an urgent need for new, reliable biomarkers that can detect ovarian cancer at an earlier stage, improving patient outcomes and enabling timely intervention. Therefore, developing technologies that can provide deep, insightful analysis to support biomarker discovery is crucial.

Methods : In this study, we developed and optimized a workflow for the isolation and multi‐omics analysis of extracellular vesicles (EVs) from different biofluids, specifically plasma and ascites. We took this approach because EVs are known to carry a rich array of molecular information reflective of their cell of origin, making them ideal candidates for biomarker discovery. Using strong anion exchange (SAX) chemistry on magnetic beads, we successfully isolated EVs and applied a single‐pot multi‐omics approach to analyse their metabolomic, lipidomic, proteomic, and surface‐enhanced Raman (SERS) profiles. EV isolations were validated by mass spectrometry, atomic force microscopy, and nanoparticle tracking analysis.

Results : Our results demonstrate the feasibility of obtaining comprehensive proteomic and Raman data from as little as 10 to 100 µL of ascites. Likewise, we were able to obtain a metabolic, lipidomic and proteomic profile from 100 µL plasma. Notably, SAX capture of EVs was able to deplete lipoproteins, TF, and albumin orders of magnitude. We also found that the use of dodecyl‐β‐D‐maltoside (DDM) significantly enhances peptide identification up to 30%, contributing to a more in‐depth characterization of EV proteomes. The workflow was initially piloted on umbilical cord plasma (human) and murine (ID8) ascites, and we are now extending the application to plasma and ascites from human patients with HGSC.

Summary/Conclusion : This work offers a promising approach for the detailed molecular characterization of EVs and may contribute to advancing biomarker discovery for ovarian cancer detection and monitoring.

Funding : Cancer Research Society and Canadian Institute of Health Research.

Protein co‐enrichment in extracellular vesicles

Presenter: Molly L. Shen

McGill University, Montreal, Quebec, USA

Introduction : EVs carry a variety of cell‐derived proteins that confer functionality. The general expression (GEx) of proteins and co‐expression (CoEx) of pairs of proteins in EVs can be measured and help map changes in EV cargo. However, CoEx measurement cannot distinguish whether two proteins co‐localize because of active biological processes or simply due to the high GEx of individual proteins and the expected CoEx as a result of stochastic co‐packageing in EVs. Indeed, we expect highly expressed proteins to be highly co‐expressed, and if they are not, then this could be ascribed to an active avoidance process, that is, co‐depletion, which has not been reported to date.

Methods : Here, we introduce and define CoEn as the co‐enrichment of a protein relative to another in individual EVs and a population of EVs, and formalize it as the CoEx normalized by the GEx of the EV population. Axiomatically, it follows that CoEn = 1 for stochastic co‐inclusion, and CoEn > 1 or < 1 for active co‐enrichment and co‐depletion, respectively. We introduce an experimental and analytical procedure to measure the GEx, CoEx and CoEn—and differential CoEn (dCoEn) between two experimental conditions—for multiple protein pairs simultaneously using antibody microarrays and universal calibrants.

Results : We validate CoEn as a biologically meaningful metric by selectively re‐routing Syntenin‐1 away from its binding partner CD63 via the use of a CD63‐C‐terminius‐mimic peptide, and observed the expected co‐depletion (i.e., reduced CoEn) of CD63 and Syntenin‐1 without change in overall GEx. Following treatment with lysosomal acidification inhibitor bafilomycin, we find that CoEn revealed numerous protein re‐routings that were not visible by GEx. CoEn of 180 protein pairs in EVs in three model cell lines reveals pan‐cell‐line cargo‐sorting relationships, where we observed clustering of presumptive exosomal and ectosomal proteins based on their CoEn patterns. Lastly, CoEn and dCoEn analysis of EVs from parental and organotropic breast cancer progeny cell lines pinpoints cargo‐sorting and organotropism‐specific up‐ and down‐regulation with functional implication.

Summary/Conclusion : Collectively, our results establish CoEn as both an analytical tool and a hypothesis generator, suggesting that CoEn and dCoEn reflect and illuminate cell physiology, particularly in EV biogenesis and cargo sorting.

A knowledge graph model for integrating microRNAs in analysing extracellular vesicles data

Ishwor Thapa 1 , Yohan Kim 2 , Fabrice Lucien 2 , Hesham Ali 1

1 College of Information Science and Technology, University of Nebraska at Omaha, Omaha, Nebraska, USA; 2 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA

Introduction : Extracellular vesicles (EVs) participate in intercellular communication by the transfer of molecular cargo that includes microRNAs. Recently, multiple studies have identified the roles of microRNAs in EV cargo as it relates to a specific disease. Web‐based tools, such as EVAtlas, were developed to browse microRNA expression in EVs and serve mostly as visual interfaces. We propose a new knowledge graph model for storage and retrieval of relationships among multiple entities, including microRNAs, EVs, disease conditions, EV sources and mRNA targets. This model allows storage of heterogenous data types and facilitates the discovery of new connections between these entities, providing valuable insights into the functions and behaviours of microRNAs within EVs.

Methods : A knowledge graph (KG) is a structured representation of different types of data that captures relationships between entities in a particular domain. Using the NCBI Gene Expression Omnibus (GEO) resource, microRNA expression data for EVs are obtained. A KG model is constructed such that its nodes represent microRNAs, EVs, disease, EV source and target mRNAs. The edges of the graph represent the associations between its nodes, such as microRNA‐EV associations, EV‐source associations and microRNA‐mRNA interactions. The microRNA expression levels are stored as edge weights, and the microRNA‐mRNA target information is obtained from miRTarBase. The KG is then implemented in the Neo4j graph database that allows retrieval of data using the Cypher query language.

Results : Using NCBI GEO, 48 non‐redundant datasets from the last 5 years were identified that contained microRNA expression profiling of EVs from human samples. This collection is associated with various diseases such as Parkinson's, oesophagitis, liver cirrhosis and many cancers. For these datasets, sources of EVs included plasma, endometrial fluid, saliva and cancer tissues. The RNA profiling was mostly performed by high‐throughput sequencing. We report various known and new associations among the integrated datasets using the constructed KG.

Summary/Conclusion : With the integration of data from multiple sources and visualizing the complex relationships between microRNAs, EVs, cell types and target mRNAs, the proposed KG model allows the identification of miRNA‐EV‐disease relations that can explain specific gene regulation in a disease condition.

The proteomic landscape of stool‐derived extracellular vesicles in patients with pre‐cancerous lesions and colorectal cancer

Emmalee J. Northrop‐Albrecht, Yohan Kim, William R. Taylor, Surendra Dasari, Jason P. Sinnwell, Douglas M. Mahoney, Patrick H. Foote, Kelli N. Burger, Shounak Majumder, Fabrice Lucien, John B. Kisiel

Mayo Clinic Rochester, Rochester, Minnesota, USA

Introduction : Colorectal cancer (CRC) is the 2nd most common cause of cancer death in the U.S. While early detection is critical for long‐term survival, screening test effectiveness is most impacted by detection and removal of precancerous lesions (PCL). Stool‐derived extracellular vesicles (EVs) are an underexplored biomarker source that could augment the detection of PCLs in non‐invasive stool‐based screening. In this study, we examined the stool EV proteome among cancer‐free controls (CTRL), PCL, and CRC patients.

Methods : EVs were isolated from stool supernatant (1 mL; n = 63 patients [15 CTRL, 32 PCL (16 adenomas and 16 sessile serrated lesions), 16 CRC] using size exclusion chromatography followed by ultrafiltration (100 kDa) after informed consent and IRB approval. Mass spectrometry was performed, and sequences were mapped to the Human Microbiome Reference Genome and SwissProt Human databases. Proteins were annotated to the human body proteome map, and differentially expressed proteins (DEPs) were identified.

Results : Mass spectrometry revealed 468 human and 5625 (139 species) bacterial‐derived EV proteins. When limiting results to those found in ≥ 75% of samples, there were only 74 human, and 223 bacterial proteins present. Many proteins were enriched/specific to the pancreas, colon, small intestine, spleen and oesophagus. Pancreatic enzymes (CELA3A, CTRC) were among the most abundant proteins identified; further exploration revealed pancreas‐derived zymogen granules are likely co‐isolated during processing. Additionally, components of neutrophil extracellular traps (ELANE, PRTN3, MPO, LTF) were over‐represented and visible by electron microscopy. There were 13 DEPs (Padj ≤ 0.05) among CRC + PCL vs CTRL patients.

Summary/Conclusion : This work highlights the biological diversity and molecular heterogeneity of the stool EV proteome. Specifically, colon‐derived EVs are detectable in stool, but they are outnumbered by pancreas and microbiota vesicles. Furthermore, we identified 10 colon‐enriched surface proteins that may serve as potential immunocapture candidates for purification of colon‐derived EVs prior to performing additional ‐omics studies. Overall, these findings serve as the framework for future stool EV biomarker discovery in CRC and other GI‐tract malignancies.

Funding : This work was supported by the National Cancer Institute under CA214679 to J.B.K. Additional support was provided by Exact Sciences (Madison, WI), under a sponsored research agreement with Mayo Clinic.

An adaptation of the Nichenet method to study targeted communication network mediated by extracellular vesicles

Presenter: Estefania Torrejón

NOVA Medical School, Lisbon, Portugal

Introduction : Extracellular vesicles (EVs) play a pivotal role in mediating cell‐to‐cell communication (CCC), yet their significance is often overlooked in CCC network modelling. Despite abundant publicly available EVs omics datasets, tailored network‐integration tools for studying EVs‐mediated CCC remain scarce.

Methods : We adapted the existing CCC analysis tool, NicheNet (Browaeys et al., 2020), to accommodate EV‐driven CCC. Nichenet predicts ligand‐target regulatory potential by combining gene expression data with signalling and gene regulatory prior knowledge. NicheNet predictions rely on ligand‐receptor interactions. However, EVs engage with the recipient cells via additional mechanisms beyond ligand‐receptor binding. In this context, we applied Nichenet's scoring algorithm not only using ligands as starting nodes, but also including intermediary signalling proteins and transcription factors, allowing a comprehensive analysis of EV‐mediated CCC that was previously unattainable.

Results : We replaced NicheNet's original ligand‐to‐target matrix (containing 1287 ligands and 22,521 target genes) with an expanded scoring matrix encompassing 19,790 proteins—spanning ligands, intermediate signalling proteins and transcription factors—while maintaining the same number of target genes. As a use case, we applied the modified Nichenet tool to in‐house and publicly available gut‐derived small EVs proteomics data from prediabetic and healthy mouse models, and single‐cell RNA‐seq data from the Tabula Muris Atlas (The Tabula Muris Consortium, 2018), focusing on Kupffer cells (KC) as the recipient cells. Our modified tool allowed us to build a gut‐to‐KC communication network mediated by EVs. We explored the top 30 EVs proteins ranked by their regulatory potential on KC, revealing unknown communication links. For instance, in prediabetes, Selenocysteine Lyase (SCLY, Uniprot‐ Q9JLI6 ), a protein involved in redox homeostasis and overabundant in prediabetic gut EVs, showed communication links with numerous KC genes, suggesting a role in manageing oxidative stress. In healthy conditions, Apolipoprotein E (APOE, Uniprot‐ P08226 ), overabundant in gut EVs from controls, exhibited regulatory potential over Interleukin 1 beta (ILB1, Uniprot‐ P10749 ), a key mediator of cytokine release and inflammation. If APOE indeed inhibits ILB1, it could prevent hepatic inflammation.

Summary/Conclusion : Our adapted tool expands the Nichenet's applicability to EV‐mediated communication networks, offering a valuable approach for investigating the impact of EVs content on targeted recipient tissues of interest.

Funding : EMBO‐short‐scientific‐exchange‐grant & iNOVA4Health‐FCT‐fellowship‐UI/BD/154345/2022.

Development of a highly sensitive, reproducible and rapid blood EV proteome analysis platform for large‐scale cohort samples

Presenter: Jun Adachi

National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan

Introduction : Extracellular vesicles (EVs) are ubiquitously secreted by every cell type and present in all body fluids, including plasma and serum. Blood EVs can be used as a promising source for biomarker monitoring and disease diagnosis. Current liquid chromatography–mass spectrometry‐based proteome analyses are becoming an excellent balance between throughput and depth. We have optimized LC‐MS‐based proteomics for blood EVs by employing an automated EV sample preparation platform that connects to a high throughput LC‐MS platform.

Methods : Automated EV sample preparation from heparin plasma was performed by combining EV isolation with proteomic sample preparation in a 96‐well format by a magnetic particle processing robot (KingFisher Apex). The EV tryptic peptide samples were loaded into Evotips that facilitate seamLess integration with LC (Evosep‐One) and mass spectrometry and analysed using data‐independent acquisition (DIA) mass spectrometry.

Results : Analysis of optimized automated EV sample preparation identified ∼3500 protein groups with a 58‐min active LC gradient with the Evosep One and DIA mass spectrometry, providing a throughput of 20 samples per day. To assess reproducibility of the workflow, we compared the quantitative value of 24 replicate EV samples. The result indicates our system has high reproducibility and stability.

Summary/Conclusion : We have successfully connected the automated sample preparation to a high throughput LC‐MS platform. This optimized platform is attractive for processing large cohort clinical samples for biomarker development.

High‐throughput and multiplexed quantification of single EVs at single‐molecule level

Juhwan Park

Kookmin University, Seoul, Republic of Korea

Introduction : Understanding the heterogeneity of extracellular vesicles (EVs) has a promising potential for disease diagnosis, but current methods for single EV analysis are limited to either low throughput or less multiplexity.

Methods : EVs labelled with multiple antibodies conjugated with RCA templates were encapsulated into agarose droplets with RCA mixture. Cleavable chemistry was used to overcome steric hindrance of RCA products (hundreds of nm) over a single EV (∼100 nm). The RCA template is conjugated to antibodies through a disulphide bond that can be cleaved by a reducing agent originally included in the RCA mixture. Once EVs are encapsulated into agarose droplets with RCA mix, cleavage of RCA templates from antibodies and the RCA reaction occur simultaneously. After the RCA reaction followed by agarose gelation, agarose beads were transferred to the aqueous phase, and RCA products within agarose beads were labelled with fluorescent detection probes.

Results : To validate the single EV assay, EVs from Mel‐624‐B7H1 cells were labelled with anti‐CD63 antibodies conjugated with an RCA templates and were encapsulated into 0.3% agarose droplets with RCA mixture. By titrating EVs concentration, the number of beads with RCA products decreases. EVs distribution over agarose beads reaches a digital regime by noting that less than 10% of agarose beads showed multiple RCA products whereas more than 90% of agarose beads did not have any RCA products. By analysing >100,000 agarose beads using a flow cytometer, the limit of detection was assessed to be 7 EVs/µL. The effect of the reducing agent was evaluated that enables single EV quantification at the single‐molecule level. To characterize the multiplex single EV assay, EVs were labelled with tetraspanin antibodies with different RCA templates. The positive ratio of each tetraspanin was measured by flow cytometry, which agrees with the EXOVIEW result. Furthermore, the co‐localization of tetraspanin was studied in a single EV level (> 10,000 EVs). We further measured PD‐L1 expression in single EV levels that play a key role in the immune checkpoint of cancer.

Summary/Conclusion : We developed a novel method to quantitatively analyse single EVs with multiplexing capabilities, high throughput, and ultra‐sensitivity, both to single molecules on an individual EV and to rare EVs suspended in complex clinical specimens, using an agarose droplet‐based RCA technique.

Identification of robust and abundant EV reference mRNA transcripts from diverse biological sources

Antje Zickler 1,2,3 , Radosław Grochowski 1 , André Görgens 1,2,3 , Erik Bäcklin 1 , Maximilian Kordes 3 , J.‐Matthias Löhr 1 , Joel Nordin 1,2,3 , Daniel Hagey 1,3 , Samir EL Andaloussi 1,2,3

1 Karolinska Institutet, Stockholm, Sweden, 2 Karolinska ATMP Center, Stockholm, Sweden; 3 Karolinska University Hospital, Stockholm, Sweden Joel Nordin, Daniel Hagey, and Samir EL Andaloussi are co‐senior authors .

Introduction : Extracellular vesicles (EVs) have garnered substantial interest as potential biomarkers and bioengineered therapeutic nanoparticles due to their ability to carry diverse biomolecules. However, reliably quantifying EVs has proven challenging, partly due to a lack of established molecular standards and standardized datasets. The heterogeneity of EV subtypes and their cellular sources further complicates consistent identification and comparison across studies.

Methods : To identify consistent reference mRNA transcripts in EVs, we analysed oligo‐dT primed RNA‐sequencing data from EVs isolated from twelve distinct cell types. EV isolation was performed using differential centrifugation followed by ultrafiltration. From these analyses, we identified the 50 most abundant mRNA transcripts across all samples. Following RT‐PCR and deeper sequencing analysis, five transcripts warranted further investigation as molecular standards: TMSB4X, ACTB, GAPDH, VIM, and FTL. To validate these, RT‐qPCR results from two oligo‐dT cDNA synthesis methods were subjected to stability assessment via the RefFinder analysis tool, with a proof‐of‐concept normalization applied to the variably expressed gene, RAB13.

Results : Our analysis revealed that two transcripts, TMSB4X and GAPDH, were consistently among the most abundant across all EV types. To judge the applicability of these genes as reference transcripts for biomarker studies, we performed RNA‐sequencing on EVs isolated from plasma by differential ultracentrifugation. Four of the transcripts assessed here were amongst the 50 most highly expressed by each donor, confirming the applicability of these genes as molecular standards for EV‐mRNA analysis.

Summary/Conclusion : Our findings establish stable reference mRNA transcripts across multiple EV types and sources, supporting their utility in standardizing EV research. Standardizing EV‐mRNA studies with these molecular references can improve consistency across experimental contexts and advance EV research by providing reliable markers for mRNA normalization in diverse applications.

Understanding MitoEVs: a Jurkat cell model of radiation‐induced senescence and lysosomal dysfunction

Iris Potakowskyj 1,2 , Gorjana Rackov 1

1 Team TDA, BioMed X Institute, Heidelberg, Germany; 2 Faculty of Biology, Heidelberg University, Heidelberg, Germany

Introduction : Extracellular vesicles carrying mitochondrial content (MitoEVs) have recently gained much attention. Studies have demonstrated that MitoEVs can deliver mitochondrial components to recipient cells and affect their function. Some models for studying MitoEV release have been developed but lack fundamental characterization limiting their application.

Methods : Here, we characterize two approaches used to produce MitoEVs in Jurkat cells. The first model uses ionizing radiation, which induces mitochondrial DNA damage; the second one inhibits lysosomal function and thereby disrupts mitophagy. We characterized mitochondrial cargo within large and small extracellular vesicles released from the cells in vitro using protein analysis techniques and mitochondrial DNA sequencing.

Results : Our results showed that each model results in a different MitoEV composition. While ionizing radiation increases the incorporation of damaged mitochondrial DNA into small EVs, bafilomycin treatment targets some mitochondrial proteins to large EVs. We show that mitochondrial DNA mutations can be detected within MitoEVs and increase upon radiation in a dose‐dependent manner. Using proteinase and DNase protection assays, we demonstrated that mitochondrial cargo is attributable to EVs.

Summary/Conclusion : This data aids in the understanding of two approaches for producing MitoEVs in Jurkat cells and presents several assays for MitoEV detection. Additionally, this protocol can be applied to detect MitoEVs produced by different cell types in vitro and in vivo.

Funding : This research is financially supported by Merck KGaA.

Detailed heterogeneity evaluation of EV preparation by anion exchange HPLC and various analytical methods

Hirotaka Nishimura, Tomofumi Yamamoto, Masato Kiyoshi, Noritaka Hashii, Akiko Ishii‐Watabe

National Institute of Health Sciences, Tokyo, Japan

Introduction : The number of clinical trials involving EV administration has been increasing in recent years, and the day when the first EV drug is launched is drawing near. However, there is still little consensus on how to evaluate the quality and ensure the safety and efficacy of EVs in the development of pharmaceutical products. In particular, it is necessary to establish a new evaluation method to assess particle heterogeneity in EV formulations, which is not used for conventional pharmaceutical products. In this study, we propose a detailed characterization method using ion‐exchange HPLC fractionation and various analytical methods as a method to evaluate the heterogeneity of particle components in EV formulations.

Methods : We used EVs derived from adipose tissue MSC and purified with TFF as a test sample. EVs were analysed using anion exchange HPLC with a multiangle light scattering detector (AEX‐MALS), and the main peaks containing particles were collected. These purified fractions and input EVs were characterized by TEM, NTA, nano‐flow cytometry, and proteomic analysis by LC‐MS/MS.

Results : AEX‐MALS revealed that the particulate contents in the EVs were classified into two major groups by their charge properties. TEM observations provided precise particle structures and accurate size distribution information for each sample, but the size distribution was quite different from those measured by NTA and nano‐flow cytometry. Detailed evaluation of expressed molecules by nano‐flow cytometry revealed significant differences in RNA loading rates and expression rates of common EV markers such as CD9 and 81 among the samples, suggesting that each fraction separated by AEX depending on the charge is composed of different types of EVs. Proteomic analysis showed that one fraction after purification contained many proteins that are considered EV markers in the database, such as Exocarta and Vesiclepedia (about 70% of the top 30 proteins and 50% of the top 100 proteins), suggesting that AEX is useful for evaluation of EV‐derived proteins in EV samples.

Summary/Conclusion : AEX‐MALS is useful not only for evaluating the charge heterogeneity of particles in EV formulations, but its use in purification will also contribute significantly to the manufacturing of EV products with higher purity.

Funding : AMED JP22mk0101218, JP24am0521007.

Investigating the interaction network of antimicrobial peptides with urine‐derived and red blood cell‐derived extracellular vesicles

Tasvilla Sonallya 1,2 , Tünde Juhász 1 , Imola Cs. Szigyártó 1 , Kinga Ilyés 2,3 , Priyanka Singh 1 , Delaram Khamari 4 , Edit I. Buzás 4,5,6 , Zoltán Varga 3,7 , Tamás Beke‐Somfai 1

1 Biomolecular Self‐Assembly Research Group, Institute of Materials and Environmental Chemistry, HUN‐REN Research Centre for Natural Sciences, Budapest, Hungary; 2 Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 3 Biological Nanochemistry Research Group, Institute of Materials and Environmental Chemistry, HUN‐REN Research Centre for Natural Sciences, Budapest, Hungary; 4 Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary; 5 HCEMM Extracellular Vesicle Research Group, Semmelweis University, Budapest, Hungary; 6 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Budapest, Hungary; 7 Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Budapest, Hungary

Introduction : Extracellular vesicles (EVs) are membrane‐enclosed structures essential for cargo transport and cellular communication. However, interactions between EVs and membrane‐active peptides (MAPs) remain underexplored, particularly regarding protein corona removal. MAPs, including antimicrobial peptides (AMPs) and cell‐penetrating peptides (CPPs), have significant roles in antimicrobial, anticancer, and cargo delivery applications. While HDPs disrupt membranes, CPPs help deliver molecules, though their interactions with EVs and impacts on protein corona remain largely unstudied.

Methods : Red blood cell extracellular vesicles (REVs) and urine extracellular vesicles (UEVs) were isolated and purified using a Sepharose CL‐2B gel column. Initial investigations of the interactions between 26 membrane‐active peptides (MAPs) and REVs were conducted using linear dichroism (LD) spectroscopy, nanoparticle tracking analysis (NTA), zeta potential, flow cytometry, and transmission electron microscopy (TEM). Peptides were then categorized based on their potential applications. From each category, five peptides were selected for further study with UEVs, with interactions characterized using flow cytometry, microfluidic resistive pulse sensing (MRPS), TEM, and proteomics. Additionally, the REV‐MAP interactions for these peptides were also characterized through proteomic analysis.

Results : Peptides were categorized into distinct functional groups based on their interactions with extracellular vesicles (EVs). At low concentrations, peptides like LL‐37 and lasioglossin‐III effectively removed the protein corona (PC) with minimal membrane disruption. Conversely, peptides such as KLA, bradykinin, histatin‐5, and most cell‐penetrating peptides (e.g., octa‐arginine, penetratin, and buforin II) showed strong cell‐penetrating abilities, maintaining structural integrity even at high concentrations. This analysis reveals potential interaction mechanisms between AMPs and EVs in vivo and suggests how specific peptides might be used to manipulate EVs for targeted applications. Five peptides—lasioglossin‐III, aurein 1.2, FK16, DHVAR 4, and dermcidin—were selected for further proteomic study on protein corona dynamics in REVs and UEVs.

Summary/Conclusion : This study explores the interactions between red blood cell‐derived extracellular vesicles (REVs) and amphipathic peptides, providing insights into their roles in the innate immune system. The proteomic analysis highlights the significance of identifying protein corona components, advancing the potential of peptides as engineering tools for EVs and other biological nanoparticles.

Funding : 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021, TKP2021‐EGA‐31 and KKP_22 project no. 144180, SA‐87/2021 and KEP‐5/2021.

Gp130‐enriched engineered small extracellular vesicles ameliorate septic acute lung injury by competitively binding IL‐6

Presenter: Feng Chen

Tsinghua University, Beijing, People's Republic of China; Southeast University, Nanjing, People's Republic of China

Introduction : Sepsis is a fatal disease with high morbidity and mortality, of which acute lung injury is the earliest and most serious complication. Excessive inflammatory response is an important mechanism in the development of sepsis‐induced acute lung injury (S‐ALI). Interleukin‐6 (IL‐6) is a pleiotropic cytokine whose abnormal activation of signal transduction plays an important role in the inflammatory cascade of sepsis, thus promoting SALI. Although extracellular vesicles derived from mesenchymal stem cells (MSC‐sEV) have anti‐inflammatory and repair promoting effects, they also have problems of low efficacy. How to develop potent EVs‐based therapeutic systems remains to be studied.

Methods : We first obtained MSC‐sEV and HEK293F‐sEV by differential ultracentrifugation combined with size‐exclusion chromatography, and then characterized their properties using transmission electron microscopy, nanoparticle tracking analysis, nano‐flow cytometry and western blot. First, we used MSC‐sEV to intervene in the mice with SALI and screened the effective active components of MSC‐sEV and their mechanisms of action by proteomic analysis. Then, we transfected HEK293F cells using plasmids to overexpress the effective active component. Further, we obtained engineered HEK293F‐sEV for the treatment of SALI mice to evaluate its real effect.

Results : We successfully isolated MSC‐sEV and HEK293F‐sEV and confirmed that their characteristics were consistent with MISEV. First, MSC‐sEV was significantly enriched in the lungs of mice with SALI and was effective in ameliorating inflammatory injury and overall survival. Second, the proteomics results of MSC‐sEV showed that its membrane was enriched in gp130, and sEV carrying gp130 could competitively bind IL‐6 when injected into mice via tail vein, thus blocking the cascade amplification of inflammation and alleviating the inflammatory injury in the lungs. To facilitate future clinical translational applications, the engineered HEK293F‐sEVs express high levels of gp130 and also show significant therapeutic effects.

Summary/Conclusion : MSC‐sEV carrying gp130 competitively binds IL‐6 and thus attenuates SALI, while gp130‐enriched engineered HEK293F‐sEV likewise exerts a significant protective effect, which facilitates future clinical translational applications.

Multi‐omics analysis of extracellular vesicles of human forebrain spheroids in a vertical‐wheel bioreactor

Chang Liu 1 , Li Sun 1,2 , Hannah Worden 3 , Justice Ene 1 , Sunghoon Jung 3 , Yan Li 1

1 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering, Florida State University, Tallahassee, Florida, USA; 2 Department of Biomedical Sciences, Florida State UniversityTallahassee, Florida, USA; 3 PBS Biotech Inc, CA, Camarillo, California, USA

Introduction : Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell‐free therapies in various diseases, including stroke. However, because of the significant amounts of EVs needed in preclinical and clinical trials, it is still challenging for EV application. Vertical‐wheel bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress.

Methods : In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3‐D aggregates (Agg) and on synthemax II microcarriers (MC) in VWBRs was investigated compared to a static aggregate culture control. The spheroids were characterized by transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and western blot. The EV cargo was analysed by proteomics and miRNA‐sequencing. The in vitro oxygen and glucose deprived stroke model and proof of concept in vivo study were performed.

Results : Human forebrain spheroids differentiated on microcarriers showed a higher growth rate than 3‐D aggregates. The VWBR culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes compared to the static culture. EVs from the three conditions had similar sizes, but the yields from high to low were microcarrier culture, dynamic aggregates, and static aggregates. The transcriptome of parent cells based on mRNA sequencing and EV proteomics data of Agg versus MC revealed a weak increasing trend of EV differentially expressed proteins (DEPs) with the parent cell differentially expressed genes (DEGs). The cargo is enriched with proteins (based on proteomics) and miRNAs (based on miRNA‐seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. The EVs demonstrated the ability to improve recovery in an in vitro stroke model and an in vivo rat model.

Summary/Conclusion : Human forebrain spheroids differentiated in VWBR significantly increased EV yields (up to 240–750‐fold) and EV biogenesis compared to static control due to dynamic microenvironment and metabolism alteration. The biomanufactured EVs from VWBRs are enriched with therapeutic cargo and functional in vitro and are promising for in vivo study.

Funding : This project is supported by NSF CAREER 1652992 and NIH R01NS125016 of the USA.

hnRNPA2b1 is a key regulator of BM‐EV‐miRNA cargo loading in the response of bone marrow to cellular stress initiated by ionizing radiation exposure

Ilona Barbara Csordás 1,2 , Tamás Visnovitz 3 , Christophe Badie 4 , Katalin Lumniczky 1

1 Unit of Radiation Medicine, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary; 2 Doctoral School of Pathological Sciences, Semmelweis University; Budapest, Hungary; 3 Semmelweis University, Faculty of Medicine, Department of Genetics, Cell‐ and Immunobiology; Budapest, Hungary; 4 Centre for Radiation, Chemical and Environmental Hazards, UK Health Security Agency, Chilton, Didcot, UK

Introduction : Bone marrow (BM) is the site of haematopoiesis, where proper function relies on interactions between BM stem and stromal cells. Extracellular vesicles (EVs) play a crucial role in this interaction by delivering bioactive cargo, such as microRNAs (miRNAs). The EV‐miRNA cargo is determined by the status of the donor cell; thus, cellular stressors, such as ionizing radiation (IR), modify its composition. In vivo, we demonstrated that IR‐modified EVs transfer radiation‐induced damage to non‐irradiated cells, with miRNAs serving as important regulators. To better understand the role of EVs in the BM microenvironment, we investigated BM‐ and EV‐miRNA cargo and EV‐miRNA loading by RNA‐binding proteins (RBPS) after IR exposure.

Methods : Nine‐ to twelve‐week‐old male CBA‐mice were irradiated, and their BM was collected 24 h after IR. BM‐EVs were isolated with the precipitation method and EV markers were detected with western blot (WB). EV and BM miRNA profiles were analysed with NCoutner and/or qPCR, then clustered into BM‐IR response‐relevant pathways: leukaemia/senescence/inflammation, and a sequence motif search was also conducted. Based on identified miRNA motifs, RBPs were selected for concentration (WB) and cellular localization (confocal microscopy) analysis. Predicted miRNA‐RBP interactions were validated in vivo by immunoprecipitation.

Results : Seventy‐one percent of the EV‐miRNAs associated with leukaemia/senescence/inflammation were linked to three RBPs: Anxa2, hnRNPQ and hnNRPA2b1. Among them, hnRNPQ and hnRNPA2b1 expression altered upon IR. hnRNPA2b1 exhibited upregulation in the BM but downregulation in BM‐EVs; the miRNAs carrying recognition motifs of hnRNPA2b1 mirrored the quantitative changes of their binding partner in EVs. We established the specific binding of miRNAs to hnRNPA2B1 through their binding motifs, as miRNAs lacking these motifs did not co‐precipitate with the protein.

Summary/Conclusion : The miRNA content of BM‐EVs following IR exposure results from a selective sorting and packageing mechanism. One observed mechanism through which IR influences this process is by deregulating RBPs, particularly hnRNPA2b1. Selectively packaged EV‐miRNAs could be involved in the development of late radiation effects, such as leukaemia development in the BM.

Funding : Funding: This research was supported by the European Union's Euratom research and training programme 2014–2018 CONCERT under the grant agreement No. 662287 and the Euratom research and training programme 2021–2025 PIANOFORTE under the grant agreement No. 101061037.

Subtopic Engineering and loading EVs 1

Coenzyme Q10 delivery to muscle cells using exosomes‐mimetic nanovesicles

Rachele Agostini 1 , Mattia Tiboni 1 , Stephanie Fondi 1 , Paola Ceccaroli 1 , Emanuela Polidori 1 , Patrick Orlando 2 , Sonia Silvestri 2 , Andrea Frontini 2 , Luca Casettari 1 , Michele Guescini 1

1 University of Urbino Carlo Bo, Urbino, Italy; 2 Polytechnic University of Marche, Ancona, Italy

Introduction : Coenzyme Q (CoQ) is an endogenous lipophilic quinone ubiquitous in biological membranes endowed with antioxidant and bioenergetic properties. CoQ deficiency can result from either a genetic defect or secondary deficiencies due to ageing, oxidative stress, or drug interaction with biosynthesis. In these conditions, CoQ supplementation represents a relevant strategy to mitigate CoQ deficit; however, numerous studies have demonstrated that the efficiency of dietary CoQ uptake is a limiting step. Exosomes and, more generally, extracellular vesicles (EVs), the endogenous nanocarriers that can deliver biological information between cells, were recently proposed as a new drug delivery system. EVs resemble liposomes in terms of size, shape, and structure. The ability of EVs to transport biomolecules to recipient cells has made them attractive for drug delivery purposes. Here, we developed bioinspired exosome‐mimetic nanovesicles to deliver ubiquinone (CoQ10) to different cell types.

Methods : The CoQ10‐loaded nanovesicles (Q10‐eNVs) were produced using a mixture of synthetic choline‐based phospholipids, membrane proteins isolated from C2C12 myoblasts, and oxidized CoQ10. Unilamellar vesicles were obtained by extrusion through cellulose acetate membranes.

Results : The Q10‐eNVs have a similar diameter to exosomes (about 110 nm), and the obtained CoQ10 packageing into Q10‐eNVs was 25 µg/mL. Human fibroblasts, HeLa, mice skeletal muscle (C2C12), and rat cardiomyocytes (H9c2) were treated with 6.25 µg/mL of Q10‐eNVs for 24 h. The obtained results showed that H9C2 ware able to take up 3 times more CoQ10 than the other cell types.

Summary/Conclusion : Taken together, these studies suggest that the bioengineered nanovesicles can serve as novel exosome‐mimetics to deliver CoQ10 to cardiomyocytes effectively.

Funding : This work has been funded by the European Union—NextGenerationEU—under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041 ‐ VITALITY—CUP [H33C22000430006].

Subtopic Assay development 1

High‐dimensional single‐cell profiling of EV recipients by tellurium‐based labelling of the EV proteome (TeLEV) identifies EV‐driven transformation of the immune microenvironment in chronic lymphocytic leukaemia

Daniel Bachurski 1 , Rahil Gholamipoorfard 1 , Yong Jia Bu 2 , Patrick Hölker 1 , Lisa Wessendorf 1 , Hendrik Jestrabek 1 , David Stahl 1 , Philipp Gödel 1 , Felix Gaedke 1 , Elias Ranjbari 3 , Selen Seyhan 4 , Ulrike Resch 5 , Luisa Marie Schmidt 1,6 , Tobias Tertel 7 , France Rose 1 , Cláudio Pinheiro 8 , Maribel L. Corona 9 , Alexander F. vom Stein 1 , Phuong‐Hien Nguyen 1 , Katrin Reiners 10 , An Hendrix 8 , Guillaume van Niel 9 , Marcus Krüger 1 , Katarzyna Bozek 1 , Lydia Meder 11 , Per Malmberg 3 , Paula Cramer 1 , Barbara Eichhorst 1 , Roland Ullrich 1 , Astrid Schauss 1 , Christian Pallasch 1 , Paul J. Bröckelmann 1,12 , Ron D. Jachimowicz 1,12 , Christian Preusser 13 , Elke Pogge von Strandmann 13 , Bernd Giebel 7 , Mark Nitz 2 , Michael Hallek 1 , Nima Abedpour 1

1 Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; 2 University of Toronto, Toronto, Canada; 3 University of Gothenburg, Gothenburg, Sweden; 4 Dokuz Eylül University, İzmir, Türkiye; 5 University of Vienna, Vienna, Austria; 6 University of Copenhagen, Copenhagen, Denmark; 7 University of Duisburg‐Essen, Essen, Germany; 8 Univesity of Ghent, Ghent, Belgium; 9 Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, van Niel's Team, Paris, France; 10 University of Bonn, Bonn, Germany; 11 University of Erlangen‐Nürnberg, Erlangen, Germany; 12 Max Planck Institute for Biology of Ageing, Cologne, Germany; 13 Philipps University Marburg, Marburg, Germany Daniel Bachurski, Rahil Gholamipoorfard and Yong Jia Bu are co‐first authors . Mark Nitz, Michael Hallek and Nima Abedpour are co‐senior authors . Daniel Bachurski is corresponding author .

Introduction : EVs play a critical role in intercellular communication within the tumour immune microenvironment (TIME), emerging as crucial in immunomodulation. However, due to methodological limitations—such as reliance on bulk EV methods and the lack of routine implementation of single‐cell (sc) techniques—the bioimmune distribution and effects of EVs on individual recipient cells remain insufficiently understood.

Methods : To address this gap and by considering the MISEV guidelines, we developed TeLEV. Our metabolic mass‐tag labelling approach using monoisotopic L‐tellurienylalanine (TePhe) to label cellular secretomes enables high‐dimensional sc analysis of EV recipient cells by (imaging) mass cytometry and nanoSIMS. Using TeLEV, we analysed the uptake and effects of the EV proteome and soluble protein fractions at the sc level in 14 million immune cells, examining EV uptake dynamics over 48 h, dose‐response relationships, and differential SEC fraction uptake. By this, we provided an immune atlas of EV uptake in 50 immune cell populations and subpopulations, comparing EVs from six primary, treatment‐naïve chronic lymphocytic leukaemia (CLL) cells purified from peripheral blood with those from nine cell lines. The findings were validated by analysing 6 million single cells from 33 CLL patient samples.

Results : We discovered that primary CLL‐TeLEVs were predominantly taken up by classical, transitional, and non‐classical monocytes and myeloid dendritic cells in a CD11c‐ and BTK‐dependent manner, leading to immune alterations in T cell effector memory and NK cells. Healthy donor myeloid cells showed a specific CD25, IL‐3R, and IL‐7R expression signature only when exposed to primary CLL and Ramos cell line TeLEVs, demonstrating the specific functional role of malignant B cell‐derived EVs compared to different EV sources. Notably, CLL‐TeLEVs induced BCL‐2 expression in immune recipient cells, promoting myeloid cell selection of the CLL TIME. Finally, we employed integrative sc multi‐omics—CITE‐seq and CyTOF—to confirm the EV‐dependent selection mechanism in CLL.

Summary/Conclusion : In conclusion, we developed TeLEV to enable comprehensive sc immune uptake studies of EVs from primary samples and cell lines. Applying TeLEV, we uncovered a novel CLL‐EV‐driven immune selection mechanism, underscoring the role of EVs as global mediators of immune cell communication.

Physiological versus traditional EV administration: improved biodistribution and reduced toxicity with slow‐release pumps

Presenter: Tomer Cooks

Ben Gurion University, Beersheba, Israel

Introduction : Extracellular vesicles (EVs) are essential mediators of intercellular communication. The method of EV administration in vivo significantly impacts their biodistribution and their effect on recipient tissues and cells. Current in vivo EV injection methods rely on direct pulses of large amounts of EVs, poorly mimicking the ‘real‐life’ chronic continuous release of EVs from cells and tumours. This study aims to compare the physiological EV secretion rates with common EV injection methodologies and assess the effectiveness of an osmotic slow‐release pump in delivering EVs continuously for substantial periods to create a chronic delivery mechanism faithfully reflecting physiological conditions.

Methods : We employed two types of osmotic slow‐release pumps (Alzet pumps as well as iPRECIO pumps) to administer EVs at a controlled, sustained, programmable rate and compared them with the traditional injection methods (intraperitoneal [ip] and intravenous [iv]). Biodistribution and concentration of fluorescently labelled EVs were monitored in various organs, including the pancreas and liver, over multiple time points. The concentration of EVs was measured using NTA and TRPS, and EM was used to verify morphology. We used gold nanoparticles as markers for EV cargo deposited in the target tissues. Additionally, the study evaluated the negative side effects associated with high‐concentration EV injections.

Results : The osmotic slow‐release pump demonstrated a more consistent and physiological distribution of EVs across the targeted tissues compared to traditional injection methods. Traditional injection methods led to a localized, short‐term, and intense cellular response with potential adverse effects, including cellular saturation and toxicity. The slow‐release method mitigated these issues, providing a more stable and prolonged delivery of EVs. When refillable pumps were used, we were able to load fresh batches of EVs and provide tissue targeting (pancreas and liver) spanning periods of weeks and months.

Summary/Conclusion : The study highlights the limitations of robust single EV injections emphasizing the benefits of using an osmotic slow‐release pump for EV delivery. This method ensures a more physiological and sustained release of EVs, enhancing their therapeutic potential and reducing adverse effects. Future research should focus on optimizing the slow‐release delivery system for clinical applications and further exploring its impact on EV‐mediated therapies.

DetectEV: a functional enzymatic assay to assess the integrity and bioactivity of extracellular vesicles

Giorgia Adamo *1 , Sabrina Picciotto 1 , Paola Gargano 1 , Angela Paterna 2 , Samuele Raccosta 2 , Estella Rao 2 , Daniele Paolo Romancino 1 , Giulio Ghersi 3 , Mauro Manno2, Monica Salamone 1 , Antonella Bongiovanni 1

1 Cell‐Tech HUB and Institute for Research and Biomedical Innovation (IRIB), National Research Council of Italy (CNR), Palermo, Italy; 2 Cell‐Tech HUB and Institute of Biophysics (IBF)—CNR, Via Ugo La Malfa, Palermo, Italy; 3 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Palermo, Italy

Introduction : The therapeutic and diagnostic promise of extracellular vesicles (EVs) as bioactive nanocarriers in cell‐free therapies necessitates robust quality control, particularly in assessing bioactivity, membrane integrity, and batch consistency. Given the typical heterogeneity of EV preparations, these assessments require functional assays that provide essential quality control metrics to complement existing physicochemical methods and ensure EV preparations meet stringent quality standards. The DetectEV assay introduced here was developed to address these limitations, offering a fast, sensitive, and quantifiable evaluation of EV functionality.

Methods : DetectEV is an enzymatic‐based assay that quantifies esterase activity within the EV lumen and assesses membrane integrity, presenting results in enzymatic units. Using microalgae‐derived EVs, or nanoalgosomes, as an EV‐model, we optimized assay parameters and validated its performance in detecting changes in bioactivity and integrity across different EV types (including human cell‐derived EVs), isolation methods, and storage conditions, requiring only minimal sampling for rapid analysis.

Results : DetectEV demonstrated high sensitivity and specificity in quantifying esterase activity in nanoalgosomes and various human‐derived EV types. The assay effectively distinguished batch‐to‐batch variability, with its readout correlating to specific functional properties, such as antioxidant capacity in nanoalgosomes. Comparative analyses showed that DetectEV could detect differences in EV bioactivity and integrity resulting from various isolation, loading, and storage methods, outperforming conventional approaches that primarily focus on physicochemical EV characteristics.

Summary/Conclusion : The DetectEV assay provides an efficient, single‐step analysis of EV functionality and membrane integrity, enabling reliable quality control for various EV applications. Its broad compatibility with diverse EV types and minimal sample requirements make it a versatile and practical tool for standardizing EV functionality assessments prior to specific EV‐based potency tests. The predictive capability of the DetectEV assay for EV functionality underscores its potential to support EV‐based therapeutic and diagnostic development.

Funding : This work was supported by the VES4US and the BOW projects, funded by the European Union's Horizon 2020 research and innovation programme, under grant agreements nos. 801338 and 952183, MUR PNRR “National Center for Gene Therapy and Drugs based on RNA Technology” (Project no. CN00000041 CN3 RNA) and by the Institute for Research and Biomedical Innovation (IRIB), National Research Council of Italy of Palermo (Internal Call@IRIB2023).

Probing extracellular vesicle interactions with lipoproteins in healthy and disease contexts

Raluca Ghebosu 1 , Jenifer Pendiuk Goncalves 1 , Nur Indah Fitri 1 , Dalila Iannotta 2 , Mohammad Farouq Sharifpour 3 , Elaina Coleborn 4 , Alex Loukas 3 , Fernando Souza‐Fonseca‐Guimaraes 4 , Joy Wolfram 1,2,

1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia; 2 School of Chemical Engineering, The University of Queensland, Brisbane, Queensland, Australia; 3 Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia; 4 Frazer Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, Brisbane, Queensland, Australia

Introduction : Recent evidence suggests that other circulating biological nanoparticles, primarily lipoproteins, can bind to extracellular vesicles (EVs) and change their biological identity. This has been demonstrated using complex techniques; however, there remains a need to rapidly and simply quantify EV‐lipoprotein binding and identify intergroup differences in EV binding to lipoproteins. Uncovering altered lipoprotein binding to certain EVs could have major implications for understanding EV biology and developing medical applications.

Methods : Here we demonstrate a first‐of‐its‐kind, simple and quick proprietary assay to assess EV binding to lipoproteins. The assay (proprietary, limiting disclosure) provides sensitive readouts that can effectively predict the extent of EV binding with lipoproteins. Non‐human derived EVs were isolated by size‐exclusion chromatography, while EVs from various human cell types, including natural killer cells, osteosarcoma and breast cancer, were isolated using tangential flow filtration. All EVs were authenticated using nanoparticle tracking analysis, western blot, and cryogenic transmission electron microscopy.

Results : The assay was validated with synthetic nanoparticles, small molecules, polymers, and proteins that display known interactions with lipoproteins. We then probed EV interactions to demonstrate that EVs from various human and non‐human (nematode and bacteria) sources bind to lipoproteins. Notably, EVs derived from cancerous cells displayed substantially greater binding to lipoproteins compared to EVs from cell type‐matched healthy cells. Additionally, EVs from a metastatic osteosarcoma line were found to bind to lipoproteins significantly more than those from corresponding patient‐matched non‐metastatic osteosarcoma cells. This trend was also shown in a breast cancer model where poorly metastatic breast cancer cell line derived EVs have significantly less lipoprotein binding when compared to highly metastatic breast cancer cell line EVs. Direct associations between osteosarcoma EVs and lipoproteins were also shown by cryogenic transmission electron microscopy, with a substantially greater number of interactions seen in the metastatic osteosarcoma cell line—mirroring the assay.

Summary/Conclusion : Taken together, we show that the developed assay is capable of measuring EV‐lipoprotein binding in a simple, rapid, and quantitative manner and can be utilized to uncover differing levels of EV binding to lipoproteins. These findings open new avenues to probe EV biology and develop novel therapeutics and diagnostics.

Bead‐based multiplex immunoassay to measure extracellular vesicle surface proteins in plasma and serum

Presenter: Erez Eitan

NeuroDex Inc., Natick, Massachusetts, USA

Introduction : All cell types release extracellular vesicles (EVs), some entering the circulation. Typically, EVs are characterized after isolation via nanoparticle tracking analysis, though this approach can introduce variability due to isolation procedures and contamination from non‐EV particles. In this study, we applied a sandwich immunoassay with antibodies targeting two distinct proteins to directly measure CD63‐, CD9‐, and CD81‐positive EVs in plasma.

Methods : We utilized a bead‐based multiplex immunoassay based on the Luminex system. EV analysis was validated following the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines and immunoassay qualification protocols to ensure specificity, sensitivity, linearity, and precision.

Results : Both singleplex and multiplex assays produced comparable signals. Specific antibodies generated at least threefold higher signals than non‐specific antibodies, and EV depletion eliminated the signal, confirming specificity. Variability between wells and plates was below 15%, and parallelism was demonstrated across 16‐fold dilutions. Using this method, we assessed CD63‐, CD81‐, and CD9‐positive EV levels in 205 individual plasma samples. CD9‐positive EVs were significantly more abundant than CD63 and CD81. In healthy individuals ( N = 80), plasma EVs had coefficients of variance of 22.4% for CD9, 65.76% for CD63, and 71.7% for CD81. Neurodegenerative patients ( N = 78) showed significantly higher CD9‐positive EVs (+10%, p = 0.002) and significantly lower CD63 and CD81‐positive EVs (−27%, p = 0.002 for CD63; −28%, p = 0.03 for CD81). Post‐COVID patients ( N = 47, 2–5 months post‐infection) showed no significant EV differences. In 17 matched plasma and serum samples, EV levels (regardless of tetraspanin) were ∼1.8‐fold higher in plasma, with a strong plasma‐serum correlation ( R = 0.71). A slight age‐related EV decrease was observed, with CD63 showing significance ( R = 0.49, p = 0.02). Analysis is ongoing in a cohort of 1200 individuals.

Summary/Conclusion : This method enables semi‐high‐throughput measurement of EV surface proteins in unprocessed plasma samples in a 96‐well format, supporting population‐level EV analysis and insights into factors driving EV variability. It is adaptable to other EV surface proteins, facilitating biomarker discovery and normalization efforts.

In vitro models of the blood brain barrier or blood tumour barrier to study the transport of exosomes and plant‐derived vesicles

Presenter: Olga Janouskova

Jan Evangelista Purkyně University, Ústí nad Labem, Czech Republic

Introduction : Extracellular vesicles (EVs), specifically exosomes (EXs) serve as biomarkers, important modulators of physiological or pathological processes, or therapeutic agents. They are utilized as a medical approach for their unique content or unique targeting possibilities. The characterization of the transport of the EVs from animal or plant sources via the blood brain barrier (BBB) or brain tumour barrier (BTB) are not a fully resolved question. We focused on the development of in vitro models of the BBB and BTB based on microchips or perfusion cultivation models to study the transport of EXs from various sources, which can influence this process and EX targeting.

Methods : EXs from primary glioblastoma tumour cell lines, blood of glioblastoma patients and plant (plant tissue cultures or calluses) derived EVs were isolated by ultracentrifugation and characterized using DLS, NTA, cryoTEM. In vitro models of BBB or BTB were prepared using the ibidi cultivation system or vertical microchips seeded with endothelial cells and astrocytes (Innoprot) or glioblastoma cell culture. EXs were fluorescently labelled. Laser scanning confocal microscopy was used for evaluation of EXs transmission and cell morphology changes.

Results : The in vitro models were optimize for cocultivation of two types of cells presented in BBB or BTB. The conventional horizontal perfusion model of BBB or BTB serves as a standard for the study of the transport the fluorescently labelled EXs or plant derived EVs via the endothelial‐astrocyte layer. The microchip vertical model can mimic more closely the in vivo conditions. Various types of EXs and EVs were able to efficiently cross the BBB and influence cell morphology.

Summary/Conclusion : These findings could bring new experimental strategies for broader application of in vitro multicellular systems in EXs research and information of EXs transmission via BBB/BTB for their therapeutic or diagnostic purposes.

Funding : Czech Health Research Agency—NU23‐08‐00307.

Advancing EV research: Novel technique for EV's surfaceome profiling

Zuzana Vince Kazmerova 1 , Jakub Janko 2 , Lucia Balazova 1

1 Biomedical Research Center of the Slovak Academy of Sciences, Bratislava, Slovakia; 2 Faculty of Medicine, Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia

Introduction : The extracellular vesicle (EV) surfaceome plays a crucial role in vesicle targeting, uptake, and functional interactions. However, its characterization remains challenging due to EV heterogeneity and small size, limiting conventional proteomics approaches. In this study, we employed a chemoproteomic approach originally designed for the identification of cell surface proteins in living cells and adapted it for the untargeted profiling of the EV surfaceome. This methodology uses selective enrichment of N‐, C‐, and O‐glycosylated proteins, which are ubiquitously present on the plasma membrane of living cells and, by extension, on the EV surface.

Methods : EVs were isolated from human primary hepatic stellate cells using size‐exclusion chromatography and treated with mild periodate oxidation to generate aldehydes from surface glycans. For unbiased surface protein capture, a trifunctional chemoproteomic reagent, pre‐quenched with glycine, was conjugated to EVs. After conjugation, EVs were lysed, and labelled proteins were enriched via a click chemistry‐based approach and identified using high‐resolution mass spectrometry.

Results : Protein identification and quantitative analysis demonstrated the efficacy of this approach, enabling the identification of 158 surface‐associated proteins on EVs, with 94 proteins detected based on the presence of at least two peptides per protein. The majority of identified proteins have been previously reported in EVs derived from various cell types, and 11 of them are among the top 100 most frequently identified EV‐associated proteins according to Vesiclepedia. These include CD63, ANXA2, CD59, CD44, Syntenin‐1, Alpha‐2‐macroglobulin, Elongation Factor 1‐Alpha 1, Albumin, CD81, GAPDH, and CD29. Moreover, several of these proteins are established EV markers, as defined by the MISEV 2023 guidelines, reinforcing the validity and specificity of our surfaceome profiling approach.

Summary/Conclusion : Profiling the EV surfaceome by capturing glycosylated proteins can play an important role in advancing both fundamental research and clinical applications. By refining our ability to map and interpret the EV surfaceome, we can unlock new diagnostic biomarkers by capturing specific EV subpopulations in circulating milieu, improve targeted therapies, and deepen our understanding of cell‐to‐cell communication in health and disease.

Funding : This work was funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project No. 09I03‐03‐V04‐00463 and APVV‐22‐0310 grant.

Subtopic Engineering and loading EVs 1

Therapeutic application of bioabsorbable nanofibre‐based EV sheet toward location‐specific targeted delivery of anti‐cancer EVs

Presenter: Akira Yokoi

Nagoya University, Nagoyo, Japan

Introduction : Extracellular vesicles (EVs) serve as promising therapeutic agents not only themselves but also biocompatible cargos that carry anti‐cancer molecules. However, delivery to targeted sites is still challenging, and systemic treatment only reaches specific sites. We recently invented the nanofibre‐based EV sheet, a novel EV isolation method that can capture EVs on organ surfaces (Yokoi A et al., Nat Commun. 2023). The potential of an EV sheet is not only for isolation but also for EV containers. This study aims to develop therapy‐oriented EV sheets (tEVsheet) that deliver anti‐tumour vesicles to a specific location.

Methods : To create a bioabsorbable EV sheet, various materials, such as nylon or polyester‐based resources, were tested to select the best fibre condition. The capability of EV loading was evaluated by nanoparticle tracking assays. We use ovarian cancer mouse models to assess the performance of tEVsheet, which is loaded with artificial vesicles, lipid nanoparticles, and encapsulated carboplatin (CBDCA), a common/key drug for ovarian cancer in clinics.

Results : We successfully created a bioabsorbable nanofibre‐based EV sheet, a significant achievement that paves the way for the practical application of this method. The tEVsheet was absorbed after 3 months from placement in the peritoneal cavity, demonstrating its safety and biocompatibility. Compared to systemic treatment, the tEV‐sheet delivered DiR labelled‐EVs to unique target sites, such as the upper or lower peritoneum. There was no difference in anti‐tumour effects between naked CBDCA and CBDCA‐tEVsheet in vitro using ovarian cancer cell lines. We also established a unique ovarian cancer peritoneal metastasis model, which creates metastatic tumours at specific locations of the peritoneum. The tEVsheet was attached to the metastatic tumours and then monitored by in vivo imaging. As the result, the tEVsheet suppressed their growth ( N = 10) without any toxicity, and growth inhibition for cancer cells was also confirmed by immunohistochemistry staining.

Summary/Conclusion : The tEVsheet has been validated as a unique tool for EV delivery, serving as a novel approach to cancer therapeutics. Its potential application extends beyond cancer fields to any disease or pre‐symptomatic state that can benefit from EV treatment.

Funding : The FOREST (JPMJFR204J) from Japan Science and Technology Agency (JST).

Rapid EV surface decoration with targeting moieties based on an FITC‐binding single‐chain antibody snorkel

Marieke T. Roefs 1 , Johanna Gamauf 1 , Madhusudhan R. Bobbili 2,3 , Michael Traxlmayr 2 , Marcelle van Mechelen 4 , Jean‐Paul Prieels 4 , Regina Grillari‐Voglauer 1 , Johannes Grillari 2,3

1 Evercyte GmbH, Vienna, Austria; 2 BOKU – University of Natural Resources and Life Sciences, Vienna, Austria; 3 Ludwig Boltzmann Institute for Traumatology, Vienna, Austria; 4 ConvEyXO, Liège, Belgium

Introduction : Extracellular vesicles (EVs) show potential as biological therapeutics and natural drug delivery vectors. However, their application is hampered by their limited reach of target tissue. One approach to generating targeted EVs is overexpression of protein fusion constructs on the EV membrane by donor cell engineering. Here, we propose a novel technology that enables the generation and isolation of EVs displaying any antibody, peptide or other targeting moiety that is connected to an FITC probe.

Methods : Recombinant HEK293 lines expressing protein fusion constructs based on our snorkel‐tag and containing CD81/CD9/CD63, a FLAG‐tag, an scFv against FITC and an HA‐tag separated by a PreScission protease (PS) cleavage site were generated and characterized for construct expression and FITC‐Ab binding by western blot and flow cytometry. EVs were isolated from serum‐free conditioned medium by 100 kDa ultrafiltration or 300 kDa tangential flow filtration and analysed for FITC‐Ab binding after anti‐tetraspanin bead capture and by nanoFCM. HER2‐targeted EVs were generated by incubation with FITC‐labelled anti‐HER2 antibody or FITC‐IgG ctrl. Free antibody was removed by subsequent anti‐HA affinity purification, followed by PS cleavage to release targeted EVs. In vitro binding to HER2‐expressing N87 cells in comparison with HER2‐low expressing MCF‐7 cells was investigated.

Results : Protein fusion construct containing CD9 showed the highest construct expression and FITC‐Ab binding on the HEK293 cell membrane compared to constructs containing CD81 or CD63. Isolated EVs also showed variable degrees of FITC‐Ab binding by the different protein fusion constructs. Anti‐HA affinity purification and subsequent PS cleavage enabled the specific isolation of targeted EVs from crude EV preparations. As a proof‐of‐concept, HER2‐targeted EVs were generated which, in contrast to IgG ctrl‐expressing EVs, showed increased binding and uptake by N87 cells compared to MCF‐7 cells.

Summary/Conclusion : Targeted EVs were generated by the expression of scFv‐containing protein‐fusion constructs capturing FITC‐labelled antibodies with a femtomolar affinity. The possibility of using this method to decorate EV membranes with any FITC‐labelled targeting moiety will prevent repeated and extensive cell engineering in the future. This technology facilitates the generation of targeted therapeutic EVs for any tissue of interest and accelerates the screening for EV tropism altering surface moieties.

Funding : Evercyte GmbH, ConvEyXO.

Extracellular‐vesicle mediated delivery of Cas9 adenine base editors by aptamer‐based loading and UV‐activated release

Omnia M. Elsharkasy 1 , Charlotte V. Hegeman 2 , Ivana Lansweers 2 , Olaf L. Cotugno 1 , Ingmar Y. de Groot 1 , Zoë E.M.N.J. de Wit 2 , Antonio Garcia‐Guerra 3,4 , Niels J. A. Moorman 2 , Willemijn S. de Voogt 1 , Jerney J. Gitz‐François 1 , Xiuming Liang 5,6,7 , Samir El Andaloussi 5,6,7 , Raymond M. Schiffelers 1 , Sander A.A. Kooijmans 8,9 , Enrico Mastrobattista 2 , Pieter Vader 1 , Olivier G. de Jong 1,2

1 University Medical Center Utrecht, Utrecht, The Netherlands; 2 Utrecht University, Utrecht, The Netherlands; 3 University of Oxford, Oxford, Oxfordshire, UK; 4 Institute of Developmental and Regenerative Medicine, Oxford, Oxfordshire, UK; 5 Karolinska Institutet, Stockholm, Sweden; 6 Karolinska University Hospital Huddinge, Huddinge, Sweden; 7 Karolinska ATMP Center, Stockholm, Sweden; 8 Wilhelmina's Children's Hospital, Utrecht, The Netherlands; 9 Regenerative Medicine Center Utrecht, Utrecht, The Netherlands Olivier G. de Jong is the presenting author .

Introduction : CRISPR/Cas9 is a prokaryotic endonuclease capable of targeting genomic sequences with high specificity and efficiency, generating double‐strand DNA breaks that may result in frameshift mutations. Additional functionalization of Cas9 with enzymatic groups, such as adenine base editors (ABE), which replace adenine with guanosine in specific targeted genomic sequences, allows for more specific genomic repairs. One major hurdle for therapeutic applications of such Cas9‐based moieties is their intracellular delivery. Extracellular vesicles (EVs) hold the potential to overcome this hurdle due to their biocompatibility and intrinsic capability of efficient intercellular transfer of RNA and proteins. Here, we present a modular strategy for EV‐mediated loading and delivery of Cas9 and Cas9‐ABE.

Methods : To facilitate loading of either spCas9 or Cas9‐ABE RNP complexes, sgRNAs with high‐affinity MS2 coat protein‐interacting aptamers were generated and expressed alongside spCas9 or ABE8e and EV‐enriched proteins fused to the MS2 coat protein (MCP). The MCP, lacking the Fg loop to prevent capsid formation, was cloned in tandem on the N‐terminus of CD9 and CD63, linked by a UV‐sensitive photocleavable protein (PhoCl). To study spCas9 and ABE8e delivery, we used previously published fluorescent Stoplight reporter systems (De Jong, Nat Commun. 2020; Öktem, Pharmaceutics 2023) and analysed endogenous genomic targets by T7E1 assays.

Results : EV loading of Cas9, as well as UV‐mediated cleavage of the PhoCl fusion proteins, was confirmed by western blot analysis and flow cytometry. Using EVs with MS2‐PhoCl‐CD63/CD9 fusion proteins, we observed efficient spCas9 and ABE8e delivery, where CD9 showed notably higher (> 2‐fold) delivery than CD63. Interestingly, ABE8e activity was hampered by the incorporation of MS2 aptamers in the second stemLoop of the sgRNA, but only in the presence of MCPs, suggesting interference by steric hindrance of the deaminase. This was resolved by solely incorporating an MS2 aptamer in the sgRNA tetraloop, resulting in high levels of EV‐mediated ABE8e delivery (> 60% reporter activation).

Summary/Conclusion : Here, we describe a novel modular platform for EV‐mediated loading and delivery of Cas9 RNPs. Our results demonstrate that EVs are capable of functional spCas9 and ABE8e RNP delivery. Moreover, these data indicate that additional modifications for regulated cargo release strongly increase Cas9‐RNP delivery.

Internalization of cell‐penetrating peptides by cells and encapsulation in extracellular vesicles

Presenter: Shinya Nakai

Osaka Metropolitan University, Osaka, Japan

Introduction : Exosomes are extracellular vesicles (EVs, approximately 30‐200 nm in diameter) characterized by containing physiologically active molecules and contributing greatly to intercellular communications. In addition, EVs are known to have high advantages from pharmaceutical perspectives and are expected to be a next‐generation drug delivery tool. In this study, we are evaluating the transfer of cell‐penetrating peptides (CPPs) into multivesicular endosomes (MVEs), which are the production sites for EVs, during their cellular uptake, and the effects on EVs secretion.

Methods : All peptides were synthesized by Fmoc‐solid phase methods. Cellular uptake of fluorescently‐labelled peptides was analysed using a confocal laser microscope. Isolation of secreted EVs was conducted by ultracentrifugation methods. In this research, CD63‐GFP‐stably expressing cells were used for assessments of intracellular locations of internalized fluorescently‐labelled peptides.

Results : Using a confocal laser microscope, we observed cellular uptake of fluorescently‐labelled octaarginine (R8‐Alexa568), a typical CPP, into HeLa cells expressing the EVs marker protein CD63‐GFP. By pretreatment with the hydrophobic anion pyrenebutyrate (PyB), highly efficient cell membrane penetration of the R8‐Alexa568 and promotion of diffusion into cytosol were observed. In addition, colocalization of the CD63‐GFP and R8‐Alexa568 was observed over time, suggesting accumulation of the peptides to the MVEs. After cellular uptake of the R8‐Alexa568 into HeLa cells, EVs secreted from the cells were isolated using ultracentrifugation. As a result of evaluating the encapsulation of isolated EVs using the quenching effect of trypan blue (TB), the R8‐Alexa568 was judged to be encapsulated within EVs. Cryo‐TEM observation showed multivesicular formation in the isolated EVs, suggesting possible induction of membrane fusion in MVEs that affect encapsulation of delivered CPPs. Furthermore, the isolated EVs were taken up by other cells; it became clear that the R8‐Alexa568‐encapsulating EVs were also taken up into other cells after their secretion. We are still studying the effects of the number of arginine residues in the peptide sequences that affect cellular uptake pathways (e.g., endocytosis and cytosolic release) and membrane curvature formation on encapsulation in EVs.

Summary/Conclusion : These results not only provide a basic technology for artificial inclusion in EVs using the CPPs but also provide basic knowledge regarding the effects of the CPPs on intercellular communication via EVs.

Encapsulation of prime editing components in extracellular vesicles using protein palmitoylation

Yaoyao Lu, Gege Zhao, Nicolas Soucy, Joel Rousseau, Jacques P.Tremblay

Centre de Recherche du CHU de Québec – Université Laval, Québec, Canada

Introduction : Prime editing (PE) is a precise genome manipulation technology based on the CRISPR‐Cas9 system, also known as the “search and replace” approach, which does not require exogenous donor DNA or DNA double‐strand breaks (DSBs) to repair the mutated gene. PE provides advantages over other nuclease gene editing technologies, including greater precision and the ability to edit different types of mutations. Nevertheless, due to its coding sequence of 6.3 kilobases, there is no effective method to deliver PE systems for in vivo treatment, which restricts its use in fundamental research and agriculture. Extracellular vesicles (EVs) are a diverse set of lipid‐ and protein‐delimited vesicles released from cells to the extracellular environment to transport nucleic acids, proteins, and metabolites between cells. Palmitoylation is an important post‐translational modification that regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interaction, protein stability, and protein conformation. Therefore, we encapsulated the components of the PE system into extracellular vesicles (EVs) through their biogenesis by using S‐palmitoylation to correct the DMD gene mutation.

Methods : To reach this goal, we incorporated a palmitoylation peptide derived from growth‐associated protein 43 (GAP43) into the N‐terminal of SpCas9 nickase and the N‐terminal of reverse transcriptase (RT). The palmitoylation peptide‐modified Cas9 nickase and RT were transfected into cells using polyethyleneimine (PEI). Afterward, the medium was changed to an EV‐depleted FBS medium. The EVs were subsequently purified by ultracentrifugation and added to EV recipient cells already containing an epegRNA permitting the correction of the G to C mutation in the EMX1 gene. The correction of this point mutation in the EVs recipient cells was analysed by Sanger sequencing.

Results : The palmitoylation peptide from GAP43 permitted the successful loading of the SpCas9 nickase and the RT into EVs. The addition of these EVs to the culture medium of EV recipient cells, permitted the correction of the G to C mutation in 10% of the EMX1 gene.

Summary/Conclusion : The incorporation of palmitoylated Cas9 nickase and reverse transcriptase proteins into EVs creates a new avenue for the safe and effective delivery of the PE components to treat hereditary diseases.

Extracellular vesicle‐mediated delivery of Let‐7a to combat peritoneal fibrosis

Po‐Chen Li 1 , Tai‐Shan Cheng 1 , Sin‐Yu Chen 1 , Hsin‐Tung Chen 1 , Wei‐Ni Tsai 1 , Chiao‐Yin Sun 2 , Ly James Lee 1 , Chi‐Ying F. Huang* 1

1 Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Hsinchu, Taiwan; 2 Department of Nephrology, Chang Gung Memorial Hospital, Keelung, Taiwan 2

Abstract unavailable

Achieving specific efficient delivery of diverse protein therapeutics with circulating exosome‐anchoring nanobody

Presenter: Gang Han

Tianjin Medical University, Tianjin, People's Republic of China

Introduction : Exosomes are promising drug delivery vehicles due to their low immunogenicity and favourable safety profile. Efficiently loading proteins or nucleic acids onto exosomes is critical for their successful application. Our team previously identified peptide‐ and DNA aptamer‐based anchoring molecules that mediate efficient loading to exosomes without modifying the source cells. However, the limited affinity of these molecules restricts their loading capacity, highlighting the need for higher‐affinity alternatives.

Methods : We employed phage display technology to screen for nanobodies with high affinity for exosomes. The best candidates were validated using single‐molecule imaging and flow cytometry. We systematically analysed the size and quantity of proteins that could be loaded by the selected nanobody.

Results : Through screening and validation, we identified the nanobody Nexo3 as having the strongest affinity for exosomes from various sources. Nexo3 facilitated the efficient loading of a range of molecules, including mCherry, Cre, double‐stranded RNA‐binding domain (DRBD)/siRNA, IgG antibodies, and CRISPR/Cas9‐gRNA (RNP). It successfully delivered DRBD/siRNA, Cre, and Cas9‐gRNA to target cells, resulting in effective interference with GAPDH mRNA and editing of reporter genes. Importantly, Nexo3 was able to bind to exosomes in both cell culture supernatants and the bloodstream of mice, leverageing the liver tropism of circulating exosomes for targeted delivery of mCherry and DRBD/siRNA to hepatic cells.

Summary/Conclusion : Nexo3 exhibits strong affinity for exosomes, enabling the efficient loading and delivery of high molecular weight proteins. Additionally, Nexo3 can modify circulating exosomes, facilitating targeted protein delivery to liver cells, thereby enhancing the potential of exosomes as effective drug delivery systems.

A supramolecular antibiotic assembly that kills E. coli by enforcing extensive release of bacterial EVs

Tamás Beke‐Somfai 1 , Tasvilla Sonallya 1 , Imola Cs. Szigyártó 1 , Zoltán Varga 1 , Dániel Molnár 2 , Kamal el Battioui 1 , Sohini Chakraborty 1 , Judit Tóth 2 , István Mándity 1 , Loránd Románszki 1 , Kata Horváti 1 , Mihály Kovács 3 , Eszter Házy 3 , Tünde Juhász 1

1 HUN‐REN Research Centre for Natural Sciences, Institute of Materials and Environmental Chemistry, Budapest, Hungary; 2 HUN‐REN Research Centre for Natural Sciences, Institute of Molecular Life Sciences, Budapest, Hungary; 3 Eötvös Loránd University, Department of Biochemistry, Budapest, Hungary

Introduction : The design and analysis of new antimicrobial solutions have been extensively investigated in the backdrop of the growing resistance towards conventional antibiotic treatments. Recently it has become clear that bacteria have an affinity to release various extracellular vesicles as part of their regular function in colonies. However, should this occur at an increased pace, that could rapidly lead to destruction of individual cells. This could be exploited by novel antibiotic strategies.

Methods : The beta‐peptide 3K was tested with both ATP and ADP on E. coli . Bacterial assays, haemolytic assays, flow cytometry, and confocal microscopy in the presence of E. coli were employed to address bioactivity. Cryo‐EM, TEM and atomic force microscopy was used to address mechanistic aspects and to get visual confirmation of bacterial EV release.

Results : We have recently designed a beta‐peptide (named 3K) [1], which here we showed that it can be triggered into extensive nanonet formation by extracellular ATP, a molecular signal of hostile microbial attacks. 3K and ATP co‐assemble into highly entangled 3D architectures of infinite fibrils, with high antibacterial activity against E. coli . The mechanism of antibacterial action was tracked by cryo‐EM, which has revealed that these fibrils not only entrap microbes by entangling them, but these fibrils also enforce various extracellular vesicles to be released from the bacteria, leading eventually to membrane disruption.

Summary/Conclusion : These mechanistic insights could provide better understanding of the natural molecular processes underlying, for example, the toxic behaviour of natural peptide nanonets, but these also suggest that enforcing increased amounts of bacterial extracellular vesicles could be a viable strategy towards the development of conceptually new antimicrobial therapies. [1] El Battioui et al., ‘in situ captured antibacterial action of membrane incising peptide lamellae,’ Nature Communications, 2024, 15, 3424.

Funding : This work was funded by the National Research, Development and Innovation Office, Hungary (TKP2021‐EGA‐31, 2020‐1.1.2‐PIACI‐KFI‐2020‐00021, KKP_22 Project n.o. 144180 and FK‐146081). Support from the Eötvös Loránd Research Network, Grant Nos. SA‐87/2021 and KEP‐5/2021, is also acknowledged. The authors acknowledge CF CryoEM of CIISB, Instruct‐CZ Centre, supported by MEYS CR (LM2023042) and the European Regional Development Fund Project ‘UP CIISB’ (No. CZ.02.1.01/0.0/0.0/18_046/0015974).

Bioengineered MSCs‐derived extracellular vesicles: a novel therapeutic strategy for targeting liver disease

Presenter: Meenakshi Mendiratta

All India Institute of Medical Sciences, New Delhi, India

Introduction : Mesenchymal stem cell‐derived small extracellular vesicles (MSCs‐sEV) are a promising candidate for cell‐free therapeutics in regenerative medicine. However, there are several challenges with the utility of sEV, including non‐specific packageing of cargo, loss in circulation, and target specificity. The current study employs surface and cargo modification strategies to overcome these challenges to enhance sEV homing, target specificity, and specific cargo enrichment for liver diseases. This research explores dual modification strategies to improve targeting precision and therapeutic efficacy of mesenchymal stem cell‐derived small extracellular vesicles, specifically emphasizing hepatic cell targeting.

Methods : Tissue‐specific MSCs (bone marrow, Wharton's jelly) were isolated with due patient consent ((IC‐SCR/140/23 (O)) and characterized as per ISCT guidelines. MSCs were cultured in serum‐free media for isolation of sEV via ultracentrifugation. The plasma‐derived sEV were isolated from healthy control, ACLF, and CLD patients and further evaluated for their content by small RNA sequencing, with a primary focus on hepatoprotective miRNA distribution. The surface of tissue‐specific sEV was bioengineered with hepato‐specific ligands using chemical conjugation methods, while the cargo of sEV was enriched with hepatoprotective miRNA using lipofectamine RNAiMAX. Naive and BioEn‐sEV were characterized as per MISEV 2023 guidelines. Surface modification was validated via the lectin‐induced aggregation method. Cargo enrichment was validated via qPCR, and hepatoprotective functionality was validated via ROS, macrophage polarization, T cell proliferation in an in vitro ACLF model.

Results : It was observed that BioEn‐sEV and Naïve sEV demonstrated a cup‐shaped morphology through TEM. Additionally, BioEn‐sEV demonstrated an increased diameter and became a positive charge compared to Naïve sEV. Furthermore, cellular uptake efficiency of BioEn‐sEV was notably higher in hepatic cells compared to non‐hepatic cells, surpassing that of Naïve sEV. BioEn‐sEV significantly reduced ROS production and T‐cell proliferation and enhanced polarization of macrophages towards M2 macrophages and Treg induction as compared to Naïve sEV. The significant hepatoregenerative capacity of miRNA (identified from CLD and ACLF patients) of BioEn‐sEV in hepatic cells further underscores their therapeutic promise.

Summary/Conclusion : This study has demonstrated precise sEV targeting through surface modification, enhancing anti‐inflammatory effects via cargo modification. Bioengineered sEVs emerge as a promising avenue for targeted therapeutic interventions in diseases, prompting further clinical exploration in this innovative field.

Ectopic expression of CD39 enhances immunomodulatory properties of ciMSC‐derived extracellular vesicles through adenosine signalling pathway

Presenter: Mohamed Elbeltagy

University of Duisburg‐Essen, Duisburg, Germany

Introduction : Mesenchymal stromal cells (MSCs) exert therapeutic effects, primarily mediated through extracellular vesicles (EVs) with immunomodulatory properties. CD73 on MSCs catalyses the conversion of extracellular ATP (eATP) into anti‐inflammatory adenosine. However, the absence of CD39 in MSCs limits this purinergic pathway. Elevated eATP, an early damage‐associated molecular pattern (DAMP), triggers severe inflammatory cascades post‐stroke. This study hypothesizes that introducing CD39 expression in immortalized MSCs (ciMSCs) will enhance the immunomodulatory efficacy of their EVs in neuroinflammatory conditions like ischaemic stroke.

Methods : ciMSCs were modified via lentiviral transduction to express CD39. EVs were isolated through ultracentrifugation and characterized per MISEV guidelines, including nanoparticle tracking analysis (NTA) and BCA assay for concentration and purity. Western blot confirmed EV markers CD63, CD9, CD81, and TSG101. Image flow cytometry quantified CD39‐fused eGFP+ EVs, and ATP hydrolysis was assessed using a colourimetric assay. Immunomodulatory potential was tested via a multi‐donor mixed lymphocyte reaction (mdMLR) with mononuclear cells from 12 donors to evaluate responses with and without eATP. The therapeutic effects of CD39+ ciMSC‐EVs were assessed in a murine middle cerebral artery occlusion (MCAO) ischaemic stroke model. Neuromuscular coordination was evaluated with the tightrope test, and infarct volume and oedema were analysed using Nissl and cresyl violet staining.

Results : Image flow cytometry and western blot analysis confirmed that ciMSC‐EVs carried CD63, CD81, and CD39. CD39+ ciMSC‐EVs demonstrated efficient ATP‐to‐adenosine conversion, a capability not observed in unmodified ciMSC‐EVs. In mdMLR assays, while both EV types suppressed T cell activation in the absence of eATP, CD39+ ciMSC‐EVs exhibited markedly enhanced suppression in the presence of eATP, supporting the role of adenosine pathways in modulating immune responses. In the ischaemic stroke model, administration of CD39+ ciMSC‐EVs significantly improved neuromuscular coordination, reduced infarct volume, and decreased oedema compared to EVs from unmodified ciMSCs, highlighting their superior therapeutic potential.

Summary/Conclusion : CD39 expression in ciMSCs enhances the immunomodulatory and therapeutic efficacy of their EVs, especially under conditions of elevated eATP. CD39+ ciMSC‐EVs are a promising therapeutic strategy for neuroinflammatory diseases such as ischaemic stroke, with further studies underway in models like acute Graft‐versus‐Host Disease (GvHD).

Funding : This project is supported by funding from the Deutsche Forschungsgemeinschaft (DFG).

Enhancement of WNT pathway‐related genes following treatment with microRNA‐31‐loaded extracellular vesicles in an inflammatory bowel disease‐mimicking organoid model

Agner Henrique Dorigo Hochuli 1,2 , Alice Zaramella 1,2 , Miriam Duci 1,2 , Raquel Moll‐Diaz ,1,2 , Paola Bisaccia 1,2 , Maurizio Muraca 3 , Konrad Basler 4 , Michela Pozzobon 1,2

1 Department of Women's and Children's Health, University of Padova, Italy; 2 Stem Cell and Regenerative Medicine Lab, Institute of Paediatric Research Città della Speranza, Padova, Italy; 3 Institute of Paediatric Research Città della Speranza, Padova, Italy; 4 Department of Molecular Life Science, University of Zurich, Zurich, Switzerland

Introduction : Inflammatory bowel diseases (IBD) are chronic inflammatory conditions with unknown aetiology, highlighting the need for advanced therapeutic strategies. Extracellular vesicles derived from mesenchymal stromal cells (MSC‐EVs) have shown promise in replicating their cells of origin's therapeutic effects, including tissue repair. Notably, microRNA‐31 (miRNA‐31) is highly expressed during IBD and plays a significant role in regulating the WNT, HIPPO, and anti‐inflammatory pathways, all crucial during the progression of IBD. A treatment strategy that delivers EVs loaded with miRNA‐31 could deepen and increase the regeneration mechanisms after IBD damage. This project aims to explore a novel regenerative approach for IBD treatment.

Methods : Colon‐derived mouse organoids were used to model IBD in vitro. IBD‐like damage was induced by exposing organoids to a pro‐inflammatory cytokine cocktail. The miRNA‐31 mimic was synthesised with a fluorescent dye. EVs were electroporated to load miRNA‐31, and the loading efficiency was verified by flow cytometry. Surface area size was monitored throughout the damage and recovery/treatment period, and immunofluorescence and qRT‐PCR were performed to analyse markers for intestinal stem cells (Lgr5+), WNT targets (Axin2 and Ccndb1), a HIPPO target (Lats2), and pro‐ and anti‐inflammatory genes (Tnf‐alpha, Il6st‐GP130 and Il10), both before and after IBD induction and subsequent treatments. Dexamethasone (100 nM) was used as a positive control.

Results : miRNA‐31 was successfully loaded into EVs, which continued to express EVs surface markers post‐electroporation. IBD‐damaged organoids showed reduced surface area after exposure to the pro‐inflammatory cocktail and recovered significantly following treatments with naïve EVs, miRNA‐31‐loaded EVs, and dexamethasone. Organoids treated with miRNA‐31‐loaded EVs exhibited significantly increased expression of Lgr5+ cells and WNT‐related genes compared to untreated IBD‐damaged organoids and those treated with dexamethasone. Lats2 and pro‐inflammatory genes (Tnf‐alpha, Il6st‐GP130): organoids treated with miRNA‐31‐loaded EVs showed a significant decrease in expression compared to those treated with naïve EVs.

Summary/Conclusion : These findings underscore the potential of miRNA‐31‐loaded EVs as a promising therapeutic strategy for IBD. This approach enhances the WNT pathway and, on the other hand, inhibits the HIPPO pathway, providing insights into mechanisms that support both tissue regeneration and anti‐inflammatory effects in IBD.

Engineered extracellular vesicles a next generation therapeutic delivery platform

Presenter: Rahmat Asfiya

Institution University of Missouri, Columbia, Missouri, USA

Introduction : Nanotechnology helps develop safer, more effective drug delivery vehicles. Liposomes, polymer micelles, and metal nanoparticles have advanced in recent decades. Nonetheless, this scientific progress could not be realized in accordance with clinical expectations. The gap has spurred drug delivery experts to build bio‐inspired nanocarriers with high compatibility, stability, and circulation time. Extracellular vesicles (EVs) represent a highly promising option for bio‐inspired nanocarriers, demonstrating significant potential for therapeutic applications. EVs, membrane‐derived structures discharged by cells into the extracellular environment, aid intercellular communication. Unfortunately, EVs' therapeutic loading efficacy is low, limiting their drug delivery potential. Researchers created hybrid EVs using nanoparticles and EV benefits. The chemically modified EV systems described here can transport a wide variety of therapeutic payloads, including small molecule inhibitors, chemotherapeutic drugs, and siRNA. We investigated its universality in diabetic retinopathy and lung cancer therapy.

Methods : The delivery vehicles were developed using EVs that were isolated through ultracentrifugation. To successfully load the therapeutic payloads, many procedures were tested, including (1) the incubation method, (2) transfection based on CaCl 2 , and (3) the heat shock method. The biophysical properties of each vehicle were examined by TEM imaging, gel retardation assay, DLS, and zeta potential measurement. Cell viability, fluorescent imaging, qPCR, western blotting, and flow cytometry were among the in vitro and in vivo techniques used to evaluate their therapeutic effectiveness.

Results : We developed multiple therapeutic EV‐hybrid systems tailored to specific applications. The EV‐G6PD‐siRNA vehicle demonstrated effective delivery to target iPSC cells. The downregulation of G6PD and alterations in cell viability confirmed the efficacy of delivery. An additional EV vehicle transporting DHEA, a small molecule G6PD inhibitor, effectively delivered DHEA to the recipient cells. A third vehicle containing metal (gold or iron) nanoparticles conjugated to B7H4 siRNA demonstrated effective uptake by recipient cells. All developed vehicles demonstrated significant therapeutic potential while exhibiting reduced toxicity.

Summary/Conclusion : In conclusion, we have developed extracellular vesicles (EVs) that effectively transport a range of therapies, establishing them as a viable alternative to synthetic nanocarriers.

Funding : US Department of Defense (W81XWH‐22‐1‐0016) and a Startup grant from MU School of Medicine.

Inclusion of recombinant proteins into extracellular vesicles by Lactococcus cremoris

Presenter: Tina Vida Plavec

Jožef Stefan Institute, Ljubljana, Slovenia

Introduction : Lactococcus cremoris is a gram‐positive bacterium and represents a well‐known system for recombinant protein expression. Gram‐positive bacterial extracellular vesicles (EVs) have recently gained more attention and have been hypothesized to have a possible physiological role. The bacteria producing recombinant proteins could be used as cell factories for the incorporation of functional and bioactive proteins into the EVs. In this study, we analysed the effect of the expression of different recombinant proteins in L. cremoris on the formation of EVs and their protein content.

Methods : EVs were isolated following a standard protocol using an ultracentrifuge at 130,000 × g for 2 h, followed by removal of the supernatant and collection of the EVs. EVs were characterised with transmission electron microscopy (TEM), flow cytometry, polydispersity index measurement and proteomic analysis.

Results : We successfully isolated EVs from recombinant L. cremoris , and characterized them by TEM and flow cytometry, which revealed differences in both the quantity and heterogeneity of the secreted EVs, depending on the recombinant protein that was expressed in L. cremoris . The size of the isolated EVs was within the expected nano‐scale range, mostly 50–200 nm. The presence and quantity of individual recombinant proteins inside the vesicle were confirmed by proteomic analysis.

Summary/Conclusion : We have shown that recombinant L. cremoris can be used to successfully produce EVs. The recombinant proteins expressed by L. cremoris were detected in the EVs‐containing ultracentrifuged pellets. Our study therefore highlights the potential of using L. cremoris for the production of EVs and for loading them with recombinant proteins.

Funding : ARRS N3‐0184 Small protein blockers of IL‐23/IL‐17 axis as intestinal inflammation inhibitors secreted by probiotic bacteria. ARRS P4‐0127 Pharmaceutical biotechnology: Science for health.

Engineered extracellular vesicles for therapeutic CRISPR‐Cas9 delivery

Silvia Fiori 1 , Alice Randon 1 , Abeer Mafoud 1 , Alessio N. Ferraro 1 , Maira Russo 1 , Manuel Albanese 1,2

1 National Institute of Molecular Genetics, Milan, Italy; 2 Department for Clinical Sciences and Community Health (DISCCO), University of Milan, Milan, Italy

Introduction : CRISPR‐Cas9 is a powerful gene‐editing tool with therapeutic potential, but its clinical application is limited by in vivo delivery challenges. Extracellular vesicles (EVs) are natural carriers of proteins, lipids, DNA, and RNA, offering protection from degradation, exhibiting low immunogenicity, and easy manipulation. Our solution is to engineer EVs to efficiently encapsulate the CRISPR‐Cas9 system by fusing Cas9 with EV‐anchored proteins. Additionally, EVs’ specificity and fusion to target cells are enhanced using binding and fusogenic molecules, respectively, making them a promising platform for targeted gene‐editing therapies.

Methods : Engineered virus‐like particles (eVLPs) and EVs (eEVs) are isolated from HEK293T cells transfected with plasmids encoding the different components. VLPs and EVs are purified from supernatant by using different purification methods. The quality of each eEVs and eVLPs preparations is assessed by several readouts, including qPCR of the gRNAs, western blotting, and nanoparticle tracking analysis. Functional evaluation was conducted in T and B cell lines as an in vitro model. Fusogenic capability and tropism were assessed using the EV fusion assay, and knockout (KO) efficiency was evaluated by flow cytometry.

Results : Our production methods allowed successful encapsulation of gRNAs and Cas9 in eVLPs/eEVs. eEVs equipped with specific ScFvs and fusogenic molecules demonstrated selective fusion to target cells compared to controls. Additionally, eVLPs achieved nearly 100% KO efficiency in SUPT‐1 cells, while EVs showed promising, though lower, efficiency.

Summary/Conclusion : Our system shows promising results, highlighting the potential of eEVs as an efficient delivery platform for therapeutic applications. With further optimization, eEVs could provide an effective approach for the treatment of cancer and latent viral infections, allowing the precise targeting of specific genes.

A comparative study of different approaches employed in the functionalization of the EVs surface with hyaluronic acid derivatives

Giulia Duca, Stefania Raimondo, Nima Rabienezhad Ganji, Salvatore Emanuele Drago, Fabio Salvatore Palumbo, Alice Conigliaro, Gennara Cavallaro, Riccardo Alessandro

University of Palermo, Palermo, Italy

Introduction : Extracellular vesicles (EVs) can be used to deliver therapeutic compounds to specific target cells by modifying the EVs surface. Based on this, focusing on finding the best methods of functionalization, the goal of our project is to develop a targeted EV‐based delivery system for anticancer therapy, functionalizing the EVs surface with hyaluronic acid (HA), whose receptor (CD44) is overexpressed in many cancer cells, by using HA derivatives bearing in the side chain hydrophobic portions able to give interactions with the phospholipid bilayer of EVs.

Methods : First, we isolated EVs from HEK293T cells by ultracentrifugation and we decorated them with synthetic Alexa fluor‐labelled HA compounds (H100kE28%C12% and H10kE26%C12%). We tried two different functionalization approaches: the overnight co‐incubation between EVs and HA and the EVs electroporation followed by 1 h of incubation with HA at 37°C. We tried different electroporation protocols, differing in voltage and pulse number; the most efficient approach was selected by evaluating the stability of the EV size through dynamic light scattering (DLS). The decoration efficiency was evaluated by measuring fluorescence emission. Finally, EVs‐HA were labelled with PKH26, and the cellular uptake of thyroid carcinoma cells was analysed by confocal microscopy.

Results : Confocal microscopy images show an extracellular deposition of HA derivatives in cells treated with EVs decorated by co‐incubation, suggesting that HA aggregates on EVs surface, also reducing their uptake. Furthermore, fluorescence quantification highlights a low EV decoration with this method. The functionalization was improved by employing electroporation at 1000 V for 5 pulses; this approach allows increasing EVs’ internalization.

Summary/Conclusion : These preliminary data suggest that electroporation can improve the EVs decoration with HA; hence, this protocol could be applied to develop a delivery system for anticancer therapy. Further experiments will be performed, loading siRNA into EVs decorated with HA and seeing the functionality of the complex.

Funding : We acknowledge the grant CN00000041 “National Center for Gene Therapy and Drugs based on RNA Technology” (concession number 1035 of 17 June 2022‐PNRR MUR‐M4C2‐Investment 1.4 Call “National Centers,” financed by EU‐NextGenerationEU), code project (CUP) B73C22000780001.

Tumour cell cytotoxicity of NK cell‐derived exosomes labelled with tumour‐targeting peptide and pro‐apoptotic peptide

Presenter: Geuna Park

Kyungpook National University, Daegu, Republic of Korea

Introduction : NK cells are natural killer cells that originated from haematopoietic stem cells and are included in the innate lymphoid cells and have the ability to inhibit tumour progression. NK cell‐derived exosomes can mimic donor cells' characterization and induce target tumour cell death by biological killing mechanisms. RELT is a cell surface receptor that is upregulated in tumour cells such as lung tumour. In this study, we surface‐modified exosomes derived from NK cells with the RELT‐biding peptide (named RELT) to target RELT‐expressing tumour cells, and also with the KLAKLAKKLAKLAK pro‐apoptotic peptide (named KLA) to induce mitochondrial damage and cytotoxicity.

Methods : NK cells were incubated in exosome‐free medium for 48 h for the isolation of exosomes. Following filtration, supernatants were pelleted by ultrahigh‐speed centrifugation twice, and exosomes were resuspended in phosphate‐buffered saline. Then, NK exosomes were surface‐modified with the RELT pep and KLA using the DOPE‐PEG‐NHS linker for 15 min at 37°C.

Results : Immunofluorescence microscopy and flow cytometry showed that A549 lung tumour cells and Panc1 pancreatic tumour cells expressed high levels of RELT on the cell surface. Both unlabelled NK exosomes and RELT‐ and KLA‐labelled NK exosomes were efficiently internalized into A549 cells. Importantly, NK exosomes dual‐labelled with both RELT and KLA showed a more efficient cytotoxicity in A549 cells and Panc1 cells than unlabelled NK exosomes and NK exosomes labelled with either RELT or KLA alone. An in vivo study using tumour‐bearing mice showed that tumour growth was reduced by NK exosomes labelled with peptides more efficiently than by unlabelled NK exosomes.

Summary/Conclusion : These results suggest that RELT‐targeted and KLA pro‐apoptotic peptide‐armed NK exosomes would be a promising tool for targeted therapy of RELT‐expressing tumours.

A novel controllable loading and releasing strategy in EV‐mediated delivery

Presenter: Houze Zhou

Karolinska Institutet, Stockholm, Sweden

Introduction : Extracellular vesicles (EVs) are naturally secreted nanoparticles that could serve as low‐immunogenic carriers for therapeutic delivery. Advances in endogenous cargo loading strategies have positioned engineered EVs as promising tools for therapeutic protein delivery. Efficient cargo loading and release are critical for achieving functional delivery to target cells. We previously developed a pH‐sensitive intein system for efficient EV‐mediated cargo loading and release, leverageing pH alterations within the multivesicular body (MVB). In this study, we present an alternative strategy that is pH‐independent, enabling active EV cargo loading and release.

Methods : In this study, we developed a controllable strategy to enhance cargo loading and release in extracellular vesicles (EVs). The system utilizes an optimized inhibitor‐sensitive protease as a controllable linker between the EV scaffold and protein cargo, incorporating cleavable peptide sites. Active loading of protein cargo into EVs was achieved by incorporating FDA‐approved small‐molecule inhibitors (A and B) to prevent the protease cleavage during EV production, theoretically increasing cargo loading capacity. Following production, the inhibitors were removed using tangential flow filtration (TFF), enabling protease‐mediated cleavage for controlled cargo release. Cleavage efficiency of the fused EV scaffold‐protease‐cargo protein complex was validated by western blot analysis. The potency and functionality of the engineered EVs were further assessed in vitro using reporter cells.

Results : Direct transfection of plasmids containing the protease constructs into cells revealed clear cleavage or non‐cleavage events, depending on the engagement of inhibitors A or B. Utilizing the protease as the cleavable peptide for engineered EVs significantly enhanced editing efficiency, achieving nearly 100% Cre‐mediated editing efficiency with 1 × 10 9 EVs, using 0.75 µM inhibitor A during EV production. For larger Cas9 Ribonucleoprotein (RNP) delivery, engineered EVs produced with 8 µM inhibitor B edited 80% of Cas9 reporter cells at the dose of 1e10 EVs.

Summary/Conclusion : Our study introduces a novel strategy for controllable cargo loading and release in EVs. This system poses a great potential for EV‐mediated delivery of protein cargos, including Cre and larger Cas9 RNPs, in vitro. This approach holds promise for the efficient delivery of more complex base editors and prime editors.

Funding : Evox Therapeutics.

Gentle harvesting, purification and labelling of plant extracellular vesicles and their loading with exogenous cargo using a µPulse tangential flow filtration system

Yu‐Hsin Chang 1 , Ani Barbulova 1 , Immacolata Fiume 1 , Priyanka Devarakonda 1 , Angela Mary Joseph 1 , Miriam Piccioni 1 , Greta Jasulaityte 2 , Stefania Crispi 1 , Gabriella Pocsfalvi 1

1 National Research Council of Italy, Institute of Biosciences and BioResources, Rome, Italy; 2 FORMULATRIX (UK) Ltd., Bedford, UK Stefania Crispi and Gabriella Pocsfalvi are co‐senior authors .

Introduction : µPulse Tangential Flow Filtration system (Formulatrix) has already been successfully used for exosome harvesting from mammalian cultures. Here we optimized the setup of the µPulse TFF system to isolate EVs from the conditioned cell culture medium (CCM) of cell suspension cultures (CSCs) of different plant species and compared it with the ultracentrifugation (UC)‐based method. Nicotiana tabacum BY2 CSCs were used to optimize the system.

Methods : Solanum lycopersicum, Phaseolus vulgaris, Humulus lupulus, Vicia faba and BY2 were grown in liquid nutrient media and sub‐cultured weekly. CCM of 7‐day‐old cultures was used as a source for EVs isolation. CCM was subjected to differential low‐velocity centrifugation to remove cells and cell debris. The supernatant of the 15000 × g (S15K) was used to harvest EVs by either UC or µPulse TFF. For TFF, disposable microfluidic filter chips containing three types of modified polyethersulfone (mPES) membrane with 300, 100, 50 kDa MWCF were tested at different return valve pressures and at different volumes. EV concentration, buffer exchange, labelling and loading experiments were optimised. The harvested EVs were characterized using the interferometric light microscopy (Videodrop, Myriade), by protein yield (Qubit) and by protein profile (SDS‐PAGE) analysis. BY2 CCM EVs were studied over 7 days alongside the growth curve.

Results : µPulse TFF system provides an efficient and gentle way to isolate plant EVs from CSCs. S15K BY2 was used to optimize µPulse TFF parameters. We have found that 300 and 100 kDa filters yield similar yields, while the 50 kDa filter resulted in higher protein concentration, most probably due to the presence of co‐purifying proteins. The size and size distribution (200–300 nm diameter particles) of EVs obtained by µPulse TFF were similar to those harvested by UC.

Summary/Conclusion : We have optimized a plant CSC‐based EV‐isolation platform on the µPulse TFF instrument.

Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs” grant agreement N. 101131175 and PRIN PNRR 2022 n. P2022RLH39 and funded by the European Union HORIZON‐EIC‐2023‐PATHFINDERCHALLENGES‐01 nutriEV project under grant agreement N. 101161353.

Engineering hybrid extracellular vesicles for mRNA delivery

Filipa Dias Louro 1 , Chuan‐En Lu 1 , Marco Alfonzo‐Gomes 1 , Inês Gomes 1 , Mohsin Khan 2 , Niek Dekker 1 , Elisa Lázaro‐Ibáñez 1

1 AstraZeneca, Gothenburg, Sweden; 2 Temple University, Philadelphia, Pennsylvania, USA

Introduction : Lipid Nanoparticles (LNPs) are effective synthetic vectors for nucleic acid delivery; however, they often trigger toxicity and immunogenicity. Naturally occurring extracellular vesicles (EVs), intrinsically biocompatible, could be alternative vehicles to LNPs, but face challenges associated with RNA loading and endosomal escape. Here, we generated hybrid nanoparticles (HEVs) from EVs and mRNA‐loaded LNPs to overcome efficacy and safety issues of natural and synthetic drug delivery vehicles.

Methods : Human induced pluripotent stem cell (hiPSC)‐derived EVs and mRNA‐LNPs were fused in bulk under controlled temperature and low pH conditions. Various LNP compositions were screened for their ability to fuse with EVs. The efficiency of hybrid formation was evaluated at the single‐particle level by nanoflow cytometry. HEVs were characterized in terms of particle size and size distribution by nanoparticle tracking analysis (NTA), their composition was assessed by automated Raman trapping analysis (SPARTA), and their morphology by cryo electron microscopy (cryo‐EM). In vitro efficacy, including uptake, endosomal escape, and mRNA delivery, was evaluated using Cy5‐labelled green fluorescent protein (GFP) mRNA as a model cargo by evaluating Cy5 and mCherry signals and GFP expression in an mCherry‐Galectin 9 reporter cell line (Huh7‐Gal9) and in hiPSC‐derived cardiomyocytes.

Results : HEV generation was dependent on LNP composition, with the helper lipid and PEG‐lipid molar percentages being critical components in driving or limiting fusion. Two specific LNP compositions enabled the generation of HEVs at high percentages (> 70%), with an mRNA encapsulation efficiency exceeding 60%. HEV samples exhibited significant heterogeneity, as confirmed by NTA and cryoEM imaging, indicating the coexistence of various hybrid particles within the final sample. Multivariate statistical analysis of SPARTA data further highlighted the compositional heterogeneity of HEVs, which nevertheless clustered independently from EVs and LNPs. Additionally, HEVs were able to escape endosomal degradation and deliver GFP mRNA to both Huh7‐Gal9 and hiPSC‐cardiomyocytes.

Summary/Conclusion : We successfully generated functional HEVs with high efficiency. Single particle characterization techniques were employed to elucidate the generation of HEVs, and, to our knowledge, we used SPARTA for the first label‐free, in vitro characterization of HEVs at a biomolecular level. Further studies are ongoing to evaluate the cytotoxicity and biocompatibility of HEVs in comparison to LNPs.

Funding : AstraZeneca R&D.

Optimization of red blood cell‐derived extracellular vesicle hybrids for therapeutic RNA delivery

Alessia Brossa 1,2 , Michela Arena 1,2,3 , Enza Di Gregorio 1,2 , Giuseppe Ferrauto 1,2 , Elena Ceccotti 5 , Stefania Bruno 4 , Benedetta Bussolati 2,4

1 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; 2 Molecular Biotechnology Center, University of Turin, Turin, Italy; 3 University of Naples Federico II, Italy 4 Department of Medical Sciences, University of Turin, Turin, Italy

Abstract unavailable

Single‐particle characterization reveals heterogeneous extracellular vesicle‐liposome fusion

Presenter: Rachel Mizenko

Northwestern University, Evanston, Illinois, USA

Introduction : Fusion of extracellular vesicles (EVs) with liposomes can be used to alter the properties of EVs to enhance their drug delivery properties. However, fusion efficiency is often characterized in bulk, clouding distribution of fusion across heterogeneous EV populations. Here we sought to use orthogonal single‐particle techniques including nanoparticle‐tracking analysis (NTA), resistive‐pulse sensing (RPS), nanoscale flow cytometry, interferometric fluorescence imaging, and laser trapping Raman spectroscopy (LTRS), each with different limitations or biases, to examine the effects of mechanical fusion techniques on physical characteristics and the distribution of fusion across single particles.

Methods : EVs were isolated via UC from HEK293Ts, and liposomes were produced via extrusion. An equivalent number, by NTA, were combined and subjected to various fusion methods (incubation, extrusion, sonication, freeze‐thaw) prior to analysis.

Results : Here we found that concentration and, to a lesser degree, size were altered differentially across different mechanical fusion techniques. In addition, fusion efficiency varied greatly across analysis techniques, with nanoscale flow cytometry and interferometric fluorescence imaging showing concordant results of freeze‐thaw and sonication having relatively high fusion efficiency. The number of remaining liposomes and EVs varied greatly across mechanical fusion techniques, with freeze‐thaw producing the ‘purest’ engineered population. LTRS identified discordant fusion efficiencies compared with other analysis techniques, which may have been due to its relatively high limit of detection.

Summary/Conclusion : Overall, this data shows that not only can single‐particle techniques be used to inform optimization of fusion technique for increasing fusion efficiencies, but also that the ingrained limitations of various equipment can change perceived fusion efficiencies.

Funding : This work was funded by the NIH (RPC: R01 CA241666, RRM: F31 NS120590) and used equipment supported by NCI P30CA093373 and R00‐ GM080249 . This work was adapted from RRM's published dissertation, “EV heterogeneity: an essential feature for intercellular communication in health and disease and an underutilized resource in the clinic,” University of California Davis (currently under embargo).

Proximity labelling and imaging of extracellular particles using membrane‐bound ascorbate peroxidases

Presenter: Kyung‐Min Kim

Hanyang University, Seoul, Republic of Korea

Introduction : We propose an innovative approach for universal labelling of cellular membrane and EP surfaces in indirect and direct manners. Our key idea is centred on cyclic ascorbate peroxidase 2 with a membrane‐accumulating peptide tag (cAPEX2P), which demonstrates strong membrane association and exceptional proximity labelling efficiency while retaining its inherent peroxidase activity.

Methods : We designed and characterized split intein‐mediated self‐splicing cyclic APEX2 variants in vitro. Then, mammalian cellular membrane labelling of modified APEX2 was performed with biotinylated substrate and evaluated using western blotting and confocal fluorescence microscopy using biotin‐streptavidin interaction. To identify protein candidates related to APEX2‐mediated universal labelling, protein lysates were analysed using LC‐MS/MS. After that, we isolated EPs secreted from APEX2‐labelled cells and visualized labelling signal with super‐resolution microscopy using fluorescent APEX2 substrate. Also, we optimized the conditions for proximity labelling, confirming with super‐resolution microscopy. Finally, we applied our universal labelling methods to other EPs derived from bacteria and plants, and the labelling efficiency was analysed using flow cytometry.

Results : We measured the peroxidase activity of cAPEX2, and the negative effect from structural alteration was not observed. Then, we optimized the universal labelling process of biotinylated substrate for mammalian cells. We could visualize the universal labelling signal, and the signal was co‐localized with plasma membrane staining. With our labelling method, we could perform spatiotemporal imaging of plasma membrane proteins and their translocation. We isolated EPs from universally labelled cells and visualized them using super resolution microscopy. LC‐MS/MS data revealed that labelled proteins were enriched and categorized as focal adhesion, semaphorin receptor families, and extracellular matrix proteins. While the EPs cannot be labelled directly with metabolic glycan labelling, our method could be applied to direct universal EP labelling to pre‐isolated mammalian, lactobacillus and lily EPs. Engineered APEX2 showed increased labelling efficiency up to 10 times.

Summary/Conclusion : Our results revealed that cyclic cAPEX2P can easily access cellular or EP membranes, maintaining its function and labelling EPs universally in indirect and direct ways. Without any genetic modification, cAPEX2P can be applied to labelling mammalian, bacterial, and plant‐derived EPs for labelling and visualizing. Our method is promising for understanding the biodistribution of donor EPs and its fate.

Subtopic EV Detection 1

Rapid plasma EV isolation and PD‐L1 mRNA detection using a CRISPR‐Cas13a‐based enzymatic hydrogel chip: immunotherapy outcome prediction

Presenter: Mingzhen Zhong

Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China

Introduction : Plasma extracellular vesicles (EVs) containing PD‐L1 mRNA are considered potential biomarkers for predicting immunotherapy efficacy. In this study, we developed an integrated chip based on interfacial lipid chemistry technology for rapid plasma EV isolation and an enzymatic nucleic acid hydrogel for mRNA detection. The predictive performance of the chip was validated in a clinical immunotherapy cohort.

Methods : The chip comprises three modules. In the separation chamber, plasma EVs are captured by lipid probes, achieving over 90% capture efficiency within 15 min. The captured EVs are then automatically transported to the signal amplification chamber, where a hydrogel composed of CRISPR‐Cas13a, catalase, and DNA nanostructures is embedded. The presence of EV‐derived PD‐L1 mRNA triggers CRISPR‐Cas13a to decompose the hydrogel, releasing encapsulated catalase. This catalase flows into the detection chamber, where it converts colourless dopamine to brown–black polydopamine under hydrogen peroxide, enabling sensitive PD‐L1 mRNA detection. The enzymatic detection signal can be observed visually or analysed via a mobile app with a simple snapshot.

Results : The chip was validated in immunotherapy patients with various tumours. Combining results with machine learning algorithms, the findings demonstrated that it can distinguish clinical stages of patients and predict immune treatment efficacy, with results consistent with computed tomography (CT) and polymerase chain reaction (PCR) data. Furthermore, the incorporation of patients’ serological immunology testing results into the analysis could potentially predict how patients respond to different immune drugs, providing strong predictive factors for clinicians.

Summary/Conclusion : By integrating the EV enrichment and analysis modules, the chip bridges Laboratory technologies with clinical diagnostics, facilitating continuous immunotherapy outcome monitoring and providing technical support for bedside testing.

Funding : National Natural Science Foundation of China (82302639). National Natural Science Foundation of China (8217081536). National Science Fund for Distinguished Young Scholars (82025024).

Ultrasensitive and absolute quantification of extracellular vesicles using the PICO digital immunoassay

Pablo Sánchez‐Martín 1 , John Atanga 2 , Irina Nazarenko 2 , Tobias Groß 1

1 Actome GmbH, Witten, Germany; 2 Institute for Infection Prevention and Hospital Epidemiology, University Hospital Münster, Münster, Germany

Introduction : Research on extracellular vesicles (EVs) is currently limited by the lack of suitable tools to analyse and quantify the heterogeneous populations of these interesting biomarkers. Based on the PICO digital immunoassay, we developed a novel assay to quantify and characterize EVs.

Methods : DNA‐labelled antibodies against different surface or intravesicular proteins were incubated overnight with intact or lysed purified EVs. In the case of intact EVs, the antibodies were selected to prevent the detection of individual solubilized proteins. The binding of at least two antibodies to their protein targets forms the detection unit of the PICO assay, named Couplex. Samples were subsequently diluted and analysed by digital PCR, which provides absolute quantification down to single EV level.

Results : Using different tetraspanins (CD9, CD63, CD81), we detected different EV subpopulations based on the presence of one or more of these markers, with absolute quantification and a sensitivity of down to 3 × 10 5 vesicles/µL, making PICO more sensitive or comparable to other available methods. EV detection was achieved from samples of different origins. The quantification values matched those determined by conventional methods (e.g., nanoparticle tracking analysis and nanoFCM), but without the need of a calibration standard. Incubating the EVs with lysis buffer allowed the detection and quantification of cytosolic markers (4EBP1) not detected in intact EVs. This enables the determination of the integrity of purified EVs, as well as the quantification of relevant biomarkers.

Summary/Conclusion : Here, we present a novel and sensitive method that provides absolute quantification of EVs and their cargo using only 1 µL of sample as input. The multiplex nature of the assay (up to 4 antibodies) also allows simultaneous analysis of multiple markers, positioning PICO as an excellent tool for the study of EVs.

Funding : The collabourative project EV‐surf (FKZ: 13GW0605B) is funded by the Federal Ministry of Education and Research (BMBF) within the funding measure “KMU‐innovativ.”

Extracellular vesicle trafficking in colorectal cancer: a deep learning approach for tracking analysis

Giulia Chiabotto 1 , Bianca Dumontel 1 , Veronica Vighetto 1 , Giorgia Savino 1 , Francesca Alfieri 2 , Luca Zilli 1 , Michela Licciardello 1 , Chiara Tonda‐Turo 1 , Gianluca Ciardelli 1 , Valentina Cauda 1,2

1 Politecnico di Torino, Torino, Italy; 2 U‐Care Medical S.r.l., Torino, Italy

Introduction : Extracellular vesicles (EVs) mediate colorectal cancer (CRC) progression and metastasis, contributing to pre‐metastatic niche formation in distant tissues, particularly the lungs. To enhance understanding of EV‐cell interactions, we developed a deep learning algorithm to track EVs in fluorescence microscopy images and quantify EV internalization in vitro.

Methods : EVs were isolated from Colo‐320DM CRC cells via sterile differential ultracentrifugation and characterized following MISEV guidelines. Nanoparticle tracking analysis, transmission electron microscopy, and cytofluorimetry were used to assess EV size, morphology, and exosomal marker expression. EV internalization studies were conducted using flow cytometry and fluorescence microscopy in in vitro colon and lung models. A tailored Dual U‐Net architecture with a dice loss function was developed for EV and cell segmentation. The network was trained on manually annotated fluorescence microscopy images, and segmentation outputs were merged for accurate EV quantification per cell. Algorithm performance was validated across diverse imaging datasets of colon and lung cells.

Results : CRC‐EVs exhibited typical exosomal size, morphology and marker expression. Cytotoxicity assays revealed no significant impact on cell viability across different doses and time points. The algorithm effectively segmented cells and EVs, revealing distinct patterns of EV internalization. Cytofluorimetric and fluorescence microscopy analyses supported these findings. CCD18Co fibroblasts showed the highest EV uptake, indicating preferential CRC‐EV interaction with healthy colon cells. Lung‐derived cells (A549 and MRC‐5) exhibited higher EV internalization than CRC cells, suggesting lung tropism and metastatic potential of CRC‐EVs.

Summary/Conclusion : The preferential uptake by lung and healthy colon cells highlights the role of CRC‐EVs in metastatic niche formation and tumour initiation. Our deep learning algorithm provides a robust, scalable tool for tracking EVs and monitoring their trafficking, and offers promising applications in therapeutic monitoring of cancer and metastasis.

Funding : This work has received funding from the European Research Council (ERC) GA No. 101081529—“AI CUrES”—ERC PoC 2021.

Visualization of extracellular vesicle DNA and CD63 in recipient cells using dual‐colour single‐molecule localization microscopy

Presenter: Venkatesh Kumar Chetty

University Hospital Essen, Essen, Germany

Introduction : Small extracellular vesicles (sEVs) are crucial players in intercellular communication in various diseases, including cancer, where they transport essential cargo molecules between cells. However, the limitations due to the lack of high‐resolution imaging techniques hindered a comprehensive understanding of sEVs, including their biogenesis and functional attributes. We addressed this knowledge gap by applying single‐molecule localization microscopy (SMLM), a powerful super‐resolution imaging technique, for the nanoscale imaging of sEVs within the recipient cells.

Methods : sEVs were isolated using a combination of tangential flow filtration (TFF), size exclusion chromatography (SEC), and ultrafiltration (UF), collectively known as TSU. According to MISEV2023 guidelines, sEVs were characterized using NTA, TEM, and western blot. 5‐ethynyl‐2′‐deoxyuridine (EdU) was employed to label DNA cargo associated with sEVs (EV‐DNA) that are released by metabolically active donor cells. Following the sEV treatment in the recipient cells, boron dipyrromethene‐azide (BODIPY‐azide) was utilized to label EV‐DNA‐EdU via click chemistry, and Alexa647‐conjugated nanobody against GFP (Alexa647‐Nb) to label CD63‐eGFP+‐sEVs.

Results : Following EV‐DNA labelling using BODIPY‐azide, we showed BODIPY's buffer‐independent blinking feature by performing EV‐DNA imaging in the recipient cells using SMLM. Next, the spatial distribution of GFP+‐sEVs in the recipient cells was visualized by performing immunostaining with Alexa647‐Nb. Using dual‐colour SMLM, we visualized the association of EV‐DNA cargo and CD63+‐sEVs in the recipient cells at nanoscale resolution. Furthermore, using dual‐colour SMLM imaging combined with cluster analysis, we revealed the intricate interactions between EV‐DNA and cytoplasmic DNA sensor cyclic GMP‐AMP synthase (cGAS) within the recipient cells.

Summary/Conclusion : Altogether, our data illustrated that dual‐colour SMLM imaging has a promising scope to study how sEVs are being taken up along with their EV‐DNA cargo in the recipient cells and their molecular interaction with different cellular compartments, which would enable us to understand the biological function of sEVs in various diseases.

Funding : This work was supported by grants to B.K.T. from the Stiftung Universitätsmedizin Essen, Deutsche Jose Carreras Leukämie‐Stiftung (DJCLS 14 R/2023), Deutsche Forschungsgemeinschaft (DFG TH 2012/1‐1), and Deutsche Kinderkrebsstiftung (DKS 2018.17). X.Z. and Q.Y. also acknowledge the support received from the Chinese Scholarship Council (CSC).

Glycan mapping of tumour‐derived extracellular vesicles: a multiplex lectin bead approach

Hema Saranya Ilamathi 1,2 , Mariana Castañeda Mesa 1,2 , Oscar P. B. Wiklander 1,2

1 Department of Laboratory Medicine, Unit for Biomolecular and Cellular Medicine, Karolinska Institutet, Stockholm, Sweden; 2 Breast Center, Karolinska Comprehensive Cancer Center, Karolinska University Hospital, Stockholm, Sweden

Introduction : The glycosylation profiles of lipids and proteins play crucial roles in cell signalling and immune response. In cancer, these patterns shift, driving tumour progression, metastasis, and immune evasion. Tumour cells release extracellular vesicles (EVs) containing various biomolecules that can serve as liquid biopsy biomarkers. However, the variation in glycan profiles of tumour‐derived EVs (tEVs) across different cancer stages remains unclear. Currently, there are limited quick and affordable technologies for glycan profiling. Our goal is to develop a rapid and cost‐effective technique to characterize these glycan profiles.

Methods : We isolated EVs from non‐cancerous and cancerous cells using size exclusion chromatography and quantified particle numbers with a Zetaview analyser. To profile glycans, we developed an in‐house bead‐based protocol utilizing lectins. EVs were pulled down using lectin‐conjugated beads and analysed via flow cytometry.

Results : Our findings reveal distinct glycan expressions on HEK EV surfaces. Using lectins such as ConA, WGA, and UEA, which target mannose, sialic acid, and fucose residues, respectively, we noted higher sialic acid levels on HEK EV surfaces with minimal mannose detection. Fluorescent‐tagged tetraspanin antibodies confirmed EV‐lectin binding. Interestingly, EVs from breast cancer cells showed altered glycan profiles with reduced sialic acid and fucose residues compared to non‐cancerous cells.

Summary/Conclusion : Our in‐house lectin‐based EV profiling technique is faster (< 24 h) and more cost‐effective than traditional glycan profiling methods like microarrays and mass spectrometry. We aim to develop a multiplex lectin‐based bead panel to fingerprint cancer cell glycan profiles and identify potential glycan‐based biomarkers for future research.

Funding : This project is funded by Cancerfonden, the Swedish Research Council fund (VR grant), and the CIMED grant.

A comparative study of magnetic bead‐based techniques for isolating plasma‐derived extracellular vesicles

Verena Pikulski, Johanna Mitterreiter, Jens‐Peter Brock, Lisa Hurler, Daniela Seidel, Gaby Marquardt

Center for Innovation in Diagnostics, Siemens Healthineers AG, Forchheim, Germany

Introduction : Since the discovery of extracellular vesicles (EVs), various isolation methods have been developed and are still in use today. Each method has its benefits, and the choice depends on subsequent analyses. Immunoaffinity selection based on surface markers followed by magnetic separation is a popular approach due to its high specificity and potential for automation. However, the firm bond between EVs and magnetic beads interferes with downstream applications, making verification and standardization technically challenging. This study aims to compare the efficiency and yield of different magnetic isolation protocols, specifically focusing on their miRNA cargo. Additionally, these methods are compared with size‐exclusion chromatography (SEC) and the precipitation method to provide a comprehensive evaluation.

Methods : Platelet‐free plasma was collected from healthy blood donors and combined into master pools. For comparative EV isolation analysis, three different magnetic bead‐based isolation kits were tested—EV Isolation Kit Pan Human from Miltenyi Biotec, EasySep Human Pan‐EV Positive Selection Kit from Stemcell Technologies and an in‐house protocol using Dynabeads from Thermo Fisher Scientific. These methods were compared to SEC columns from Izon Science and a precipitation approach from Thermo Fisher. Additionally, to optimize miRNA retrieval, different RNA extraction methods were tested post EV isolation procedures and evaluated by selected miRNA PCRs.

Results : First results showed all bead‐based isolation protocols to be equally robust and reproducible with comparable yield in EV‐derived miRNA content. EV isolation by SEC or precipitation only resulted in slightly higher miRNA yield. Binding experiments, however, detected unspecific miRNA binding to Dynabeads, rendering this method unsuitable for plasma EV‐derived miRNA analysis. Different miRNA extraction methods tested had little effect on miRNA yield.

Summary/Conclusion : Overall, the bead‐based EV isolation methods tested in this study offer a specific, fast and robust isolation of EVs with high promise for future automation approaches. Small compromises as observed in slightly lower miRNA yield are often acceptable depending on downstream applications. The inclusion of suitable controls is pivotal to exclude potential unspecific binding to magnetic beads. The development of new verification methods is needed to guarantee a better standardization of bead‐based EV isolation in the future.

Funding : This project is supported by the IHI.

Detection and characterization of extracellular vesicles from platelet‐poor plasma samples using a nanoscale‐dedicated CytoFLEX nano analyser

Presenter: Katily Ramirez

Beckman Coulter Life Sciences, Bangalore, India

Introduction : Extracellular vesicles (EVs) are a heterogenous family of membranous particles released by all cells, which provide crucial insights into a variety of fields and diseases. To better understand EVs, researchers must characterize EVs. However, their heterogeneity, size, refractive index, and complex nature make their analysis challenging. For instance, the EVs can range in size from 20 nm up to 5 µm for the bigger apoptotic bodies. The CytoFLEX nano analyser is an RUO flow cytometer with high sensitivity capable of detecting nanoparticles via violet side scatter at least as small as 40 nm, as well as providing better sensitivity and resolution for the 6 fluorescent channels. Here we present the immunophenotype characterization using a designed 5‐colour panel to identify different types of EVs in platelet‐poor plasma (PPP) samples.

Methods : Platelet‐poor plasma (PPP) EVs were prepared from fresh human blood as follows: blood was centrifuged for 5 min at 200 × g . One millilitre of the PPP was removed from the top and filtered through a 200 nm syringe. PPP was further purified using Izon size‐exclusion columns (qEV70nm SEC column). Selected fractions were screened and used for single‐colour stain and multi‐colour stain for immunophenotyping. PPP was then stained with the following antibodies: CD81 PB, CD9 FITC, CD63 APC, CD61 PC7 and CD235a PE. Samples were acquired on the CytoFLEX nano flow cytometer and analysed in CytExpert nano software.

Results : Based on the size distribution and fluorescence staining of the fractions collected from the qEV 70 nm and 35 nm SEC columns, we were able to identify the fractions where EVs reside. The CytoFLEX nano flow cytometer was able to detect and distinguish more than one FL signal on single PPP EV particles, including PB, FITC, PE, PC7, and APC.

Summary/Conclusion : We demonstrated that at the proper antibody concentration and with the use of proper controls, we avoid antibody aggregates and antibody background interference. Finally, we demonstrated that the system was able to compensate for the spillover between different fluorescent dyes, allowing analysis of the 5‐colour staining simultaneously.

Optimization and challenges of isolation‐free single‐vesicle measurement in diabetic patient plasma using high‐sensitivity flow cytometry

Miłosz Majka 1 , Karolina Soroczyńska 1 , Anna Nowak‐Szwed 2 , Leszek Czupryniak 2 , Małgorzata Czystowska‐Kuźmicz 1

1 Chair and Department of Biochemistry, Medical University of Warsaw, Warsaw, Poland; 2 Department of Diabetology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland

Introduction : The diagnostic use of extracellular vesicles (EV) is limited by the labour‐intensive isolation from biofluids. High‐sensitivity flow cytometers (HSFC) enable multiparametric characterization of EVs, but their direct analysis in unprocessed plasma remains challenging. This study aimed to develop a protocol and identify key steps for isolation‐free single‐vesicle measurement in platelet‐poor plasma (PPP) from diabetic patients using HSFC.

Methods : PPP was obtained from blood samples of patients with type 2 diabetes. Plasma aliquots were incubated with pre‐diluted fluorescently labelled antibodies at room temperature for 1 h to ensure optimal binding. After incubation, samples were diluted to final volume and analysed using the Amnis CellStream in autosampler mode. Multiple assay controls were included in accordance with MIFlowCyt‐EV guidelines. Detergent lysis controls were prepared using ionic and non‐ionic detergents diluted in PBS. Where necessary, EVs were isolated from 1 mL of plasma using size‐exclusion chromatography, and particle size and concentration were assessed via nanoparticle tracking analysis. EV‐specific marker expression was validated using Western blotting.

Results : Antibody concentrations and fluorochrome‐particle ratios significantly affected EV concentration measurements and varied between antibody lots. We observed a critical impact of detergent type on lysis controls, with some antibodies exhibiting increased fluorescence following detergent treatment.

Summary/Conclusion : The proposed protocol enabled isolation‐free detection of EVs in patient plasma. Our findings highlight the importance of reporting lysis controls for each antibody in the panel and incorporating detergent plus staining buffer assay controls.

Funding : This research was funded by the Polish Ministry of Science and Higher Education under the programme “Studenckie koła naukowe tworzą innowacje” (grant number SKN/SP/601502/2024) and the programme “Perły Nauki” (grant number PN/01/0095/2022).

Fluorescence imaging and qPCR analysis for assessing biodistribution of extracellular vesicles

Presenter: Hyeonseung Lee

Korea Basic Science Institute, Daejeon, Republic of Korea

Introduction : Extracellular vesicles (EVs) have emerged as promising therapeutic targets, and understanding their biodistribution is essential for evaluating potential effects. This study presents a novel quantitative polymerase chain reaction (qPCR) assay to detect unmodified EVs by targeting mitochondrial DNA (mtDNA) unique to human EVs.

Methods : We focused on specific mtDNA regions with distinct variations from rodent mtDNA to enhance specificity. We developed primers and probes for both human and rodent mtDNA, resulting in a sensitive and specific qPCR method. We also found that the quantification of EVs varied based on the cell source and manufacturer.

Results : To assess biodistribution, we administered IRDye 800CW‐labelled Expi293F EV mimetics to animals via tail vein injection and compared imaging results with mtDNA‐qPCR data.

Summary/Conclusion : This study established a novel qPCR‐based molecular platform that can analyse EVs without manipulation using certain mtDNA primers and probes. The method demonstrates high sensitivity and allows quantitative assessment of EV particles at the level of individual particles in the body. The technique also facilitates the analysis of EVs present in blood samples that are difficult to visualize using an imaging approach. In addition to mtDNA, additional nucleic acid targets that can distinguish EVs should be identified, and optimal quantification methods tailored to each EV should be devised.

Versatile EV surface modification for selective uptake

Barbara Krönigsberger 1,2,3 , Sivun Dmitry 1,2,3 , Madhusudhan Reddy Bobbili 1,4,3 , Johannes Grillari 1,4,3 , Jaroslaw Jacak 1,2,4,3

1 Ludwig Boltzmann Institute for Traumatology, The Research Centre in Cooperation with AUVA, Vienna, Austria; 2 Department of Medical Engineering and Applied Social Sciences, University of Applied Sciences Upper Austria, Linz, Austria; 3 Department of Biotechnology, Institute of Molecular Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria; 4 Austrian Cluster for Tissue Regeneration, Vienna, Austria

Introduction : Efficient and standardized EV internalization remains a challenge. To address this, we aim to develop a versatile strategy for EV modification to target specific cellular uptake pathways.

Methods : We employed the biotin‐streptavidin system to modify eGFP‐tagged EVs derived from HEK293T cells. The first step involved the incorporation of cholesterol‐lipids into the EV membrane. A polyethylene glycol (PEG) linker of approximately 2.45 nm length conjugated cholesterol to biotin, forming a cholesterol‐PEG‐biotin (CPB) construct. For functionalization, we added Alexa Fluor 647‐labelled streptavidin (SA) to the sample, creating a tight bond to the biotinylated EVs. To optimize the labelling of unpurified EVs, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy (SMFM), enabling precise characterization of surface modifications at a single‐molecule level. Building on this approach, we plan to introduce a biotinylated ligand, such as transferrin (Tf), to coat the EV surface as a test system. This strategy is intended to exploit the Tf‐transferrin receptor (TfR) pathway for targeted uptake of small EVs. Linking EVs to Tf enables specific binding to TfR, facilitating cellular internalization via the canonical clathrin‐dependent uptake mechanism.

Results : Using SMFM, we confirmed the incorporation of CPB into the EV membrane and the subsequent binding of streptavidin (SA). Through dual‐colour 2D imaging, we observed that approximately 80% of the eGFP‐positive EVs also carried the CPB‐streptavidin construct, with free SA accounting for about 35% of the total detected SA in the solution.

Summary/Conclusion : The proposed EV's surface modification protocol (cholesterol‐biotin‐streptavidin) is a versatile tool for wide‐range customization of EVs. Targeted delivery into cells via canonical clathrin‐dependent pathways using modified EVs may represent the first standardized method for cellular internalization.

Funding : This project is supported by Interreg AT‐CZ NanoPrecMed ATCZ00052.

Development of an NHS ester‐based lipid staining method for small extracellular vesicles and particles under low pH conditions

Presenter: Jiyoung Goo

Korea Institute of Science and Technology, Seoul, Republic of Korea

Introduction : Small extracellular vesicles (sEVs) are difficult to detect due to their nanoscale size and inner lumen, which result in weak scattering signals. To address the heterogeneity of sEVs, lipophilic dyes targeting lipids—an essential component of sEVs—have been utilized. However, these dyes have notable limitations. In this study, we demonstrate that NHS esters, traditionally employed for protein labelling, can effectively stain PE lipids within sEVs. Moreover, we propose a simple strategy to minimize the staining of lysine ε‐amine groups, which could potentially interfere with protein functionality, among amine‐containing substances in sEVs.

Methods : sEVs were purified from MCF7 cells using differential centrifugation and tangential flow filtration (TFF), followed by ultracentrifugation in line with MISEV2018 guidelines. sEV samples were then resuspended in PBS and labelled with NHS esters under controlled pH conditions (e.g., pH 6.0 or 8.5) to optimize the selective staining of phosphatidylethanolamine (PE) lipids and proteins. Post‐labelling, the samples underwent purification using a G‐25 desalting column to remove excess dye, ensuring only labelled sEVs remained for analysis.

Results : Liposome experiments confirmed that NHS esters effectively label PE lipids under low pH conditions. Moreover, western blot analysis revealed that while the known limitation of NHS esters reducing antibody binding efficiency exists, this drawback was minimized under the low pH conditions we proposed. Compared to conventional lipophilic dyes, the low pH NHS ester‐based staining conditions exhibited superior efficiency, reduced self‐aggregation, and achieved higher colocalization rates with EV markers. Additionally, the versatility of this staining method was highlighted by its successful application to exomeres and supermeres.

Summary/Conclusion : In summary, we developed a low‐pH NHS ester‐based staining method that effectively labels PE lipids and proteins in sEVs, overcoming the limitations of conventional lipophilic dyes such as low efficiency and self‐aggregation. This approach minimizes the impact on protein functionality while achieving superior staining efficiency and high colocalization rates with EV markers. Furthermore, the versatility of this method was demonstrated by its successful application to EPs like exomeres and supermeres, highlighting its potential for broad use in EV and EP research.

Spectrally separated dual‐label upconversion luminescence lateral flow immunoassay for simultaneous detection of CD9+ and CD63+ extracellular vesicles

Imran Mahmud*, Md. Khirul Islam, Iida Martiskainen, and Janne Leivo

Department of Life Technologies, University of Turku, Turku, Finland

Introduction : Extracellular vesicles (EVs) are highly enriched with tetraspanins like CD9 and CD63, which are widely used for EV analysis and characterization and serve as key markers for the development of EV‐based detection technologies. This study aimed to develop a highly sensitive dual‐label lateral flow immunoassay (dual‐LFIA) for multiplex detection of CD9+ and CD63+ EVs using upconverting nanoparticles (UCNPs) as reporters. This dual‐LFIA employs two different UCNPs as luminescent reporters, enabling spectrally distinct and simultaneous detection of CD9 and CD63 targets within a single assay.

Methods : EVs were isolated from prostate cancer Du145 cell culture medium (CCM) by using ultracentrifugation (UC) and size exclusion chromatography (SEC). To capture EVs, anti‐CD9 and anti‐CD63 antibodies were printed onto the nitrocellulose membrane of lateral flow cards as test lines. The cards were assembled with sample and absorbent pads and cut into 4.8 mm strips. Next, the isolated EVs were added on strips for their simultaneous detection using the same CD9 and CD63 antibodies, which were conjugated to erbium‐doped (NaYF4:Yb3+, Er3+) and thulium‐doped (NaYF4: Yb3+, Tm3+) UCNPs, respectively. These UCNPs emit spectrally distinct, narrow emission peaks, enabling optical separation. Specifically, thulium‐doped UCNPs produce a blue emission at 450 nm, while erbium‐doped UCNPs emit green light at 540 nm, facilitating multiplex EV detection and differentiation.

Results : This developed proof‐of‐concept dual‐LFIA was able to identify both CD9‐ and CD63‐positive EVs in Du145‐CCM, ucEVs, and secEVs. It demonstrated high sensitivity and linearity for EV detection.

Summary/Conclusion : Our preliminary studies demonstrate that this novel UCNP‐based dual‐LFIA approach offers superior sensitivity due to the simultaneous detection of CD9+ and CD63+ EVs. Future studies will focus on comparing the sensitivity of this dual‐LFIA with our previously developed single‐label LFIA and investigating disease‐specific markers for clinical applications.

Profiling the biochemical composition of extracellular vesicles in breast cancer using Raman spectroscopy

Presenter: Arianna Bonizzi

University of Milan, Milan, Italy

Introduction : Extracellular vesicles (EVs) are a subgroup of the circulating particles, released by cells in both normal and disease conditions, carrying active biomolecules. Over the past decade, EVs have gained significant attention as a potential source of tumour‐related information, especially in breast cancer (BC). However, exploring the biochemical profile of EVs is challenging due to their low abundance and complex composition. Additionally, EVs can interact with other plasma components, like lipoproteins (LPs), forming functional complexes whose significance is not yet fully understood. Here, Raman spectroscopy (RS) could be a promising tool for assessing EVs chemical composition. In this work, we focused on the RS analysis of EVs as well as of LPs in order to investigate the biochemical distinctions of these extracellular particles (EPs) present in blood. Once we had proven RS's to identify EVs, we investigated RS's ability to distinguish between EVs isolated from the plasma of healthy controls (HC) and BC patients.

Methods : EVs were obtained from human plasma by differential ultracentrifugation (UC), while LPs were isolated using density gradient UC. Both types were analysed by RS, with results compared to those obtained using traditional biochemical assays. The compositional differences in large EVs (lEVs) and small EVs (sEVs) extracted from human plasma were assessed between 30 healthy controls (HC) and 34 BC patients using RS.

Results : Our results demonstrated that consistent Raman bands can be identified across all circulating EPs and that the intensity of these bands can be utilized for their effective discrimination. Additionally, RS revealed a distinct biomolecular profile in EVs from BC patients compared to HC, with particularly pronounced differences in sEVs, associated with elevated levels of nucleic acids and lipids in cancer patients.

Summary/Conclusion : Overall our data strongly support the use of RS as a rapid and reliable method for studying the biochemical composition of circulating EPs. This technique reveals distinct biochemical signatures associated with tumour disease, highlighting its potential as a valuable diagnostic tool for BC.

Optimizing cryopreservation conditions to protect the integrity of red blood cell‐derived extracellular vesicles

Kinga Ilyés 1,2 , Tasvilla Sonallya 1,2 , Anikó Gaál 1 , Zoltán Varga 1,3

1 HUN‐REN Research Centre for Natural Sciences, Budapest, Hungary; 2 Eötvös Loránd University, Budapest, Hungary; 3 Budapest University of Technology and Economics, Budapest, Hungary

Introduction : Extracellular vesicles (EVs) are lipid membrane‐enclosed spherical structures that play a crucial role in biomass transport and intercellular communication. They hold promise as drug carriers and biomarkers, reflecting the physiological and pathological conditions of their cells of origin. Despite extensive research, challenges in EV isolation and storage remain, with standardization still lacking. This study seeks to establish a standardized cryopreservation protocol to maintain the structural and biochemical integrity of red blood cell‐derived EVs (REVs) by optimizing freezing conditions, including buffer composition and concentration.

Methods : To identify optimal cryoprotective conditions, REVs were stored at ‐80°C for 1 month in different dilutions in PBS and PBS supplemented with cryoprotective additives, albumin and trehalose. To investigate the effects, the concentration and diameter of REVs were measured by size exclusion high‐performance liquid chromatography and dynamic light scattering. REVs were immunofluorescently stained with erythrocyte‐specific (CD235a) antigens and analysed by flow cytometry too. Additionally, protein and lipid content were determined by colourimetric bicinchoninic acid protein assay and the sulpho‐phospho‐vanillin method.

Results : The results demonstrate that storing REVs in PBS without cryoprotective additives and at lower concentrations leads to a decreased recovery rate. Flow cytometry analysis also indicates a change in vesicle composition, with reduced structural complexity and the appearance of a disintegrated vesicle population. The best preservation results were achieved in PBS supplemented with albumin and trehalose.

Summary/Conclusion : The optimal conditions for preserving the structural and biochemical integrity of REVs while storing at −80°C were identified as freezing concentrated samples in PBS supplemented with albumin and trehalose.

Funding : This work was funded by the National Research, Development and Innovation Office (NKFIH), Hungary, under grant numbers K 131594 (JM), 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021, and TKP2021‐EGA‐31.

Construction and application of a highly sensitive detection technology for extracellular vesicle membrane proteins based on droplet microfluidics in the early diagnosis of gastric cancer

Presenter: Xueying Mai

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Gastric cancer has become the third leading cause of cancer incidence worldwide, posing a serious threat to national health. Therefore, accurate diagnosis and early treatment are crucial for prognosis. Although diagnostic methods used in clinical practice can be employed for cancer screening, non‐invasive and accurate diagnosis of gastric cancer remains a significant challenge in clinical settings. Single‐molecule quantitative analysis of specific proteins on extracellular vesicles is essential for cancer diagnosis. Although many emerging technologies have achieved single‐molecule protein detection, developing a simple and cost‐effective single‐molecule protein analysis method to facilitate clinical application remains challenging.

Methods : Here, we constructed a droplet microfluidics‐based single‐molecule detection platform for specific proteins on EV, which directly captures plasma‐derived exosomes using functionalized magnetic beads and employs antibody‐nucleic acid complexes for specific capture of target proteins. We then add an RNA enzyme for the cleavage of nucleic acid strands within the complex and finally collect the cleaved template strands for subsequent droplet digital PCR quantification analysis. This method integrates the high specificity of immuno‐PCR and the high sensitivity of droplet digital PCR to analyse EV surface proteins, achieving absolute quantification of EV surface protein molecules.

Results : By simultaneously detecting EGFR and MUC1 on the EV membrane, we validated their diagnostic efficacy in clinical cohorts. The results indicated that the single‐molecule quantification of specific proteins on EVs can significantly distinguish gastric cancer, benign gastric diseases, and healthy control cohorts. Moreover, the area under the curve for distinguishing the healthy cohort from the gastric cancer cohort with the combination of two markers reached as high as 0.99. Furthermore, using mass spectrometry technology, we identified two early gastric cancer markers, TGF‐B1 and TNN, and validated them in gastric cancer clinical cohorts. The results demonstrated that the combined detection of the two markers can significantly distinguish early gastric cancer, benign gastric diseases, and healthy control cohorts.

Summary/Conclusion : Therefore, this study provides a very promising strategy for single‐molecule quantification of proteins on EVs for cancer diagnosis.

Liquid biopsy‐based depression detection and response prediction using EV‐SERS‐XAI

Seungmin Kim 1,2# , Youbin Kang 3# , Hyunku Shin 4# , Eun Byul Lee 4 , Byung‐Joo Ham 3* and Yeonho Choi 1,2,4,5 *

1 Department of Biomedical Engineering, Korea University, Seoul, Republic of Korea; 2 Interdisciplinary Program in Precision Public Health, Korea University, Seoul, Republic of Korea; 3 Exopert Corporation, Seoul, Republic of Korea; 4 Department of Life Science, University of Seoul, Seoul, Republic of Korea; 5 School of Biomedical Engineering, Korea University, Seoul, Republic of Korea

Introduction : The diagnosis of depression typically relies on subjective patient self‐reports and clinical observations, and treatment response can only be confirmed after a 5–7 week course of antidepressants. This highlights the need for diagnostic techniques using objective indicators of medical decision‐making. We developed a novel method to detect depression and predict treatment response by analysing plasma extracellular vesicles (EVs) using nanoplasmonic spectra and deep learning.

Methods : We isolated plasma EVs, which can penetrate the blood‐brain barrier and carry psychiatric information. EV Raman signals were obtained to detect molecular information from depressive patients ( n = 98), and non‐depressive controls, including healthy controls ( n = 42) and panic disorder patients without depression ( n = 6). These signals were used to develop a deep learning‐based diagnostic and predictive algorithm. The algorithm consisted of two main steps: (1) diagnosing depression and (2) predicting treatment response.

Results : Our algorithm successfully distinguished depression patients not only from healthy individuals but also from panic disorder patients with an area under the curve (AUC) accuracy, sensitivity, and specificity of 0.95, 0.91, and 0.95, respectively. Subsequently, our algorithm classified responders and non‐responders to antidepressants with a high AUC accuracy, sensitivity, and specificity of 0.91, 0.89, and 0.93, respectively. The clear identification of individuals demonstrates the potential of the model to proactively predict the treatment response before encountering medication failures. To confirm the basis of our diagnostic outcomes, we applied explainable AI (XAI) to the algorithm. Consequently, we identified the key decision‐positive values, allowing us to interpret the critical features driving the model's classification performance.

Summary/Conclusion : Our technique demonstrates the potential of liquid biopsy‐based depression detection and prediction of antidepressant response for companion diagnostics. We address an unmet need in depression diagnosis by offering a more objective method and showcasing its potential for personalized treatment strategies. This approach reduces unsuccessful treatment attempts and improves therapeutic outcomes, enabling individualized strategies that maximize treatment efficacy.

Funding : This study was supported by the National Research Foundation of Korea grant (RS‐2023‐00277719) and the Technology Development Program (RS‐2022‐TI016650) funded by the Ministry of SMEs and Startups.

Microfluidic device to monitor brain tumours using extracellular vesicles from cerebrospinal fluid

Sara Cavallaro 1,2 , Sahbra Eldosougi 1 , Uma Paithankar 1 , Sara I. Veiga 1,2 , Raheel Ahmad 1,2 , Cecile Riviere‐Cazaux 3 , Terence C. Burns 3 , Brian V. Nahed 1,2 , Shannon L. Stott 1,2 .

1 Massachusetts General Hospital, Boston, Massachusetts, USA; 2 Harvard Medical School, Boston, Massachusetts, USA; 3 Mayo Clinic Rochester, Rochester, Minnesota, USA

Introduction : Liquid biopsies represent a revolutionary approach in brain cancer management, being significantly less invasive than brain biopsies while also enabling real‐time tumour monitoring. However, a blood test for brain tumours has not translated into clinical practice due to the low abundance of circulating tumour biomarkers. While less accessible than blood or other biofluids, cerebrospinal fluid (CSF) is viewed as one of the most valuable sources of tumour‐specific biomarkers, particularly for glioblastoma (GBM), due to its proximity to the brain and direct access to molecules that cannot easily cross the blood‐brain barrier. Several studies have successfully identified GBM biomarkers in CSF, including extracellular vesicles (EVs). Microfluidic devices are especially well‐suited for analysing CSF, as they can handle incredibly small volumes of fluid (10 µL to 1 mL) with exquisite precision and sensitivity and relatively low cost.

Methods : In this work, we present a microfluidic technology that can isolate cancer‐specific EVs from the CSF of brain cancer patients. Inspired by the ‘backbone’ of our EV isolation technology, the EV‐HB‐Chip, our device will use herringbone patterns and immunoaffinity capture to isolate brain cancer‐specific EVs. The developed platform will be specifically designed for CSF and optimized for small biofluid volumes (∼50 µL–1 mL). Following isolation, the EV RNA content will be analysed using ddPCR.

Results : To demonstrate the feasibility of our approach, we processed 500 µL of CSF obtained from adult brain tumour patients (one of which was serially sampled at three time points). For these experiments, EVs were captured into the EV‐HB‐Chips using a cocktail of antibodies targeting EGFR, podoplanin, PDGF and Axl. The results showed successful detection of some of the brain tumour specific genes (EGFR, MYCN) using ddPCR following EV capture. For the patient serially monitored, the data showed high signals of GFAP gene expression that decreased over time, mimicking the patient's clinical response (decreasing tumour burden).

Summary/Conclusion : The preliminary data suggest the feasibility of the proposed technology, while also indicating areas for further optimization.

Funding : National Cancer Institute under Grant R01‐ CA226871 (S.L.S.); National Cancer Institute under Grant F32‐ CA236417 (D.C.R.); American Cancer Society under Grants 132030‐RSG‐18‐108‐01‐TBG (S.L.S.) and PF‐23‐1151433‐01‐CCB (S.C.); V Foundation (S.L.S.).

In vivo investigation of whole body biodistribution of tumour cell‐derived exosomes

Presenter: Nadin Bedikyan

Koç University, Istanbul, Turkey

Introduction : Exosomes are vesicles that vary in size from 30 to 100 nm and contribute significantly to the progress of cancer by including a large amount of released cell DNA and RNA. The biodistribution of tumour‐derived exosomes are critical for understanding their role in cancer progression and metastasis. Exosomes, are nano‐sized vesicles secreted by tumour cells, carrying bioactive molecules like proteins, lipids, and so forth, which can modulate the tumour microenvironment and influence distant organs. This study investigates the whole‐body biodistribution of fluorescent labelled tumour‐derived exosomes following intravenous injection in mice, leverageing advanced imaging techniques for comprehensive analysis. First, appropriate amounts of fluorescently stained MDA‐MB‐231 (breast cancer cell line) and U87 (GB cancer cell line) tumour‐derived exosomes were injected intravenously into C57BL6 mice. The injected tumour‐derived exosomes will then be used for imaging with IVIS. In our in vivo experiments, non‐toxic exosomes obtained from HEK293T will be used as a control, and MDA‐MB‐231 and U87 will be used, and the organs in which they accumulate their biodistribution will be compared, and micrometastasis will be examined in our future experiments. This comparison may provide insight into how different tumour‐derived exosomes contribute to organ‐specific metastasis and provide clues for targeted therapy, as well as the opportunity to examine in which organs non‐toxic HEK‐derived exosomes and tumour‐derived exosomes accumulate.

Methods : Cell culture experiment exosome isolation exosome labelling with Alexa Fluor 647 exosome characterization, IVIS imagaing, and analysis of the biodistributıon.

Results : In the study, successful isolation of non‐toxic exosomes from HEK cells was achieved. The isolation method resulted in high efficiency while preserving the biological activity of exosomes. The results obtained with the IVIS imaging system show that injected exosomes significantly The images revealed how the distribution of exosomes in different organs changed over time, which is important for understanding their behaviour in vivo and accumulation in certain organs, especially in organs such as the liver, spleen and lung. This suggests that exosomes have organotropic properties.

Summary/Conclusion : our study will investigate whether injected tumour‐derived exosomes promote the formation of secondary tumours or affect the tumour microenvironment in specific organs, which will be the subject of subsequent research in vivo after biodistribution is observed.

Fully integrated microfluidic systems for EVs analysis and applications to cancer identification with AI

Presenter: Bifeng Liu

Huazhong University of Science and Technology, Wuhan, People's Republic of China

Introduction : Cancer is a leading cause of global mortality. Recent studies have demonstrated that EVs play an irreplaceable role in cancer progression. To develop an efficient method for EVs analysis would be greatly helpful for cancer identification in clinics. The emerging microfluidic technology is anticipated to become an ideal approach for EVs analysis due to its advantages in miniaturization, automation, integration, and so forth. In this presentation, we interestingly demonstrated fully integrated microfluidic systems for EVs analysis. Coupled with AI, those methods have been successfully applied to cancer identification.

Methods : Here, we present a novel fully integrated lab‐on‐a‐disc (iExoDisc) that isolates exosomes directly from blood samples. This platform consists of three integrated modules: plasma separation, highly abundant protein removal, and nanopore membrane‐based total isolation module. We applied iExoDisc to isolate exosomes from clinical samples and validated its performance by high‐throughput MALDI‐TOF‐MS and lectin microarrays for glycan profiling of plasma exosomes. For enhancing EVs detection, a rapid and visually detectable method termed freeze‐thaw‐induced floating patterns of gold nanoparticles (FTFPA) is proposed, which surpasses current state‐of‐the‐art technologies by achieving a 100‐fold improvement in the limit of detection of EVs.

Results : The findings from the microfluidic systems indicated that galactosylation and sialylation exhibit potential as diagnostic indicators for triple‐negative breast cancer (TNBC). FTFPA could accurately identify EVs derived from subtypes of breast cancers with AI algorithms. Intriguingly, learning the freezing‐thawing‐microstructures of EVs with a random forest algorithm is not only able to distinguish their original cell lines (with an accuracy of 95.56%) but also succeeds in processing clinical samples ( n = 156) to identify EVs by their healthy donors, breast lump and breast cancer subtypes (Luminal A, triple‐negative breast cancer, and Luminal B) with an accuracy of 83.33%.

Summary/Conclusion : The iExoDisc for Evs analysis paves a new avenue to early cancer detection and point‐of‐care diagnosis. The AI‐empowered micro‐visualization method establishes a rapid and precise point‐of‐care platform that is applicable to both fundamental research and clinical settings.

Funding : National Key Research and Development Program of China (2021YFA1101500, 2023YFF1205002); National Natural Science Foundation of China (22074047, 32171248, 12102142).

Single‐molecule protein interaction coupling (PICO) for absolute quantification of intact individual extracellular vesicle protein markers

John Atanga 1 , Pablo Sánchez‐Martín 2 , Siobhan King 3 , Tobias Gross 2 , Irina Nazarenko 1

1 Faculty of Medicine, Institute for Infection Prevention and Control, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany; 2 Actome GmbH, Freiburg, Germany; 3 ONI UK, Linacre House, Oxford, UK

Introduction : Extracellular vesicles (EVs) hold great potential in liquid biopsy applications, especially in disease diagnostics. However, translation of EV‐based findings from the bench to the bed faces significant drawbacks due to a lack of methods that allow for precise EVs quantification and the value of surface biomarkers on EVs. Here, we present a novel method using protein interaction coupling (PICO), enabling precise EV quantification and surface biomarker evaluation on intact EVs at the single‐vesicle level. The PICO assay utilizes oligonucleotide‐labelled antibodies, which are incubated with a sample to form a ternary complex comprising at least two antibodies and a target (e.g., a protein or EV), referred to as a ‘couplex’ in the so‐called ‘binding reaction,’ which will be the reference point for all later quantitative calculations. The assay is entirely homogeneous, requiring no washing or purification steps, as none of the reactants are bound to a solid phase or bead. In order to detect the complexes and antibodies, the oligonucleotide of the antibodies must be amplified and detected using a workflow that is analogous to a digital polymerase chain reaction.

Methods : The present study utilised small extracellular vesicles (sEV) isolated from cell‐conditioned media supernatant employing a combination of tangential flow filtration (TFF) and size exclusion chromatography.

Results : PICO enables absolute quantification, as validated through single‐molecule specific quantification of EV‐membrane proteins (CD9, CD63, CD81). Additionally, PICO allows for EV subpopulation analysis. This was evaluated using a 3‐plex antibody panel allowing for colocalization analysis as demonstrated by simultaneously quantifying the proportion of sEV harbouring cancer‐specific markers (HER2/HER3, HER2/CD9, HER2/CD81, HER2/CD63 and HER2/CD9/CD63) on tumour‐derived sEV isolated from the C4.2B cell line. We further illustrate PICO clinical applicability by quantifying HER2 and HER3 on single vesicles isolated from blood plasma of prostate cancer patients.

Summary/Conclusion : PICO holds tremendous promise for robust, standardized, and precise EV surface biomarker quantification, providing valuable insights into EV‐protein biology and liquid biopsy applications.

Funding : Federal Ministry of Education, Germany.

Study of the properties of modified vesicles as therapeutic vehicles

Maribel Lara Corona 1 , Anaïs Bécot 1 , Pierre‐Michaël Coly 1 , Olivier de Jong 2 , Pieter Vader 2 , Frederik Verweij 2 and Guillaume van Niel 1,3

1 Institut de Psychiatrie et Neurosciences de Paris, Paris, France; 2 Universiteit Utrecht, Utrecht, The Netherlands; 3 Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers (CRCI 2 NA), Angers, France

Introduction : The capacity of extracellular vesicles (EVs) to target and deliver material to recipient cells has positioned EVs as appealing nanovehicles for therapy for numerous diseases. Currently, several loading strategies exist to load biomolecules in newly formed EVs, mainly RNA. Yet the mechanisms of action of the delivering capacities of EVs are still poorly understood, and so is the cell biology supporting this activity. To better understand these processes, we developed innovative strategies to load specific cargo in the lumen of EVs and investigate their fate in recipient cells equipped with a delivery reporter.

Methods : Here, we developed a system that does not require pharmacological reagents but rather allows cargo loading into EVs through light, by transient interactions. We adapted the optogenetic moieties eMagA and eMagB to reversibly load the Cre protein into CD63 vesicles upon blue light stimulation, to target a Cre‐loxP system in recipient cells in vitro and in the zebrafish embryo. This strategy uses fluorescently tagged CD63, which has been previously developed in the lab to study exosome biogenesis and fate, and allows us to follow EVs at a single scale in vitro and in vivo in the zebrafish embryo.

Results : We show that the optogenetic system using eMags promotes a translocation of cargo from the nucleus to the cytoplasm upon light stimulation. In addition, EVs obtained upon light stimulation contain a higher level of Cre inside them without affecting vesicle size or morphology. As others before us, we confirm that functional protein delivery does not occur naturally. Endosomal escape seems to heavily rely on massive amounts of EVs and the addition of fusogenic proteins to the EV surface, such as VSV‐G.

Summary/Conclusion : We present here an innovative loading strategy into EVs that we have used to refine their capacity as delivery vehicles. While we can promote functional protein delivery in recipient cells by overexpressing the VSV‐G protein, our results point to a poor capacity of EVs to deliver by membrane fusion with the recipient cell. This prompts us to reconsider the way we want to use EVs as therapeutic nano‐vehicles.

Funding : French National Research Agency (Agence Nationale de la Recherche—ANR).

Heteronuclear MRI of targeted extracellular vesicles

Galisova A. 1,2 , Havlicek D. 1,2 , Zahradnik J. 2 , Tunca A. 2 , Androvic L. 3 , Sedlacek O. 2 , Laga R. 3 , Jirak D . 1,2

1 Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 2 Charles University, Prague, Czech Republic; 3 Czech Academy of Science, Prague, Czech Republic

Introduction : Imaging of extracellular vesicles (EVs) is crucial for understanding their role in intercellular communication and disease mechanisms. We aim to develop an array of tracking strategies for genetically‐engineered EVs by magnetic resonance imaging (MRI). Here, we encapsulate synthetic biocompatible polymer probes composed of different MR‐active isotopes, as 19F and 31P, in EVs and then monitor the EV distribution in tissues in vivo. Such a heteronuclear approach allows specific tracking of labelled EVs compared to conventional 1 H MRI that provides only relative signals.

Methods : HEK293 cells stably displaying a cancer‐targeting peptide (RGD) were used for EV isolation by a standard ultracentrifugation method (100,000 × g , 2 h) combined with ultrafiltration (100 kDa). RGD‐displaying EVs were characterized by western blot and NTA. Binding to cancer cells (4T1) was tested in an uptake experiment by flow cytometry. Isolated EVs were labelled with MR‐active macromolecular agents: fluoropolymers (TFEAM‐based) and phosphopolymers (pMPC‐based) using microporation (1000 V, 2 pulses, 20 ms) or extrusion (5 µm, 1 µm, 400 nm). Polymer‐labelled and unlabelled EVs were tested on a 7T MR scanner. Then, fluorescently and magnetically labelled EVs were intravenously injected into tumour‐bearing Balb/c mice and visualized by fluorescence and MR imaging.

Results : Vesicles originated from the engineered cells contain the full‐length of the targeting peptide on the surface, as confirmed by western blot. Preferential uptake of engineered RGD‐EVs in 4T1 cells compared to unmodified EVs was proved by flow cytometry and in vivo fluorescence imaging in mice. Polymer‐labelled EVs in solution were specifically visualized by 19 F and 31 P MRI showing specific MR signals on a background 1 H MR image. Targeted 19F‐labelled EVs were visualized in a tumour by ex vivo MRI.

Summary/Conclusion : We implemented protein engineering in combination with cutting‐edge MRI technology to create efficient EV‐based drug delivery and diagnostic systems for cancer therapy. MRI, as non‐invasive, three‐dimensional and clinically translatable imaging method, allowed visualization of magnetically labelled EVs in solutions and in tissue. Heteronuclear MRI (19F/31P) showed promise for specific in vivo detection of EVs, although the sensitivity needs to be tailored by improvement of chemical synthesis of probes or enhancement of labelling efficiency.

Funding : This work was supported by the Czech Science Foundation (GACR)—Grant No. 22‐13334I.

Metabolic glycoengineering‐based bioorthogonal extracellular vesicle surface engineering for tumour targeting

Samar Ahmad 1 , Pradnya Patil 1 , Seppo Vainio 1, 2

1 Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; 2 Kvantum Institute, Infotech Oulu, University of Oulu, Oulu, Finland

Introduction : Extracellular vesicles (EVs) are secreted entities that mediate intercellular communication. The ability of EVs to cross various cellular barriers reflects their potential as a drug delivery vehicle in therapeutic applications. However, the therapeutic efficacy of EVs as a drug delivery tool is limited due to the unpredictable in vivo biodistribution of EVs. Therefore, effective strategies are required to increase the cellular uptake of EVs in recipient tumour cells.

Methods : Here we report our preliminary results about the feasibility of a universal metabolic glycoengineering technique, a method frequently used to label cell surface glycans, to install a chemical functional group on the surface of EVs. Subsequently, the labelled EVs were detected using bioorthogonal click chemistry.

Results : Cell and EV surface glycans of a non‐tumour and various tumour cell lines were tagged with an azido functional group, which was detected on the EV surface through click chemistry‐based fluorescence measurement. This surface chemical tag can provide a flexible system to conjugate diverse moieties using non‐toxic copper‐free bioorthogonal click chemistry for efficient tumour targeting.

Summary/Conclusion : Thus, EV surface engineering using click chemistry to enhance selective EV uptake in tumour cells can prove to be an efficient tumour‐targeting approach.

An automated, high‐throughput method for genetically engineering and phenotyping yeast extracellular vesicles

Kathleen Hon, Curt Logan, Christopher L. Brett

Department of Biology, Center for Applied Synthetic Biology, Concordia University, Montreal, Quebec, Canada

Introduction : Extracellular vesicles (EVs) represent a promising new modality for drug delivery. We use Saccharomyces cerevisiae (baker's yeast)—an organism used for drug biomanufacturing—as a platform to design, build, and test engineered EVs for therapeutic applications. This involves modifying their contents and surfaces by adding human or yeast proteins with diverse functionalities. We aim to test thousands of modifications (individually or in combination) to optimize EV cargo loading, cell targeting, and content delivery. To support our studies, we sought to establish high‐throughput cloning and phenotyping protocols to generate libraries of genetically modified S. cerevisiae strains.

Methods : We employed Golden Gate and Gateway cloning strategies based on the modular Yeast Toolkit, enabling use of new constructs by the synthetic biology community. Candidate genes were introduced into donor plasmids and then integrated into expression vectors containing a strong promoter (TDH3) and the Nanoluc luciferase (Nluc) gene. PCR, genetic assemblies, bacterial transformations and colony selection were conducted in 96‐well plate format by robotic equipment housed in Concordia University's Genome Foundry. After assembled vectors were validated by pooled nanopore sequencing, robots were used to transform them into S. cerevisiae (e.g., wild type BY4741), to select clonal transformants, and to prepare furimazine‐based assays for detection of candidate proteins tagged with Nluc within whole cell lysates or EV‐containing samples, measured using a plate‐reading luminometer.

Results : As proof of concept, we implemented an automated procedure to generate an initial set of 96 yeast strains, each expressing a candidate protein fused to Nluc. To limit cost and accommodate high‐throughput screening, we—for example—determined minimal reaction volumes required for efficient cloning and minimal numbers of yeast cells required for reliable protein‐Nluc detection. Despite using a single promoter, we observed variable expression levels of human and yeast candidate proteins within S. cerevisiae cells. Most candidate proteins studied were observed in EVs. These included human proteins (e.g., CD81), suggesting that the mechanism(s) underlying EV protein loading are conserved.

Summary/Conclusion : We developed an automated yeast genetic engineering and phenotyping research pipeline for high‐throughput screening of strategies to improve EV properties required for use as next‐gen drug delivery vehicles.

Efficient labelling and purification of extracellular vesicles using an aptamer to CD9 and PKH‐26 dye for enhanced cellular uptake

Presenter: Satendra Kumar Jayswal

Deakin University, Geelong, Australia

Introduction : Extracellular vesicles (EVs) are key mediators of intercellular communication, and their labelling, capture, and release are essential for studying their biological functions. PKH‐26, a lipophilic fluorescent dye, integrates efficiently into EV lipid bilayers but forms free dye nanoparticles during the labelling process. These nanoparticles can cause false‐positive results in downstream applications and are difficult to remove without compromising EV integrity or yield. This study introduces a novel method using a DNA aptamer against CD9 to capture EVs, label them with PKH‐26 dye, and wash and elute the labelled EVs, followed by the evaluation of their cellular uptake.

Methods : Biotin‐labelled CD9 aptamer was conjugated to streptavidin‐coated magnetic beads to form bead‐aptamer complexes. These complexes were incubated for 16 h with 2.0 × 10⁸ EVs derived from SW480 cell culture media, forming bead‐aptamer‐EV conjugates. Control samples without EVs were used to assess selectivity and specificity. After labelling the EVs with PKH‐26 dye on beads, the washed EVs were eluted in PBS. To verify the removal of free dye nanoparticles, the eluted EVs were recaptured with CD9 antibody‐coated beads and analysed by flow cytometry. The eluted bead‐free EVs were incubated with HepG2 cells, and their uptake was visualized after 24 h using the EVOS FL Auto Imaging System with an RFP filter.

Results : Flow cytometry assay showed a significant difference in median fluorescence intensity (MFI) and positive parent percentages between labelled EVs and non‐labelled controls. After elution and recapture, the MFI and positive parent percentages of labelled EVs remained significantly higher compared to controls, confirming effective removal of free dye nanoparticles. Labelled EVs introduced into HepG2 cells displayed positive red fluorescence after 24 h. In sharp contrast, there was hardly any red fluorescence observed for the non‐labelled EVs, indicating successful uptake.

Summary/Conclusion : This study demonstrates a robust method for EV labelling using CD9 aptamer‐conjugated beads and PKH‐26 dye, ensuring the removal of free dye nanoparticles while maintaining EV integrity. This approach advances EV research for diagnostic and therapeutic applications.

Funding : Deakin University.

Ultra‐sensitive detection of breast cancer‐derived extracellular vesicles using single molecule array technology: a comparative study of capture strategies

Presenter: Arianna Bonizzi

University of Milan, Milan, Italy

Introduction : Extracellular vesicles (EVs) have gained significant interest as promising liquid biomarkers for breast cancer (BC). However, many aspects of their biological roles remain unexplored due to technical limitations in current EVs isolation and characterization protocols. A critical challenge is identifying markers enriched in cancer‐released EVs to distinguish them from the general EVs population. Cancer‐specific markers are present on EVs at minimal concentrations, underscoring the need for sensitive, user‐friendly methods to unlock their full clinical potential.

Methods : Here we leverage single molecule array (SiMoA) technology, enabling ultra‐sensitive biomarker detection, to detect HER2‐positive EVs in BC patients. Two distinct SiMoA immunobeads were developed: magnetic beads functionalized with either an anti‐CD63 antibody or a membrane‐sensing peptide (MSP) with specific affinity for EVs curvature. These approaches were evaluated and compared as strategies for capturing EVs in biological samples, using CD9 and HER2 detector antibodies to differentiate between general and tumour‐specific EVs populations, respectively.

Results : The two assays were tested on EVs isolated from patient‐derived organoids (PDOs) lines: one from a healthy sample and the other from a tumour sample. Both assays exhibited a clear dose‐dependent response to the loaded EVs. Importantly, the MSP/HER2 assay demonstrated superior performance compared to the CD63/HER2 assay, effectively distinguishing EVs from the two PDOs lines and revealing significantly higher HER2 expression in tumour‐derived EVs compared to those from the healthy sample. The two detection strategies were also evaluated directly on plasma samples collected from metastatic breast cancer (MBC) patients and healthy controls (HC). The results indicate that the signals obtained with MSP‐functionalized beads were higher than those obtained with CD63‐functionalized beads across HER2 assays. In both assays, general EVs levels appeared lower in MBC patients compared to HC, and HER2‐expressing EVs showed an enrichment trend in MBC patients.

Summary/Conclusion : This study demonstrates the feasibility of SiMoA technology for detecting cancer‐released EVs, with its high sensitivity, and highlights how the use of magnetic beads conjugated with MSP can further improve the specificity of the assay. Coupling SiMoA technology with MSP thus offers a rapid and effective method for the detection of cancer‐released EVs in BC patients.

Funding : PRIN, EV‐PRINT 2022CS9H53

Optimizing downstream processing of enveloped virus‐like particles and extracellular vesicles using fluorescently labelled particles

Presenter: Malena von Elling‐Tammen

Cologne University of Applied Sciences, Cologne, Germany

Introduction : Enveloped virus‐like particles (VLPs) are of significant biopharmaceutical interest due to their high immunogenicity and non‐infective nature. These particles lack viral genomes and contain only viral structural characteristics such as surface or capsid proteins. Likewise, extracellular vesicles (EVs) are emerging as promising therapeutic candidates. Both EVs and VLPs share structural similarities, arising from analogous budding mechanisms. Notably, human immunodeficiency virus (HIV)‐derived VLPs tagged with GFP have been utilized as reference material for EVs, underscoring their structural and functional parallels. The downstream processing of VLPs and EVs presents similar challenges of achieving high recoveries while ensuring high purity. Due to their structural similarities, quantifying intact particles necessitates complex analytical methods, complicating their detection using common high‐throughput techniques. Additionally, both entities face stringent purity requirements for therapeutic applications, such as the FDA mandate for less than 10 ng of residual DNA.

Methods : In this study, we employed fluorescently labelled HIV‐derived VLPs as a model for enveloped VLPs. Their fluorescent labelling makes them invaluable tools for optimizing downstream processing platforms, as they can be directly and rapidly quantified using various fluorescence‐based techniques. Anion exchange chromatography was used for the purification step. We monitored the process, focusing on EV, VLP, and DNA analytical methods.

Results : Initially, we developed methods to distinguish between EVs and VLPs to implement different membrane chromatography and filtration techniques in the next step. To optimize the purification step with anion exchange chromatography, we particularly focused on host cell DNA in the form of chromatin as the main contaminant. By degrading residual DNA using a nuclease treatment, we succeeded in separating residual DNA from VLPs in the subsequent chromatography step.

Summary/Conclusion : Distinguishing between enveloped VLPs and EVs, as well as optimizing their purification processes, presents numerous challenges. Our findings address these challenges and contribute to developing a scalable downstream processing platform based on anion exchange chromatography for enveloped VLPs, thereby enhancing their potential for biopharmaceutical applications.

Funding : This work has received funding from the German Federal Ministry of Education and Research, funding programme Forschung an Fachhochschulen, contract number 13FH048KX1.

A reporter system to study adipocyte‐derived extracellular vesicles in vivo

Didde Riisager Hansen 1,2 , Rugivan Sabaratnam 1,2,3 , Lasse Bach Steffensen 1 , Per Svenningsen 1

1 Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark; 2 Steno Diabetes Center Odense, Odense University Hospital, Odense, Denmark; 3 Department of Clinical Research, University of Southern Denmark, Odense, Denmark

Introduction : Adipocytes secrete extracellular vesicles (EVs), but their effect on whole‐body metabolism is unclear due to tracking and isolation limitations. We hypothesized that viral delivery of CD63 fused to nanoluciferase and hemagglutinin (CD63‐NanoLuc‐HA) to adiponectin‐Cre mice induces adipocyte‐specific NanoLuc expression, creating a sensitive reporter system. Furthermore, we hypothesized that obesity increases EV secretion from adipocytes.

Methods : We designed an AAV vector with a CD63‐NanoLuc‐HA construct, and a control with an IgK‐NanoLuc‐HA vector, reflecting EV and protein secretion, respectively. Both vectors were packed in AAV9 capsids and given to adiponectin‐Cre mice via intraperitoneal injection (5 × 10 11 vg). For the diet experiment, mice were put on a chow diet or HFD for 2 weeks. At sacrifice, organs, plasma and urine were harvested for evaluation of adipocyte‐specific NanoLuc and HA expression by western blot, immunohistochemistry, luciferase assay and qPCR.

Results : qPCR confirmed selective reporter expression in adipose tissues, and the HA‐tag was detected in interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (iWAT) of both models by western blotting. By histological evaluation, HA was observed in adipose tissues with the highest signal in eWAT and iBAT. Also, the liver, kidney and small intestine showed a signal. The control model had low signal in adipose tissues, but higher in the kidney and small intestine. NanoLuc activity was detected in plasma, urine, and tissues, with the highest levels in eWAT, iBAT, and iWAT. The EV reporter showed higher tissue levels, while the control model had the highest plasma and urine levels. Between diets, a significant increase in NanoLuc activity was observed in iBAT, eWAT, urine, kidney and lungs of the EV reporter model, but not the control model.

Summary/Conclusion : Opposite our control model, high tissue levels and low body fluid levels, emphasizes high cellular protein release, but a small EV fraction entering body fluids. HFD‐induced obesity seems to increase EV secretion from adipose tissue, renal filtration of EVs, and pulmonary EV uptake. In conclusion, our model enables investigation of adipocyte‐derived EVs in vivo to unveil their role in normal physiology and during metabolic disturbances.

Funding : Steno Diabetes Center Odense, University of Southern Denmark, Aase & Ejnar Danielsen Foundation, A. P. Møller Foundation.

Small RNA‐sequencing (miND) enables discovery of EV‐specific microRNA signatures that support EV drug development

Presenter: Marianne Pultar

TAmiRNA GmbH, Austria

Introduction : Extracellular vesicles (EVs) are promising drug products or drug delivery tools. Information about the biodistribution of native or engineered EVs is important to validate drug delivery and to make decisions on target organs and indications. We hypothesize that the identification of EV‐specific or ‐enriched microRNA (miRNA) cargo can provide valuable insights into the biodistribution and uptake of EVs in target cells.

Methods : To test this hypothesis, we performed a comprehensive NGS‐based analysis of human Wharton's Jelly (WJ) MSC‐EV RNA cargo as well as lung, liver, bone, and blood (mouse) as a panel of common target tissues (Figure 1). We utilized the miRNA Next‐Generation‐Sequencing Discovery Assay (miND), which uses spike‐ins to enable absolute quantitation of small RNAs, including miRNAs.

Results : We identified a cluster of miRNAs with significant enrichment in WJ‐MSC‐derived EVs in comparison to the selected target tissues. We developed a miRNA RT‐qPCR assay for targeted analysis of 5 WJMSC‐EV‐specific miRNAs and added four control miRNA assays consisting of a liver (miR‐122‐5p), lung (miR‐150‐5p), bone (miR‐140‐3p), and blood‐enriched (miR‐451a) miRNA. Initially, NGS results were verified by RT‐qPCR using the same samples. We went on to validate the utility of miRNA as tracers of EV biodistribution by spiking defined amounts of WJ‐MSC EVs (100 to 109, measured by NTA) into homogenized mouse tissues corresponding to 1/6 to 1/3 of total organ mass. We find that 105 WJ‐MSCs are sufficient to obtain a significant increase in signals for the EV signature, while control miRNA levels remained unchanged.

Summary/Conclusion : These results demonstrate that information on EV miRNA composition may be utilized to develop biomarker signatures for tracing the distribution and accumulation of EVs in vivo. A small RNA‐sequencing workflow that enables absolute quantification of miRNAs is a prerequisite to perform such analyses.

Measuring the dynamics of RNA cargo invasion from breast cancer extracellular vesicles on immune cells

Panagiotis Papoutsoglou 1 , Alain Joliot 2 , Louise Merle 2 , Lorena Martin‐Jaular 2,3 , Anna Almeida 1 , Clotilde Théry 2,3 , Antonin Morillon 1

1 ncRNA, Epigenetic and Genome Fluidity, CNRS UMR3244, Sorbonne University, PSL University, Institut Curie, Centre de Recherche, Paris, France; 2 INSERM U932, Institut Curie Centre de Recherche, PSL Research University, Paris, France; 3 Institut Curie Centre de Recherche, CurieCoreTech Extracellular Vesicles, Paris, France

Introduction : Extracellular vesicles (EVs) contain heterogeneous nucleic acid content. Their role as transporters of biomolecules is well‐established. However, there is a lack of knowledge regarding the dynamics of EV RNA delivery and whether a specific transcript can preserve and exert its functionality during its intercellular travel. Here, we aim to analyse the EV RNA content of donor triple negative breast cancer (TNBC) cells, and the dynamics of its transfer into recipient monocytes. The ultimate goal is to identify functional long RNAs, transferred via EVs, that impact immune cell physiology.

Methods : To study EV‐mediated cargo transfer from donor (MDA‐MB‐231) to recipient (THP‐1) cells, we first utilize a split nanoluciferase‐based (Split NanoLuc) technology to decipher the timing of EV cargo cytosolic release. We constructed donor cells expressing high‐affinity small subunit (HiBiT) tagged to a lipid‐binding domain for targeted localization to the EV inner membrane. Recipient cells expressing the large subunit (LgBiT) were also established. Small EVs were purified from cell‐conditioned medium using size exclusion chromatography (SEC) (qEV2 column, 35 nm) and used for time course uptake experiments, whereby EV cytosolic release was estimated by luciferase assays in live cells. Inhibition of EV uptake was performed by pre‐treating recipient cells with an endocytosis inhibitor. EV RNA staining was performed using SYTO RNASelect green fluorescent cell stain, and the efficiency of staining was evaluated by nano‐flow cytometry.

Results : Purified MDA‐MB‐231 HiBiT‐EVs were positive for CD63, CD9, syntenin‐1, Alix and Hsc‐70 and enriched for HiBiT, compared to parental cells. The presence of HiBiT in EVs was also verified by increased NanoLuc activity in the presence of exogenous LgBiT and under mild lysis conditions. Cytosolic release into THP‐1 was observed after 1 h and peaked after 24 h of incubation with EVs. An endocytosis inhibitor greatly reduced EV uptake. Fluorescently stained EV RNA was detected in approximately 55% of isolated EVs and used for complementary EV uptake experiments.

Summary/Conclusion : In summary, we demonstrate that the timing of EV uptake/cytosolic release can be efficiently assessed using Split NanoLuc technology. Moreover, TNBC EVs are taken up by monocytes mainly via endocytosis.

Funding : Institut National du Cancer (INCA), grant numbers PLBIO21‐100 and PLBIO22‐222.

Endothelial cell‐derived extracellular vesicles as future drug delivery systems—from bioreactor production to in vitro label‐free uptake visualization

Kamil Wawrowicz 1,2 , Martyna Durak‐Kozica 1,2 , Marcin Marzejonc 3 , Maciej Trusiak 3 , Ewa Ł. Stępień 1,2

1 Faculty of Physics, Department of Medical Physics, M. Smoluchowski Institute of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland; 2 Center for Theranostics, Jagiellonian University, Kraków, Poland 3 Institute of Micromechanics and Photonics, Warsaw University of Technology, Warsaw, Poland

Introduction : Produced by different types of cells, extracellular vesicles (EVs) have many important functions. The natural characteristics of these protein‐lipid structures make them extremely interesting potential drug delivery systems, combining precise interaction with the target cell and a carrier function for substances with therapeutic effects. Designing vesicle‐based carriers, however, involves addressing two major requirements: large‐scale production of EVs (1) and understanding the natural behaviour of these structures (2) under different clinical conditions. Current methods for producing EVs are well understood, but due to a number of limitations, they require further improvements. The most desirable direction for the development of these technologies should address more efficient production resulting in a highly concentrated product. Thus, the purpose of our study was to verify whether EVs produced by endothelial cells in a bioreactor exhibit biological activity and native internalization capacity in target cells.

Methods : We implemented and evaluated the applicability of hollow fibre membranes (HFMs) bioreactors as a potentially beneficial approach for large‐scale EVs production. The obtained EVs were comprehensively characterized with transmission electron microscopy (TEM), spectral flow cytometry and tunable resistive pulse sensing. Subsequently, we examined the obtained small and large EVs uptake by target cells. For this purpose, state‐of‐the‐art label‐free imaging techniques were implemented in order to assess intercellular uptake (optical diffraction tomography) and morphological changes in cells after EVs internalization (Fourier ptychography microscopy).

Results : Morphological and physiological evaluation of the resulting vesicles confirmed the expression of their characteristic proteins, such as CD9, CD63 or CD81, and typical structure. Using modern label‐free imaging techniques, an increased accumulation of small (< 200 nm) and large (> 200 nm) vesicles was observed in target cells.

Summary/Conclusion : The results provide a promising introduction to further research into the development of methods for producing extracellular vesicles and their application as drug carriers. A new EV visualization technique based on optical diffraction tomography allowed for obtaining new types of microscopic images.

Funding : This work was supported by the National Science Centre of Poland through grant no. NCN OPUS 2022/45/B/NZ7/01430 to E.Ł.S. and the Jagiellonian University via projects CRP/0641.221.2020, SciMat and qLife Priority Research Areas budget under the programme Excellence Initiative—Research University.

Nanobody‐based biomarker detection on individual extracellular vesicles using the capture‐EVQuant assay

Suzan Glänzer 1 , Jenny Ge 1 , Mana Nedjabat 1 , Natasja Dits 2 , Joke Veldhoven‐Zweistra 2 , Ardalan Mansouri 2 , Aline Carvalho Tahyra 2 , Alba Martin Barreiro 2 , Guido W. Jenster 2 , Martin E. van Royen 1*

1 Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands; 2 Department of Urology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands Martin E. van Royen is the presenting author .

Introduction : High‐throughput detection of disease biomarkers on individual EVs in clinical samples is challenged by the low abundance of disease‐derived EVs in clinical samples and the low number of biomarkers on the surface of individual EVs. To address these challenges, we aim to develop an EV‐capture variant of the EVQuant assay where we use biomarker‐specific immune capture to enrich for biomarker positive EVs and use high fluorescence nanobody‐bacteriophage conjugates for the detection of low‐abundance EV surface markers on individual EVs using high‐throughput confocal microscopy.

Methods : Using biotin conjugated CD9/CD63 or PSMA antibody coating of 96‐well imaging‐compatible well plates, we aim to capture specific subpopulations of EVs from conditioned culture medium and clinical samples. Sensitive detection of scare EV surface markers was performed using custom nanobody‐M13 bacteriophage conjugates that were fluorescently labelled using Atto488 NHS‐ and Alexa647 NHS‐ester dies. The large size of the M13 bacteriophage allows sensitive detection of individual nanobodies using the Opera Phenix high‐throughput confocal screening microscope platform (Revvity). Nanobody‐bacteriophage binding of EVs was further evaluated using dSTORM super resolution microscopy.

Results : After protocol optimization and evaluation of experimental parameters, we were able to quantitatively detect CD9 on individual DU145 and LNCaP derived EVs. In contrast, PSMA was only quantitatively detected on PSMA+LNCaP derived EVs, but not on PSMA‐DU145 EVs. The EV origin of the CD9 and PSMA signals was confirmed in colocalization analysis with fluorescently labelled EVs and the efficiency of immune detection was evaluated in multi‐colour colocalization analysis using Alexa 488. dSTORM analysis showed surprisingly mobile nanobody‐bacteriophage conjugates bound to individual EVs.

Summary/Conclusion : We have successfully developed a microscopy based high‐throughput analysis of individual EVs using biomarker dependent immune capture and sensitive nanobody‐bacteriophage detection of EV surface markers. We will further develop this assay towards more disease‐specific EV biomarkers in more complex clinical samples also by introducing innovative labelling strategies.

Funding : This work was supported by the Health–Holland TKI‐LSH (Match) grant EMCLSH22011, the Dutch Cancer Society (“IMMPROVE,” EMCR2015‐8022) and a Daniel den Hoed Foundation grant on Cancer Nanobodies.

Subtopic EV enrichment 1

A fed‐batch chemically defined hMSC‐EV bioprocess medium enabling 2‐4× EV yield improvements in bioreactor culture

Terri Willstaedt, Amy Walde, Elie Zakhem, Jon A. Rowley

RoosterBio, Inc., St. Louis, Missouri, USA

Introduction : Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (hMSC‐EVs) have been studied in over 200 preclinical applications and dozens of human clinical trials, underscoring the need for scalable production processes compatible with GMP environments. Most existing 2D and 3D bioreactor hMSC‐EV production processes require a cell expansion stage utilizing undefined components, followed by a wash and medium exchange to remove expansion medium impurities prior to an EV collection phase in a defined medium. Simplifying this 3D process to include cell expansion and EV collection in one medium requires chemically defined growth conditions, a fed‐batch medium design, and an efficient process to maximize cell and EV yield. This study evaluates hMSC‐EV production and EV quality across multiple donors and tissues in 0.1 L microcarrier spinner flask cultures using a traditional cell expansion, wash, and collect process versus the single‐step production process, including scale‐up to a 3 L stirred tank bioreactor.

Methods : MSC‐EVs were produced from hMSCs (hBM and hUC RoosterVial, 1 M) in either RoosterNourish‐MSC‐XF/RoosterReplenish/RoosterCollect‐EV or the new highly productive, chemically defined fed‐batch system (RoosterHD‐EV) and conditioned medium was collected at set times. MSC‐EV yields were quantified by NTA, and MSC‐EV CQAs, including size distribution, tetraspanin expression, RNA content and CD‐73 activity, were assessed.

Results : Elimination of the medium exchange and wash steps resulted in utilization of fewer raw materials, retention rather than disposal of EVs produced during cell growth, and significant reductions in total media used and total cost per billion EVs. Additionally, EV quality attributes including size distribution, tetraspanin expression and lipid content are preserved in the HD‐EV system. Scale‐up in a 3 L Eppendorf bioreactor showed comparable results to those observed in 0.1L spinner cultures. The fed‐batch process for hMSC‐EV production increased the EV collection window from healthy hMSCs, resulting in a 2–4× increase in hMSC‐EV yield over traditional EV production processes.

Summary/Conclusion : We have developed a chemically defined, scalable fed‐batch bioreactor production medium to enable the streamLined and highly efficient production of hMSC‐EVs. This highly productive chemically defined EV medium is a simplified, time and cost saving solution for the large‐scale production of higher purity hMSC‐EVs necessary for extensive clinical investigations.

Development of chemistry, manufacturing and control (CMC) framework for bioengineered extracellular vesicles derived from HEK293T for a phase I clinical trial in oncology

Choon Keong Lee, Janice Ou, Claudine Ming Hui Lim, Marielle Cham, Winnie Faustinelie, Ellenmae Leong, Amy Tan, Reshma Pillai, Desy Silviana, Xiangliang Lin

Esco Aster Pte Ltd, Singapore, Singapore

Abstract unavailable

A canonical targeted approach to detect and purify exosomes for standardized diagnostic workflows

E. Hammer 1 , R. Napieralski 1 , W. Hammerschmidt 3 , R. Zeidler 3 , A. R. Brisson 4 , Ch. Krisp 5 , G. Kruppa 5 , G. Schricker 1 , B. Höchst 2 , V. Bergant 6 , A. Pichlmair 6 , P. A. Knolle 2 , O. G. Wilhelm 1

1 Therawis Diagnostics GmbH, Munich, Germany; 2 Institute of Molecular Immunology, TUM University Hospital, TUM, Munich, Germany; 3 Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany; 4 UMR‐5248 CBMN CNRS‐University of Bordeaux‐IPB, Pessac, France; 5 Bruker Daltonics, Bremen, Germany; 6 Institute of Virology, Technical University of Munich, Germany

Introduction : Extracellular vesicles (EVs) display significant heterogeneity and diversity, which reflects the state of their cell of origin. Thus, analysis of EVs may enable insights into whether cells or tissues are healthy or diseased. Accurate identification, isolation and analysis of EVs from blood without loss of information are major challenges. The lack of standardization and reproducibility hampers the development of EV‐based in vitro diagnostic workflows. Currently applied methods for EV isolation, such as ultracentrifugation, size exclusion chromatography, or electrical charge‐based methods, do not specifically purify EVs but also enrich for lipoproteins, which have a similar size and charge as EVs and are abundantly present in the blood. Immunocapture with antibodies against surface antigens isolates only subpopulations of EVs. We developed a fluorochrome‐labelled biopeptide named ELAEXIA targeting phosphatidylethanolamine, which is part of the vesicles membrane and can be found on the outer leaflet.

Methods : We use multiparametric flow cytometry to characterize EVs from different cell lines and from plasma of healthy volunteers. ELAEXIA‐isolated EVs from human plasma were analysed by mass spectrometry, nanoparticle‐tracking‐analysis (NTA) and cryo‐electron microscopy (cryo‐EM).

Results : Fluorophore‐labelled ELAEXIA allowed for flow cytometry‐based identification of EVs in cell culture supernatant and in plasma from healthy volunteers. ELAEXIA did not label lipoproteins, and their binding to EVs is reliably detected in the blood of fasted or non‐fasted healthy volunteers. ELAEXIA‐isolated EVs showed a size of 30–300 nm using nanoparticle‐tracking‐analysis (NTA). Ultrastructural analysis by cryo‐electron microscopy revealed EVs with the characteristic lipid bilayer membranes. Mass spectrometry analysis confirmed significant enrichment of exosome proteins in ELAEXIA‐enriched samples.

Summary/Conclusion : ELAEXIA is highly efficient at identifying, isolating, and analysing EVs. The enrichment strategy is independent of variable surface marker expression, charge and size of EVs and separates them from common contaminants, such as lipoproteins. This novel technology is a promising way to standardize EV detection and isolation to facilitate sample preparation for EV‐based diagnostic workflows.

Selective enrichment of tumour‐derived extracellular vesicles by targeting oncofoetal chondroitin sulphate using antibody fragments

Emma Boerrigter 1 , Amalie Mundt Jørgensen 2 , Mandy Los 3 , Chi Hau 3 , Caroline Bang Løppke 2 , Robert Dagil 2 , Tobias Gustavsson 2 , Ali Salanti 2 , Rienk Nieuwland 3 , Mette Ørskov Agerbæk 2 , Peter ten Dijke 1 and Agustin Enciso‐Martinez 1,3

1 Oncode Institute and Department Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands; 2 Center for Translational Medicine and Parasitology, Department for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark; 3 Amsterdam Vesicle Center and Laboratory of Experimental Clinical Chemistry, Department of Clinical Chemistry, Amsterdam UMC, Amsterdam, The Netherlands

Introduction : Extracellular vesicles (EVs) carry biomolecular cargo from their cells of origin, making tumour‐derived EVs (tdEVs) promising cancer biomarkers. However, tdEVs are rare in blood compared to non‐tumour particles. Therefore, selective enrichment of tdEVs is essential to enable early cancer detection and downstream characterization of tdEV cargo. tdEV enrichment requires targeting of tumour‐specific markers present on EVs. In this study, we target oncofoetal chondroitin sulphate (ofCS), a malignancy‐associated glycosaminoglycan present on tdEVs. Using anti‐ofCS antibody fragments (scFv) coupled to magnetic beads, we aimed to isolate tdEVs from cancer cell line‐conditioned medium, as a first step towards isolation and characterization of tdEVs from cancer patient plasma.

Methods : To validate the binding of anti‐ofCS scFv to ofCS+ tdEVs, A549 lung adenocarcinoma EVs were labelled with fluorophore‐conjugated scFv, isolated by size exclusion chromatography (SEC), and analysed by super‐resolution fluorescence microscopy and flow cytometry. For selective isolation of ofCS+ tdEVs, magnetic beads were coated with anti‐ofCS scFv, incubated with SEC‐purified A549 EVs and stained with MemGlow membrane dye. Enrichment of tdEVs was evaluated by flow cytometry and western blot analysis for tetraspanins. Specific capture of ofCS+ EVs was confirmed by chondroitinase pretreatment to digest ofCS chains.

Results : ofCS+ tdEVs were successfully labelled with anti‐ofCS scFv, validated by fluorescence microscopy and flow cytometry. Furthermore, selective isolation of ofCS+ EVs from A549‐conditioned medium using scFv‐coated beads was confirmed by flow cytometry. Chondroitinase treatment resulted in reduced fluorescence intensity, validating of CS specificity, while uncoated beads showed minimal nonspecific EV capture. Membrane dye controls verified the capture of membranous particles. Western blot analysis showed that approximately 75% of EVs were captured in the bead fraction compared to the supernatant.

Summary/Conclusion : This study showed the selective capture of tdEVs from conditioned medium by targeting ofCS using anti‐ofCS scFv in a bead‐based isolation method. This method has the potential to enrich rare tdEVs from cancer patient plasma, facilitating selective detection and downstream characterization.

Funding : A.E.M. and E.B. acknowledge funding from the Oncode Institute and the Dutch Cancer Society (KWF) grant 15079. A.E.M. acknowledges funding from the Dutch Research Council (NWO), VIDI grant 19724. C.L. and M.Ø.A. were funded by the Innovation Fund Denmark (grant number 9090‐00024B).

Lab‐on‐chip technologies for extracellular vesicle enrichment and detection

Presenter: Alessia Foscarini

CNR NANOTEC—Institute of Nanotechnology, Bologna, Italy

Introduction : Liquid biopsy and the use of selected biomarkers from biological fluids, greatly enhance an increasingly patient‐centred approach, avoiding invasive tests and tissue biopsies. Extracellular Vesicles (EVs) act as a snapshot of the cells from which they originate and as a repository of crucial information, facilitating the direct extracellular transfer of proteins, lipids, and miRNAs/mRNAs/DNAs. Despite EVs showing great potential as powerful biomarkers, their isolation and characterization remain challenging. Lab‐on‐Chip (LoC) technologies represent innovative tools to overcome the limits of standard methods, and we aim to implement these technologies for EV investigation.

Methods : We design customizable LoC devices based on the experimental needs, starting from fluid dynamic simulations and using microfabrication techniques (micro‐milling and 3D printing). We develop two different LoCs exploiting the microfluidic approach and electrochemical detection for EV enrichment and characterization. Large and small EVs were isolated by ultracentrifugation and characterized by high‐resolution flow cytometry, western blot, and transmission electron microscopy.

Results : We developed an in‐flow device using moulded‐plastic substrates assembled on a glass slide to create a microfluidic chamber for dynamic cell culture. Fluorescent neuroblastoma cell lines (SH‐SY5Y) were seeded into the device to allow a complete replacement of the medium in the dynamic condition within 24 h at a controlled flow rate. We observed increased EV production and decreased EV size in dynamic versus static conditions. Moreover, we realized a biosensing platform for EVs electrochemical characterization. Functionalized microelectrodes were used for differential pulse voltammetry measurements to build a calibration plot considering different EVs membrane proteins. With this method we were able to demonstrate (i) the possibility to detect very low concentrations of EVs and (ii) the retention of sample integrity.

Summary/Conclusion : The key goals of the LoC technologies are to eliminate high‐impact procedures, reduce time and cost, and preserve EV morphology. Our systems allow us to study the release of EVs under dynamic cell culture conditions that mimic the physiological scenario at the cell surface, and to identify arrays of biomarkers associated with EV subclasses. The next step is to integrate microfluidic sorting and electrochemical characterization into a benchtop device that can be customized to meet clinical needs.

Improved EV isolation and analysis using multimodal SEC and enhanced chemiluminescent ELISA

Tal Gilboa 1 , David Freedman 1 , Steve Scherr 1 , Patricia Delgado 1 , Gregg Lithgow 1 , Luca Musante 2 , George Daaboul 1

1 Everest Biolabs, Waltham, Massachusetts, USA; 2 University of Pennsylvania, Philadelphia, Pennsylvania, USA

Abstract unavailable

Impact of culture media and size‐based sorting methods on extracellular particle subpopulations

Barbora Popova, Aiste Jekabsone

Lithuanian University of Health Sciences, Kaunas, Lithuania

Introduction : Extracellular particle (EP) populations of varying sizes, such as large extracellular vesicles (lEVs), small extracellular vesicles (sEVs), and non‐enveloped extracellular particles (NVEPs), have distinct molecular profiles and roles in intercellular communication. Isolating specific EP populations enables a more detailed molecular and functional characterization in research and clinical applications. It has been observed that cells grow in a monolayer in DMEM media and exhibit cluster growth in EMEM, while variations in molecular composition under different media conditions have also been reported. This study aims to sort EP subpopulations from differently cultured breast cancer cells using ultracentrifugation and filtration‐based techniques. By comparing the size and yield of populations, it aims to identify the most effective sorting method while assessing how nutritional conditions shape the EP profile.

Methods : MCF7 cells were grown in EMEM + insulin or DMEM (4× more nutrients than EMEM) supplemented with 10% FBS. EPs were isolated from 2‐day serum‐free MCF7 conditioned media using differential ultracentrifugation and 3‐step dead‐end or tangential flow filtration (TFF). EP fractions were characterized using dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA). Protein content in each fraction was quantified using a BCA assay, while TRIzol purified RNA using absorbance.

Results : All three methods successfully fractionated EPs to varying degrees, as confirmed by DLS, NTA, and TRPS. Filtration‐based methods achieved higher particle recovery than ultracentrifugation, with TFF providing the purest fraction (based on the protein‐to‐particle ratio) and the highest lEV enrichment relative to smaller EPs. Across the three isolation methods tested, DMEM‐cultured cells released more lEVs, whereas EMEM‐cultured cells released more NVEPs. RNA quantification revealed that the NVEP fraction had a higher RNA content than sEVs or lEVs.

Summary/Conclusion : The observed differences suggest the potential to modulate EP production through tailored culture media, offering a strategy to selectively enhance specific EP subpopulations for targeted applications. The enrichment of RNA in the < 50 nm EP fraction aligns with prior findings. This study also highlights the efficiency of filtration‐based fractionation, particularly TFF, which enables higher EP yields and improved molecular cargo recovery in less time compared to ultracentrifugation.

Funding : Research Council of Lithuania, P‐PAD‐22‐105.

Evaluation of 12 extracellular vesicle (EV) isolation methods with focus on proteome composition, reproducibility, yield and size distribution

Scheila Werle, Marie Louise N. Therkelsen, Mads Groenborg, Dres Damgaard

Novo Nordisk, Bagsværd, Denmark

Introduction : Extracellular vesicles (EVs) present a valuable approach for measuring surrogate target engagement (TE) biomarkers. They offer a new way of obtaining deeper information from plasma as a liquid biopsy, providing potential as a tool for mitigating risks in drug development. However, the yield and purity of EVs isolated from plasma can vary depending on the method used, making isolation efficiency a crucial parameter to establish this technology. This study compares different isolation techniques with respect to EV yield, size distribution, tetraspanin profile, and proteome composition.

Methods : We prepared EV‐containing preparations from pooled plasma obtained from 5 healthy donors using a total of 12 different isolation methods, including filtration, size exclusion chromatography (SEC), precipitation, and centrifugation, as well as membrane affinity binding methods. The size and concentration of isolated particles were acquired on a NanoFCM using two size settings for small (68–155 nM) and large (155–850 nM) vesicles. Tetraspanin presence and distribution were assessed using a commercial immunoassay for CD9, CD63, and CD81. Proteome composition was analysed through liquid chromatography‐mass spectrometry (LC‐MS).

Results : The isolation methods resulted in diverse particle concentrations and size distributions, with up to a 5‐fold difference in yield among methods. By assessing 6 technical replicates, we observed strong differences in reproducibility between isolation methods regarding particle size and yield. The immunoassay experiment confirmed the presence of tetraspanin markers and demonstrated that different isolation methods isolate EV‐containing preparations with distinct profiles of CD9, CD63, and CD81 on their surfaces. LC‐MS analysis revealed varying amounts of identified proteins across isolation methods, with some methods showing an abundance of unique proteins. Furthermore, these methods also differed in their ability to provide additional protein profiles compared to our standard plasma proteomics analysis, with certain methods enhancing sensitivity and identifying up to 30% more unique proteins.

Summary/Conclusion : We show that different isolation methods yield distinct EV‐containing subpopulations with varied sizes and protein compositions, including tetraspanins. Some methods demonstrated higher reproducibility, crucial for reliable biomarker discovery and analysis. Overall, it is important to be aware of the introduction of biases, as different approaches produce varying results from the same input material.

Funding : The work was funded by Novo Nordisk.

Characterization of EV from synovial fluid

Sergio Lucio‐Gallego 1 , Rocío Mato‐Basalo 1 , Lama Berjawi 1 , Javier De Toro 1 , Alba González‐Rodríguez 1 , Pablo Fernández‐Pernas 2 , Alberto A Jorge‐Mora 3 , Juan Fafián‐Laboura 1 , María C. Arufe 1

1 Therapy Cellular & Medicine Regenerative Group, Universidade da Coruña (UDC), Interdisciplinary Center for Chemistry and Biology (CICA), INIBIC—Complejo Hospitalario Universitario de A Coruña (CHUAC), A Coruña, Spain; 2 Centre of Biological Sciences, University of Veterinary Medicine of Vienna, Vienna, Austria; 3 Servicio de Cirugía Ortopédica y Traumatología. Complejo Hospitalario Universitario de A Coruña (CHUAC), A Coruña, Spain

Introduction : Osteoarthritis (OA) is a chronic, debilitating joint disease that affects hundreds of millions worldwide, with significant pain and socioeconomic costs. OA is involved with ageing and is characterized by cartilage degradation and chronic inflammation of the affected joints. In this work, we are going to find out the proteomic fingerprint of each OA stage through label‐free proteomic analysis. Our aim in this initial step of the investigation was to evaluate various EV isolation methods from the synovial fluid of OA patients to determine the most suitable approach for our subsequent proteomic study.

Methods : The following EV isolation methods were compared: ultracentrifugation, size exclusion chromatography (SEC) using 70 nm columns, OptiPrep Density Gradient Medium, and EXO‐NET Pan Exosome Capture Tool. NTA and western blot were used for characterization of EV from the different isolation techniques. A study was conducted on the internalization of EVs in mesenchymal stem cells through LeonHeart FX/LX (Agilent) after labelling with DiI and the cells were measured for 16 h.

Results : The OptiPrep (F5: 1.2 × 10 10 particles at 4.5 µg/µL) and SEC (F6, F7, and F8: 1 × 10 12 particles at 4.1 µg/µL; 1.3 × 10 12 particles at 4.5 µg/µL; 1.4 × 10 12 particles at 5 µg/µL, respectively) methods yielded the best results in terms of concentration and purity of EV by NTA and western blot analysis. Among these, the SEC method provides the highest quality functional EV, which was efficiently internalized by target cells.

Summary/Conclusion : Based on these results, we will use the SEC method to isolate the EV from fluid synovial patients with OA for the subsequent proteomic analysis.

Enrichment and quantitation of small platelet‐derived extracellular vesicle subpopulations with field‐flow fractionation

Johanna Puutio 1 , Mari Palviainen 1 , Saara Laitinen 2 , Pia R.‐M. Siljander 1,2

1 Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland; 2 Finnish Red Cross Blood Service, Helsinki, Finland

Introduction : Platelets release heterogenous platelet‐derived extracellular vesicle (PEV) subpopulations in response to pathophysiological stimuli. These subpopulations can be classified based on size and structural properties as large EVs (lEVs, > 200 nm), small EVs (sEVs, < 200 nm) and other extracellular particles, such as exomeres (< 50 nm). Quantitation of the subpopulation composition (number of populations, measurement of size and particle number) < 100 nm remains challenging due to technological limitations. Additionally, storage and isolation conditions affect the subpopulation composition of EV samples. We fractionated and analysed differently induced and isolated PEVs with asymmetrical flow field‐flow fractionation (AF4) to evaluate the formation and retention of sPEV populations.

Methods : PEVs were generated by stimulating human platelets from platelet concentrates with different agonists and isolated with either iodixanol cushion ultracentrifugation (C‐UC), size exclusion chromatography (SEC) or concentrated with ultrafiltration (UF) without additional purification. PEVs released by unstimulated platelets were used as controls. Subpopulation composition was analysed with AF4 after short (< 6 months) and long‐term (> 12 months) storage at −80°C. Four sPEV subpopulation peaks of interest were identified and fractionated with AF4. Expression of PEV markers and absence of co‐isolated lipoproteins was investigated with ELISA, western blotting and SP‐IRIS from total populations and pooled subpopulation fractions to evaluate sPEV enrichment.

Results : AF4 analysis of PEVs isolated with UC, SEC and UF showed that 1) sPEVs were the most abundant subpopulation, and 2) the isolation method significantly impacted their enrichment. SEC isolation removed up to two CD41+, CD63 and CD9+ sPEV populations < 100 nm and masked variations between differently induced PEVs, whereas C‐UC retained the original subpopulation composition of UF samples. A similar loss of sPEVs was observed during long‐term storage at −80°C.

Summary/Conclusion : A comprehensive analysis of all EV subpopulations is often overlooked due to methodological challenges. We propose AF4 as a method for monitoring sEV loss and characterising them from complex media. AF4 analysis of PEVs isolated using C‐UC or UF enables quality control and the identification of subtle differences in sPEV subpopulation composition that may have biological significance.

Funding : This project is a part of the extracellular vesicle ecosystem (EVE) consortium funded by Business Finland.

Evaluation of a novel magnetic bead‐based method for isolation of intact EVs or EV‐RNA

Simon Hertlein 1 , Tobias Tertel 2 , Karolin Spitzer 1 , Bernd Giebel 2 , Martin Schlumpberger 1

1 QIAGEN GmbH, Hilden, Germany; 2 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany Bernd Giebel and Martin Schlumpberger are co‐senior authors. Simon Hertlein and Tobias Tertel are co‐first authors .

Introduction : Extracellular vesicles (EVs) and their cargo, including EV‐derived RNA, are essential tools for diagnostics and therapeutic development. Efficient, reproducible workflows for isolating intact EVs and their RNA content remain a key challenge. This study evaluates a novel magnetic bead‐based workflow, focusing on its relevance for clinical applications, its unbiased performance across diverse EV subpopulations, and its potential for high‐throughput scalability.

Methods : EVs were isolated from cell culture supernatant and human plasma using a novel magnetic bead‐based approach. EV size, concentration, and subpopulations were analysed using imaging flow cytometry (IFCM) and nanoparticle tracking analysis (NTA). Protein levels were quantified using the BCA assay, while EV‐RNA recovery and specificity were assessed via qPCR and digital PCR. To evaluate reproducibility, isolations were performed on multiple days with different kits. Donor variability was addressed by analysing plasma samples from multiple individuals.

Results : The workflow demonstrated robust RNA recovery and high reproducibility, with consistent results across multiple days and kits. Imaging flow cytometry revealed that the method preserved EV heterogeneity, capturing over 20 distinct EV subpopulations in plasma without bias, including markers such as CD9, CD31, CD41, CD63, and CD235a. RNA recovery and specificity for EV‐associated miRNA were comparable to established spin column‐based methods but achieved with significantly reduced processing time. The method enables parallel processing of large sample numbers with minimal hands‐on time and is well‐suited for automation, addressing scalability challenges in clinical and translational research.

Summary/Conclusion : This novel magnetic bead‐based workflow combines methodological rigour with practical applicability, ensuring reproducible and unbiased EV and RNA isolation. By capturing the complexity of EV subpopulations and offering a streamLined, scalable solution, this method is ideally suited for clinical and high‐throughput applications. These findings provide a solid foundation for further functional studies and support the translational potential of EV‐based diagnostics and therapeutics.

Extracellular vesicle and nanovectors production, engineering, and characterization for therapy and diagnosis

Presenter: Fahima Di Federico

NABI, Paris, France

Introduction : IVETh, at Université Paris Cité, is a cutting‐edge platform for studying and characterizing extracellular vesicles. It offers high‐tech equipment and expertise for their isolation, analysis, and quantification. Through multidisciplinary collabourations, IVETh advances research and innovation in health, biotechnology, and biomedicine.

Methods : Single‐vesicle imaging techniques: nanoscopy of single vesicles, interaction with cells, cargo delivery, and EV morphology; AFM‐based EV fractionation and characterization from cell secretome biofluids, bioproduction, or engineered EVS (e.g., TFF, asymetric flow field flow fractionation). Characterization of size and concentration (nanoparticle tracking analysis NanoSight video‐drop delivery)—EV phenotyping and biological identity: introduction to nanoparticle flow cytometry (NanoFCM), ExoView platform, Raman spectroscopy for EV and biofluids biomolecular characterization; potency tests using high‐content screening microscopy; machine learning.

Results : Dual‐THP1 treatment with EV‐hASC from 2D starvation increased inflammation via IFN and NFkB activation, whereas 3D turbulence‐derived EV‐hASC showed lower immunogenicity. A proof‐of‐principle study using ML classification on RS data successfully differentiated EVs from two HeLa cell lines (WT vs. HSP70‐GFP) using our GMP‐compatible turbulent flow method in bioreactors. ILM‐NTA enabled nanoscale viscosity measurements in Newtonian and non‐Newtonian environments, surpassing standard EV characterization tools. It revealed EV size distribution in complex poloxamer matrices and highlighted EV‐poloxamer‐407 interactions, as evidenced by altered diffusion coefficients and size distributions compared to beads.

Summary/Conclusion : Our proposed characterization toolbox integrates advanced techniques within the IVETh platform, including Raman spectroscopy, AF4, high‐content screening, and classical methods like NTA and phenotypic analysis. ILM‐NTA shows strong potential as a nanorheometer for assessing EV transport in viscous matrices, relevant for drug delivery and regulatory standards. Immunomodulation assays on Dual‐THP1 revealed that starvation‐derived EVs induce more inflammation than turbulence‐derived EVs. These findings support the development of a robust multimodal characterization approach.

Funding : Région Ile de France ANR.

Methods for EV enrichment workflow characterization

Presenter: Thomas Maslanik

Cellarcus Biosciences, San Diego, California, USA

Introduction : Enrichment of extracellular vesicles (EVs) is required for many studies. Screening methods, functional studies, and analysis of rare subsets of EVs all require some form of EV enrichment. However, it is important to characterize any enrichment method to understand how it impacts the composition of EVs in a given sample and validate the approach to EV enrichment for downstream applications. A rigorous analytical approach for evaluating enrichment methods is needed.

Methods : Vesicle Flow Cytometry (vFC) and BCA measurements were used to characterize media from hollow‐fibre bioreactor (HFB) DiFi cell cultures pre‐ and post‐enrichment.

Results : There were 8.5E9 total EVs/mL in HFB DiFi cell culture media. Fifty‐nine percent of EVs were positive for EGFR above the limit of detection (25 antibodies per vesicle). Enrichment by UF (100k MWCO) resulted in 52% recovery of total EVs with similar EGFR positivity and a significant increase in EV/protein ratio. Other pore sizes differed in total EV recovery (300 kDa MWCO – 35%, 30 kDa MWCO – 25%) and EGFR positivity (300 kDa MWCO – 67%, 30 kDa MWCO – 54%). SEC enrichment resulted in a > 90% recovery of total EVs across all fractions (41% EGFR+). Sixty‐five percent of total EVs (86% of EGFR+ EVs) were present in fractions 7 and 8. Selecting appropriate fractions also significantly increased the EV/protein ratio.

Summary/Conclusion : In this study, we demonstrate workflows for enrichment method characterization. This study demonstrates a broadly applicable analytical approach to characterizing enrichment methods for a given sample. Adopting such an approach in workflows requiring EV enrichment will improve our understanding of input material in any downstream application and will enhance rigour and reproducibility of these studies.

Funding : Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R44CA272098. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Advancing extracellular vesicle research: an integrated suite for high‐purity isolation, standardized quantification, and single‐vesicle biomarker detection

Presenter: Irina Nazarenko

University of Freiburg, Freiburg, Germany

Introduction : Extracellular vesicles (EVs) hold great promise for liquid biopsy applications. However, challenges in high‐purity EV isolation, precise quantification, and molecular characterization hinder clinical translation. We address these limitations by introducing an integrated suite of novel methodologies: (1) Superose 6‐based size exclusion chromatography (SEC) for high‐resolution EV isolation, (2) PHoNUPS, an advanced software for standardized EV size and concentration analysis, and (3) protein interaction coupling assay (PICO) for absolute EV biomarker quantification at the single‐vesicle level.

Methods : We developed and validated a Superose 6 FPLC‐based SEC method to obtain highly pure small EVs (sEVs) from biofluids. To improve NTA and DLS data analysis, we created PHoNUPS, an open‐source R‐based software enabling standardized, high‐throughput EV sizing and concentration analysis across multiple platforms, ensuring broad accessibility. Finally, we optimized the PICO assay, enabling absolute single‐vesicle quantification of EV biomarkers in a reagent tube without requiring specialized infrastructure.

Results : Superose 6‐based SEC enabled precise separation of EV‐rich fractions from biofluids, significantly reducing lipoprotein contamination and facilitating easy detection of disease‐related EVs, including tumour‐derived vesicles. PHoNUPS efficiently processed NTA and DLS data, delivering automated, standardized particle size distribution analysis, enhancing reproducibility across platforms. The PICO assay provided absolute EV biomarker quantification with high specificity and reproducibility. This integrative suite was evaluated on cell models and clinical samples.

Summary/Conclusion : Our integrated suite provides a transformative approach to EV research, overcoming key challenges in isolation, quantification, and biomarker detection. The advantages include high‐purity EV isolation, standardized size and concentration analysis, and a breakthrough single‐vesicle biomarker quantification method that requires no specialized equipment or costly infrastructure. These innovations pave the way for next‐generation EV‐based liquid biopsy diagnostics and precision medicine applications.

Funding : Supported by the German Federal Ministry of Education and Research (EV‐Surf 13GW0605F, Nanodiag 03ZU1208CA, Next‐GenNTA KK518401 ) and the European Union HORIZON‐EIC‐2021‐TRANSITIONOPEN‐01 (Nexus 101058200).

A novel aptamer‐based approach for lipoprotein removal to achieve ultra‐pure blood EV isolation

Presenter: Soyoung Jeon

Korea University, Seoul, Republic of Korea

Introduction : Extracellular vesicle (EV) isolation from blood is crucial for disease diagnosis and treatment, with growing demand in basic and applied research. However, plasma lipoproteins, similar to EVs in size, density, and zeta potential, make contamination hard to avoid with current technologies. UC and SEC in combination remain the only effective but costly solution. This study introduces a novel approach for efficient removal of HDL and (v)LDL from plasma, followed by a performance comparison of EV extraction using SEC and ExoFilter.

Methods : We developed an aptamer‐based filter (LipoFilter) for selective lipoprotein removal. By targeting ApoB100 and ApoA1, markers for low‐density lipoproteins (LDL) and high‐density lipoproteins (HDL), we conjugated these aptamers onto a porous nylon mesh with an average pore size of 5 µm, allowing minimal resistance and natural flow. Lipoprotein removal and EV purification efficiencies were assessed via EM, NTA, BCA assay, and ELISA.

Results : When applying solutions containing HDL or LDL separately to the LipoFilters, over 99% of each lipoprotein was removed. Applying LipoFilter to 3 mL of plasma resulted in 98.9% removal of lipoproteins with less than 1% of EV loss. Furthermore, subsequent application of SEC or ExoFilter to LipoFilter‐treated samples allowed for effective EV isolation with a purity level of 99.9%.

Summary/Conclusion : This study presents LipoFilter, a high‐throughput, aptamer‐based filter that rapidly removes plasma lipoproteins, addressing a significant challenge in EV isolation. When integrated with existing EV extraction methods, LipoFilter significantly enhances EV purity and promises to improve the accuracy of blood‐based EV research. The ultra‐pure EVs obtained can serve as valuable standardized materials for biomarker discovery and therapeutic applications in diagnostics and industry.

Funding : This project was conducted with the support of the Alchemist Project of the Korea Evaluation Institute of Industrial Technology (KEIT 20018560/NTIS 1415184668) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and the National Research Foundation of Korea (NRF) Grant funded by the Korean Government, MSIP (RS‐2023‐00207833).

Probiotic‐derived engineered extracellular vesicles as delivery vehicles for health‐promoting xenometabolites

Hannah J. O'Toole 1 , Trina A. Knotts 1,2 , Neona M. Lowe 1 , Dustin Hadley 1 , Luis Valdiviez 1 , Jean Debédat 1,2 , Oliver Fiehn 1 , Sean H. Adams 1,2 , Randy P. Carney 1

1 University of California, Davis, California, USA; 2 UC Davis Health, Davis, California, USA

Introduction : Probiotic bacteria‐derived extracellular vesicles (bEVs) are a promising therapeutic and preventative modality for metabolic and gut health. bEVs containing natural microbial metabolites (xenometabolites), such as anti‐inflammatory indole derivatives and peroxisome proliferator activated receptor (PPAR) activating cyclopropane fatty acids (CpFAs), are examples of probiotic‐derived molecules with potential gastrointestinal (GI) health benefits. Here, we engineered bEVs (e‐bEVs) to augment the natural cargo of xenometabolites (i.e., CpFAs) to assess their bioactivities in vitro and in vivo.

Methods : Probiotic Lactobacillus reuteri ATCC PTA 6475 was cultured under low pH (3–4 pH) conditions to optimize bEV yield. bEVs were isolated via differential ultracentrifugation from bacterial cell culture media. bEV size, concentration, and morphological confirmation were performed via nanoparticle tracking analysis and cryogenic electron microscopy. Protein concentration was analysed via BCA assay. CpFA loading into isolated bEVs was performed via a novel microfluidic mixing approach. Size‐exclusion chromatography was used to remove free CpFA, then gas chromatography time of flight mass spectrometry (GC‐TOF(MS)) was performed to quantify native indole and CpFA metabolite cargo and determine the absolute concentration of target CpFA loading into e‐bEVs. Cell media and native bEVs were analysed as negative controls.

Results : As evidenced by MISEV2023 guidelines, L. reuteri bEVs were successfully enriched. As expected, MRS media did not contain detectable particles. bEVs isolated from low pH conditions had ∼10‐fold higher number of particles from an equivalent number of cells when compared to cultures grown under standard conditions. GC‐TOF(MS) metabolomics analysis of native L. reuteri bEVs confirmed the presence of indole‐3‐lactate and indole‐3‐butyric acid. GC‐TOF(MS) analysis of CpFA‐loaded e‐bEVs had > 40‐fold enrichment in dihydrosterculic acid CpFA in comparison to native bEVs, confirming our ability to engineer bEVs to contain xenometabolites of interest. Notably, our high‐throughput microfluidic CpFA loading scheme did not compromise the native indole cargo, nor majorly alter EV morphology or concentration.

Summary/Conclusion : This work demonstrates that native probiotic bEVs are carriers of xenometabolite cargo, which can be further increased by exogenous loading of target molecules. These e‐bEVs and our microfluidic loading workflow will have a broad impact in developing more robust therapeutics and probiotic‐derived supplements to improve GI health.

Platelet‐free plasma does not (yet) exist

Jillian W. P. Bracht 1 , Mendel Engelaer 1,2 , Adam Ceroi 3 , Marco Guadagnoli 3 , Edwin van der Pol 1,2 , Rienk Nieuwland 1

1 Department of Clinical Chemistry, Amsterdam Vesicle Center, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands; 2 Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands; 3 Miltenyi Biotec B.V., Leiden, The Netherlands

Introduction : Human blood plasma contains clinically relevant information that can be used to develop disease biomarkers. Despite intensive research, >99.9% of initially promising biomarkers never reach the clinic. A possible explanation is the inconsistent composition of plasma samples stored in biobanks. Although believed to be “cell‐free”, human plasma still contains 1E5‐1E8 erythrocyte ghosts (ery‐ghosts) and platelets per mL, even after two centrifugation steps. Moreover, the concentration of remaining cells varies, and depends on both the operator and centrifugation protocol (> 200 existing protocols). Although for some analytes the presence of remaining cells will not matter, for others, such as extracellular vesicle (EV) concentration and EV‐miRNA analyses, remaining cells disturb measurement and interpretation of results (Bracht et al. J Extracell Vesicles 2023). Since the concentration of remaining cells is too low for visual confirmation and because downstream effects on other analytes (DNA, proteins) are unknown, this is an unrecognized and possibly underestimated problem.

Methods : We developed a procedure to specifically and simultaneously sort and concentrate ery‐ghosts and platelets from double centrifuged human plasma (MACSQuant Tyto, Miltenyi). Cell concentrations pre‐ and post‐sorting were measured by flow cytometry (Northern Lights, Cytek). The presence of ery‐ghosts and platelets was confirmed by super‐resolution microscopy (dSTORM, ONi), using specific capture‐ and fluorescent detection antibodies. We also developed a filtration method to remove remaining cells and cell ghosts from plasma.

Results : Using a novel sorting procedure, the concentration of ery‐ghosts and platelets increased 78‐fold and 37‐fold post‐sorting, respectively. We confirmed that the sorted particles were ery‐ghosts and platelets using dSTORM imaging. Ery‐ghosts were both membrane dye and CD235a positive, whereas platelets showed characteristic pseudopods and were positively labelled with a membrane dye and CD63. Our filtration method removed > 98% of the remaining cells from plasma, whereas the detectable concentration of EVs by flow cytometry remained unaffected.

Summary/Conclusion : Taken together, we have developed methodologies to (1) remove remaining cells from plasma and (2) isolate remaining cells, allowing us to characterize and quantify their effects directly on downstream analyses. By standardizing the quality and composition of plasma samples, a more uniform playing field is being prepared for enhanced biomarker development.

Funding : Marie Sklodowska‐Curie (No. 101067982).

An integrated microfluidic device for neutrophil extracellular vesicle analysis and gastric cancer diagnosis

Presenter: Dan Yu

Jiangsu University, Zhenjiang, People's Republic of China

Introduction : Neutrophil‐derived extracellular vesicles (NEVs) are critically involved in tumour progression, carrying miRNAs that may be potential biomarkers for gastric cancer liquid biopsy. However, the non‐invasive assay is largely hampered by the tedious process of NEVs separation and detection from heterogeneous body fluids.

Methods : Herein, we presented an integrated microfluidic chip for NEVs (IMCN) analysis, which achieved immune‐separation of CD66b/CD63+ NEVs and multiplexed detection of their contained miRNAs (termed as NEV signatures) by using 10 µL serum samples. After recognition of the captured NEVs by a specific CD63 aptamer, an on‐chip rolling circle amplification (RCA) reaction was triggered by the released aptamers and miRNAs from the heat‐lysed NEVs, and the resulting RCA products were subsequently detected by molecular beacons (MBs), initiating allosteric hairpin structures and amplified ‘turn on’ fluorescence signals (RCA‐MBs assay). The fluorescence signals were further analysed by an AI‐based ensemble classification system.

Results : The optimized microchannel and flow rate enable a highly efficient capture of NEVs (> 90%). Moreover, the IMCN chip enabled highly sensitive detection of CD66b/CD63+ NEVs and the containing miRNAs (miR‐223‐3p and miR‐425‐5p). High diagnostic accuracy was achieved via the IMCN chip for healthy controls (HC) and gastric cancer (GC) (80.95%), benign gastric diseases (BGD) and GC (76.11%), as well as GC of different stages (91.20% for I/II stages of GC, 82.93% for III/IV stages of GC). The high diagnostic accuracy enabled the differentiation between HC and GC, reaching 83.91% with a cocktail of five biomarkers (NEV signatures, CEA and CA199) and further increasing to more than 90% with an AI‐based ensemble classification system. Moreover, the high levels of miR‐223‐3p and miR‐425‐5p in NEVs were found to correlate to poor prognosis in GC patients.

Summary/Conclusion : The IMCN chip enabled sensitive quantification of CD66b/CD63+ NEVs abundance and miRNAs by consuming only 10 µL of serum sample in less than 4 h. We envision that the proposed IMCN chip with NEVs signatures may show great potential for the diagnosis and prognosis evaluation of gastric cancer and promote the clinical applicability of NEVs miRNA‐based liquid biopsy.

Development of a single reagent for sequential separation and detection of extracellular vesicles from complex biological fluids

Presenter: Dario Brambilla

National Research Council of Italy—Institute of Chemical Sciences and Technology, Rome, Italy

Introduction : Extracellular vesicles (EVs) have emerged as a promising source of novel biomarkers for disease diagnosis and prognosis. However, their full potential in clinical diagnostics remains underutilized, primarily due to the limitations of current separation strategies. Among these, immunoaffinity approaches provide unmatched selectivity and enable the isolation of specific EV subpopulations. Despite their advantages, these methods are hindered by certain intrinsic drawbacks. To address these issues, we developed a single reagent—a novel aptamer‐antibody conjugate (AAC)—that overcomes these challenges by facilitating both the separation of EVs from complex matrices and their detection on a microarray platform.

Methods : AACs were synthesized by tagging antibodies targeting EV surface markers with a dual‐domain single‐stranded DNA (ssDNA). The first domain, a streptavidin‐binding aptamer, allows for the reversible immobilization of the AAC on magnetic beads. The second domain, a barcode sequence, is responsible for recapturing the EV‐antibody complexes on the surface of DNA microarrays. Using AACs, EVs were first separated from human plasma and subsequently captured on the DNA microarray surface. After labelling the EVs with gold nanospheres, they were detected using single particle interferometric reflectance imaging sensor (SP‐IRIS) technology.

Results : AACs demonstrated an exceptional ability to efficiently separate EVs from complex biological media under mild conditions, preserving their integrity as confirmed by transmission electron microscopy (TEM) and western blot analysis. The choice of capturing EVs on the microarray substrate enabled their multiplexed analysis. Using this combined separation and detection strategy, multiple tumour markers were successfully detected from the plasma of cancer patients.

Summary/Conclusion : We present a novel immunoaffinity separation strategy for EVs that addresses several limitations of traditional antibody‐based methods. The integration of EV separation using magnetic beads with microarray‐based detection opens new avenues for EV analysis in diagnostics. This system is inherently flexible, allowing for the evaluation of different biomarker panels by simply modifying the antibodies used in AAC synthesis, making it adaptable to a wide range of diagnostic applications.

Funding : NEXUS (European Union's Horizon—Transition to Open Research and Innovation Programme under grant agreement no. 101058200).

EV‐zone: AI‐enhanced platform for standardized reporting and advanced literature search in EV research

Rodolphe Poupardin 1 , Martin Wolf 1,2 , Nicole Maeding 1 , Gregor Fuhrmann 3 , Dirk Strunk 1,2

1 Cell Therapy Institute, Paracelsus Medical University, Salzburg, Austria; 2 Austrian Red Cross Research, Vienna, Austria 3 Department of Biology, Friedrich‐Alexander‐University Erlangen‐Nürnberg, Erlangen, Germany

Introduction : The rapidly expanding extracellular vesicle (EV) research field necessitates rigorous reporting standards and efficient tools for navigating the growing body of information. Our EV‐Zone platform (www.ev‐zone.org) addresses these challenges through two innovative applications: EV‐Checklist and EV‐PMC Search.

Methods : We developed EV‐Checklist, a digital tool designed to facilitate rapid standardized reporting on EV studies during manuscript preparation. Our second tool, EV‐PMC Search, provides an EV‐focused comprehensive searchable database of constantly updated open‐access publications. We are integrating Large Language Models (LLMs) into EV‐PMC Search to enhance its capabilities for complex, context‐aware literature exploration.

Results : EV‐Checklist quickly guides researchers through a comprehensive checklist covering nomenclature, source, isolation, characterization, and functional studies of EVs, compliable in under 30 min. It generates a concise two‐page PDF‐formatted table for easy integration into manuscripts and stores the information in a searchable database. The checklist is adaptable to evolving research needs, as demonstrated by the MiBlood EV checklist variant. EV‐PMC Search offers advanced search functionalities, including Boolean and regular expressions, enabling efficient exploration of full‐text or specific sections within over 23,500 open‐access EV publications. To enhance its capabilities, we are integrating Large Language Models (LLMs) into EV‐PMC Search, allowing for more nuanced and context‐aware literature exploration. This AI‐powered feature will enable researchers to perform complex semantic searches, extract relevant information from papers, and generate summaries of multiple publications.

Summary/Conclusion : The open access EV‐Zone suite of tools streamLines the reporting and discovery process in EV research. EV‐Checklist complements existing registries with unmet time‐saving efficiency, and the AI‐augmented EV‐PMC Search revolutionizes literature exploration, enabling researchers to navigate the rapidly growing EV knowledge base with unparalleled precision.

Study and validation of the metabolic signature of extracellular vesicles as a prognostic and predictive marker in pancreatic cancer

Pilar Espiau‐Romera 1 , Oihane E. Albóniga 2 , María Blasco‐Montolio 1 , Beatriz Parejo‐Alonso 1 , Alba Royo‐García 1 , Isabel Villaoslada 1 , Martin Nørmark Thesbjerg 3 , Allan Stensballe 3 , Juan Manuel Falcón 2 and Patricia Sancho 1 .

1 IIS Aragon, Zaragoza, Spain; 2 CICbioGUNE, Donostia, Spain; 3 Aalborg University, Aalborg, Denmark

Introduction : Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal disease caused by late diagnosis, aggressiveness and lack of effective therapies. Nowadays, PDAC models can be classified according to molecular or metabolic features, with a strong correlation between them: the classical molecular signature relates to the lipogenic subtype, whereas the highly aggressive basal profile correlates with glycolytic tumours. The properties of extracellular vesicles (EVs; released into biological fluids, cell‐specific content of origin) let us hypothesise that the identification of their metabolic signature in liquid biopsies can be used for the proper stratification of PDAC patients into metabolic subtypes.

Methods : Materials—Primary cultures of PDAC patient‐derived xenograft models (PDXs), established cell lines and patient samples (informed consent was obtained). Methods—Quantitative PCR. western blot. EVs isolation techniques: ultracentrifugation, ultrafiltration, immunoprecipitation, anti‐CD9‐based affinity column, size exclusion chromatography (SEC). nanoparticle tracking analysis. Metabolomic analysis. Statistical analysis: ANOVA and Kruskal–Wallis H test.

Results : Based on a pre‐defined metabolic PDAC signature, we classified established cell lines, PDXs and patient samples into glycolytic and lipogenic subgroups. Moreover, we compared different EV isolation techniques to subsequently characterize enzyme and metabolite content. We obtained sufficient EVs by ultracentrifugation, combined ultrafiltration and immunoprecipitation, an anti‐CD9 affinity column, and SEC from in vitro and in vivo samples. According to the specificity, efficiency, phenotype and grouping of obtained EVs and their use in downstream applications, the anti‐CD9 affinity column allowed us to specifically isolate EVs of interest in the proper concentration for further analysis. In parallel, we studied the uptake and internalization of EVs in cancer cells and in cells from the tumour microenvironment to confirm their role in communication. Finally, we showed by Western blot and metabolomics analyses that enzymes and metabolites useful for the proper classification of PDAC samples can be detected in EVs.

Summary/Conclusion : Our results experimentally support previous molecular analyses suggesting the existence of different metabolic PDAC subtypes. Furthermore, the anti‐CD9 affinity column seems the most suitable technique for us. Consequently, we are developing a customized affinity column to use specific PDAC antibodies present in EVs. Additionally, distinctive metabolites can be detected in EVs, which is useful for the metabolic PDAC classification.

Scalable, high‐throughput isolation of extracellular vesicles using electrokinetic‐assisted mesh filtration

Presenter: Jae Hyeon Lee

Korea University, Seoul Republic of Korea

Introduction : As extracellular vesicles (EVs) gain recognition for their therapeutic potential, there is a critical need for scalable EV isolation technologies to support clinical and industrial applications. Traditional EV isolation methods are generally optimized for small‐scale Laboratory use, limiting their scalability and efficiency for larger production needs.

Methods : We developed an electrokinetic‐assisted filtration system, ExoFilter, which combines charge interaction with physical mesh flow filtration to achieve high‐throughput EV isolation. This hybrid filtration system employs a coarse physical filter to electrokinetically capture EVs as they flow through the filter lattice, providing a robust and scalable approach.

Results : The ExoFilter system demonstrated exceptional high‐throughput performance, achieving a flux of approximately 750 mL/min while isolating EVs. Compared to tangential flow filtration (TFF), ExoFilter showed significant improvements in both yield (40%–100%) and purity (110%–214%) across various sample types, including human plasma, saliva, urine, and cell culture media. ExoFilter also enables efficient EV isolation from small sample volumes as low as 200 µL, facilitating rapid blood‐based EV research. Additionally, low‐molecular‐weight albumin was effectively removed from plasma samples, further enhancing EV purity.

Summary/Conclusion : ExoFilter's high‐throughput, high‐efficiency capabilities make it a promising solution for scalable EV production. This innovative filtration system supports convenient and rapid EV isolation, offering significant benefits for clinical applications and advancing large‐scale EV research.

Funding : This project was conducted with the support of the Alchemist Project of the Korea Evaluation Institute of Industrial Technology (KEIT 20018560/NTIS 1415184668) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and the National Research Foundation of Korea (NRF) Grant funded by the Korean Government, MSIP (RS‐2023‐00207833).

Rapid and high‐yield recovery of plasma‐derived extracellular vesicles using modified chromatography with soluble protein depletion for biomarker discovery

Presenter: Yang You

Mayo Clinic, Rochester, Minnesota, USA

Introduction : Extracellular vesicles (EVs) are important mediators in intercellular communication by transferring proteins, lipids and nucleic acids. EVs in biofluids, such as blood, have gathered interest as potential biomarkers for disease diagnosis. However, isolating EVs from blood poses a challenge due to the high concentration of plasma proteins, which obscure the detection of less abundant EV‐associated proteins. Here, we employed size exclusion chromatography (SEC: qEV column) combined with optimized flow‐through chromatography (Capto Core 700 beads: CC) to isolate intact, high‐purity EVs with significantly reduced plasma protein contamination.

Methods : Human plasma‐derived EVs were isolated using five different methods: qEV alone, CC followed by qEV (CC+qEV), qEV followed by 1‐hr incubation with CC (qEV+CCL), qEV followed by 5‐min incubation with CC (qEV+CCS) and Mag‐Net (Resyn, a hyper‐porous magnetic bead technique that captures membrane‐bound particles). The particle number and size distribution were characterized by nanoparticle tracking analysis (NTA) and nano‐flow cytometry (NanoFCM). Label‐free and data‐independent‐acquisition mass spectrometry (DIA‐MS) was performed to analyse the proteomic profiles of plasma‐EVs isolated from different methods, and the proteins of interest were further validated by super‐resolution microscopy.

Results : The combination of SEC and Capto Core beads significantly increased EV purity and reduced plasma protein contamination. Across methods, 1240 proteins were identified: 778 (neat plasma), 870 (qEV), 908 (CC + qEV), 925 (qEV + CCL), 941 (Resyn), and 1,153 proteins (qEV + CCS), including 589 shared proteins. Proteomic profiling of plasma‐EVs further revealed a marked reduction in abundant plasma proteins (e.g., ALB, IGG1 and APOA1) and a higher enrichment of common EV markers (e.g., CD9, CD81 and CD63) in the qEV+CCS method. Notably, cell‐type enrichment analysis of the plasma‐EV proteome revealed a higher presence of brain‐specific proteins, including those strongly associated with neurodegenerative diseases (e.g., TSPAN9, APP, APOE), in the qEV + CCS group, which were validated at a single‐EV level by super‐resolution microscopy. Pathway analysis indicated that proteins uniquely identified in the qEV + CCS group were involved in RNA binding, cell adhesion, metabolism and synaptic function.

Summary/Conclusion : Our findings present an improved chromatography method and an efficient strategy for the purification of plasma‐derived EVs, providing a valuable tool for advancing plasma EV‐based biomarker development.

Funding : CurePSP Pipeline and Pathway Grant Award (691‐2024‐01‐Pipeline).

Development of tissue‐derived EV enrichment from body fluids

Presenter: Satoshi Muraoka

National Institutes of Biomedical Innovation, Health and Nutrition, Shizuoka, Japan

Introduction : Extracellular vesicles (EVs) are ubiquitously secreted from almost all tissues and cells and carry many cargoes, including proteins, RNAs and lipids, which are involved in various biological processes. EVs are released from tissues into the blood and are expected to be used as biomarkers for disease diagnosis. We identified tissue‐specific proteins in plasma and serum EVs by deep EV proteome analysis (Muraoka et al. iScience 2022). Those proteins are promising markers to isolate tissue‐derived EVs from body fluids. We developed a quantitative assessment method for an immunoprecipitation system to enrich tissue‐specific EVs from plasma and serum EVs. Specifically, we introduced stable isotope labelling of amino acids in cell culture (SILAC)‐labelled EVs as an internal standard for quantitative evaluation of EV enrichment using capture reagents.

Methods : We performed cell passages with growth medium prepared where natural ‘light’ lysine and arginine were replaced with isotope‐labelled ‘heavy’ lysine and arginine for MS‐based quantitative proteomics. SILAC‐labelled EVs were isolated from the cell culture medium of SILAC‐labelled cell lines using the MagCapture exosome isolation kit. Then a dilution series of those EVs was spiked into plasma or serum EVs. Endogenous and SILAC‐labelled EVs were immunoprecipitated using CD9 antibody and quantified by LC‐MS/MS.

Results : After five cell passages, the naturally occurring lysine and arginine were found to be completely replaced by heavy lysine and arginine in cultured cells and cell‐derived EVs. After EV immunoprecipitation using CD9 antibody, the amount of endogenous ‘light’ CD9 and SILAC‐labelled ‘heavy’ CD9 from plasma or serum EVs was quantified. The ‘heavy’ CD9 amount was quantified in the particle‐number‐dependent (dilution series), while the ‘light’ CD9 amount was quantified in almost the same range.

Summary/Conclusion : The immunoprecipitation quantitative proteome analysis using SILAC‐labelled EVs enabled us to assess the performance of the capture antibody or aptamer quantitatively. Our developed method is theoretically applicable to immunoprecipitation experiments for any tissue‐derived EV enrichment.

EVring: a new platform enabling isolation‐free enrichment and size fractionation of extracellular vesicles for enhanced clinical analysis

Presenter: Rebecca Mayer

University of California, Davis, California, USA

Introduction : Effective surveillance for many cancers, including head and neck cancer (HNC) and ovarian cancer (OvCa), is limited by current biomarker assays, which often lack specificity and sensitivity, particularly in early‐stage cases. To address these limitations, we introduce EVring technology, enabling isolation‐free enrichment of extracellular vesicles (EVs) directly from whole biofluids by exploiting Marangoni flow during biofluid drop drying. This approach preserves EV heterogeneity and optimizes subpopulation analysis, advancing EV‐based diagnostics for cancer detection and surveillance.

Methods : EVring leverages apparatus‐free coffee ring dynamics and Marangoni flow to enrich EVs directly from diluted patient serum (IRB‐approved protocol with informed consent), using only simple solution and surface treatments to control separation and enhance EV subpopulation resolution at the droplet edge. Raman spectroscopy was employed to analyse EV metabolite profiles within the ring, capturing protein, lipid, and metabolite profiles relevant to cancer progression and recurrence. Machine learning models trained on Raman spectral data classified EV features and linked them to metabolic pathways associated with cancer.

Results : The EVring platform achieved size‐specific separation of EV subpopulations without any additional isolation. Control analyses using reference bead size standards within the size range of small EVs (50–200 nm) confirmed a high degree of spatial subfraction by size within the ring. Raman spectra revealed metabolic signatures in EVs associated with recurrence, including cholesterol, proline, and fatty acid esters. Subsequent machine learning models classified recurrence vs. remission states with 100% sensitivity and 94.3% specificity. Additionally, EVring demonstrated the ability to maintain EV integrity, providing direct insights into EV‐driven metabolic reprogramming in cancer.

Summary/Conclusion : This approach represents a major advancement in EV analysis, enabling the first isolation‐free, clinically relevant analysis of EVs directly from whole biofluids. The isolation‐free approach dramatically simplifies clinical workflows by eliminating labour‐intensive purification, reducing sample processing time, and minimizing known artefacts associated with traditional isolation methods. With broader implications beyond cancer surveillance, this platform can be used for isolation‐free enrichment of EVs directly from human biofluids for numerous downstream applications across many indications.

Crafting clarity on synergy between clinical sample preparation and extracellular vesicle biomarker analysis for metastatic prostate cancer precision care

Prima Dewi Sinawang 1 , Priyanka Multani 1 , Mehmet O. Ozen ,1 , Jodie Wong 2 , Demir Akin 1 , Claire Hanson 3 , Matt Larsen 3 , Liang Wang 2 , Brian T. Cunningham 4 , Manish Kohli 3 , Utkan Demirci 1

1 Stanford University, Stanford, California, USA; 2 H. Lee Moffitt Cancer Center, Tampa, Florida, USA; 3 University of Utah, Salt Lake City, Utah, USA; 4 University of Illinois at Urbana‐Champaign, Urbana, Illinois, USA Manish Kohli and Utkan Demirci are co‐senior authors .

Introduction : Extracellular vesicles (EVs) offer minimally invasive means for cancer detection and monitoring. However, the lack of standardized methods for clinical biospecimen preparation and EV isolation hampers the clinical utility of EV‐based biomarker assessments. Our study investigates the impact of clinical sample preparation and our ExoTIC device on the quality of plasma‐derived EVs in metastatic castration‐resistant prostate cancer (mCRPC) patients.

Methods : We assessed sample preparation variables, including blood anti‐coagulant choice (EDTA or sodium citrate), type of plasma platelet fraction (platelet‐rich or platelet‐poor), and use of protease inhibitors. EVs were isolated using the ExoTIC device, followed by EV characterizations and biomarker analysis using nanoparticle tracking analysis, cryogenic electron microscopy, Western blot, and digital PCR. Blood was collected from all patients after obtaining written consent, adhering to an institutional IRB‐approved protocol for specimen collection.

Results : We detected mCRPC‐relevant proteins ARv7 and PSMA in EVs from all plasma sample types. Our findings indicate that platelet‐poor plasma (PPP) from EDTA tubes without protease inhibitors, or sodium citrate tubes with protease inhibitors, is optimal for detecting EV‐ and biology‐associated mCRPC biomarker miR‐375. Elevated EV miR‐375 levels from PPP samples correlate with the poor therapeutic response to docetaxel chemotherapy.

Summary/Conclusion : Optimal biospecimen preparation for EV analysis could enhance detection accuracy and patient management, highlighting detection of plasma EV‐associated mCRPC‐specific marker proteins (e.g., ARv7 and PSMA) and microRNA miR‐375.

Funding : NIH R01EB029805 and R01CA212097; P.D.S. acknowledges support from the James D. Plummer Graduate Fellowship, EDGE Doctoral Fellowship Program, Summer First Fellowship Program, Dean's Office of the Stanford School of Engineering, Cancer Imaging & Early Detection Award, Canary‐ACED Graduate Fellowship, and Stanford Bio‐X Interdisciplinary Initiatives Program Seed Grant.

Multi‐instrumental analysis of extracellular vesicle (EV) heterogeneity—experience of the Centre for Applied Nanomedicine (CAN)

Nadim Tawil 1 *, Laura Montermini 1 , Mahsa Jalali 2 , Molly L. Shen 2 , David Juncker 2 , Sara Mahshid 2 , Thupten Tsering 1 , Julia Burnier 1 , Hélène Pagé‐Veillette 1 , Ekaterina Iourchenko 1 , Dongsic Choi, Brian Meehan 1 , Anthoula Lazaris 1 , Peter Metrakos 1 , Janusz Rak 1,2 *

1 Research Institute of the McGill University Health Centre, Montreal, Québec, Canada; 2 McGill University, Montreal, Québec, Canada

Introduction : Current and emerging nanotechnologies offer diverse and often incomplete perspectives on the complexity of extracellular vesicle and particle (EVP) landscapes. In order to obtain a more comprehensive picture, we employed complementary technologies available at the Center for Applied Nanomedicine (CAN) at the Research Institute of the McGill University Health Centre (RI‐MUHC) in Montreal, Canada. Here we describe the capacities and results that can be generated through the use of orthogonal particle counters and single EVP analysis platforms using fluorescent probes and label free detection techniques as tools that enable dissection of unsuspected EVP subsets released by a range of donor cells.

Methods : CAN features a robust, high‐resolution EV research pipeline that includes nanoparticle sizing and counting, nanoflow cytometry, imaging cytometry, super‐resolution microscopy, and chip‐based immunofluorescence for molecular EVP profiling. These technologies facilitate detailed EVP characterization including surface marker detection, cargo analysis, and localization of EVP‐associated moieties.

Results : Leverageing CAN's capabilities, a series of pioneering studies has emerged. Using well characterised cellular sources of EVPs we have conducted instrumental and molecular analysis of particle subpopulations in cellular conditioned media and biofluids. Using the integrated CAN pipeline enabled identification of unsuspected subsets of EVPs carrying signalling membrane receptors, tetraspanins and functional markers. Chip technologies revealed shifts in EVP subpopulations following disruption of biogenic mechanisms dependent on syntenin‐1, neutral sphingomyelinase, and other effectors. Extending CAN capacity to support novel platforms enabled fingerprinting of cancer EVPs at single particle resolution using molecular Surface‐Enhanced Raman Spectroscopy (MoSERS) and machine learning‐based detection of hallmarks associated with progression of glioblastoma (GBM). Multi‐instrumental approaches afforded by CAN have also propped the development and assessment of novel EV isolation microfluidic devices as well as various fusion‐protein systems that take advantage of membrane‐localizing peptides as tools for prolonged and non‐disruptive EV tracking in both in vivo and in vitro settings.

Summary/Conclusion : Integrated technology platforms, such as CAN, enable objective and orthogonal analysis of complex EVP populations and benchmarking novel nanotechnologies aimed for biomedical applications. Our recent experience with CAN infrastructure illustrates how essential the multi‐instrumental approach is for EV‐focused discovery and facilitating cross‐disciplinary collabourations in nanomedicine and nanotechnology.

Helping development of new projects on extracellular vesicles through a dedicated core facility at Institut Curie

Presenter: Nicolas Ansart

Institut Curie, Paris, France

Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed particles, which can transfer bioactive molecules to target cells, modulating their physiology. EVs are therefore the object of rising interest from scientists interested in all possible physiological or pathological questions. However, EVs are very heterogeneous and co‐secreted by cells with various other extracellular factors. Demonstration that EVs are involved in a given function thus requires several steps of isolation and analysis, for which the International Society for EVs has published guideline articles (MISEV2023, MISEV2018). Based on our long‐term expertise in EVs, and the frequent requests from colleagues for help on EV‐related questions, we created in 2020 at Institut Curie a core facility dedicated to EVs.

Methods : The CurieCoretech EVs are co‐led by the head of the Cytometry Coretech (CG), the head (CT) and a senior scientist (L.M.J.) of the ‘EV, Immune responses and Cancer’ research team, and experiments are run by engineers of the facility and the team (N.A., A.Z., N.N.). It disposes of 2 EV‐specific instruments: Zetaview (ParticleMetrix), and FlowNanoAnalyzer (nanoFCM). It offers access to instruments of the CurieCoretech Cytometry: Aurora spectral and ImageStream Imaging flow cytometers (Cytek) and CytoFLEX (Beckman). It also provides access to various devices for EV isolation (differential ultracentrifugation, size exclusion chromatography) and analysis (Western blotting, MacsPlexExo).

Results : The first step for any new user is a discussion with one of the senior leaders of the core facility, to define the scientific question and determine the best approaches which the facility can offer. The user will then bring their own biological samples to be trained by an engineer of the facility to perform isolation and characterization of EV preparation. The CurieCoreTech can perform these experiments for the users. The CurieCoreTech also develops protocols and pipelines for single EV analysis, in particular by one‐ or two‐colour staining for nanoFCM analysis, and for EV staining for downstream analysis of interaction with target cells.

Summary/Conclusion : By providing advice on how to isolate and characterize EVs following the MISEV guidelines, and by developing novel protocols for EV analysis, the CurieCoreTech EVs aim to promote reproducible and interpretable research on EVs.

Proteomic profiling of breast cancer cells and their secreted extracellular vesicles upon drug treatment

Ahmad Maswadeh 1 , Florent Dingli 2 , Damarys Loew 2 , Henri Guilhon 3 , Michael Richard 3 , Patrick Poullet 3 , Nathalie Névo 1 , Clotilde Théry 1 , Lorena Martin‐Jaular 1

1 Institut Curie, PSL Research University, Paris, France; 2 Institut Curie, PSL Research University, CurieCoreTech Mass Spectrometry Proteomics, Paris, France; 3 Institut Curie, PSL Research University, CurieCoreTech Bioinformatics, Paris, France

Introduction : Extracellular vesicles (EVs) are very heterogeneous in terms of their intracellular origin, release mode, size, composition, and functions. Despite efforts to identify markers specific to EV sub‐populations, no such markers are currently known. We have previously applied an unbiased quantitative protein‐profiling approach to Jurkat cell‐derived EVs (Martin‐Jaular, 2021, EMBO Journal, 40: e105492) and thus identified networks of proteins with almost identical association to EVs, meaning with very similar pelleting properties, and studied their changes upon HIV infection. Here, we further developed this workflow, making it adaptable to different cell types and biological samples, to partially separate and analyse (a) secretome components, including populations of EVs, non‐vesicular extracellular particles (NVEPs) and secreted soluble proteins, and (b) intracellular components, using differential ultracentrifugation (dUC) followed by label‐free data‐independent acquisition (DIA) quantitative proteomics.

Methods : We performed five consecutive and increasing speeds of ultracentrifugation on MDA‐MB‐231 breast cancer cells and their secretome, obtaining five sub‐cellular and five sub‐secretome fractions with similar protein amounts, followed by quantitative proteomics. We assessed the ability of this workflow to compare between two conditions by treating MDA‐MB‐231 cells or not with homosalate, a compound we previously found to increase MDA‐MB‐231‐derived EVs and enhance their pro‐cancerous properties (Grisard, JEV 2022, 11: e12242).

Results : This workflow yielded sub‐cellular and sub‐secretome fractions with varying protein abundances across the fractions, clearly seen with different gene ontology (GO) terms associated with the different fractions. By looking at organellar marker profiles, this workflow partially but robustly separates intracellular compartments, while the sub‐secretome fractions show a higher degree of overlap between markers of some organelles. The analysis is underway to identify protein neighbour networks in cells and extracellular particles (EPs), proteins specific to either EVs or NVEPs and proteins shifting in cells and EPs between the two conditions.

Summary/Conclusion : This workflow will be used to (a) identify novel protein networks, potentially identifying novel EV or NVEPs sub‐populations, (b) identify proteins with altered trafficking in cells and presence in EVs between two biological conditions, and (c) study their correlation with the observed alterations in EVs to possibly identify targets playing a role in EV secretion, content and/or functions.

Size‐specific isolation and profiling of extracellular vesicles to create an atlas of their cargo in prostate cancer

Prima Dewi Sinawang 1 , Fernando J. Garcia‐Marques 1 , Abel Bermudez 1 , Chenchen Zhu 1 , Demir Akin 1 , Emily Ding 1 , Lars Steinmetz 1 , James D. Brooks 1 , Manish Kohli 2 , Andrew J. Gentles 1 , Sharon J. Pitteri 1 , Utkan Demirci 1 *

1 Stanford University, Stanford, California, USA; 2 University of Utah, Salt Lake City, Utah, USA

Introduction : Extracellular vesicles (EVs) play a crucial role in medical diagnostics. However, the size heterogeneity and the lack of convenient size fractionation methods pose challenges in identifying size‐specific vesicles with distinct markers. Common techniques, such as ultracentrifugation and size‐exclusion chromatography, are often time‐consuming and labour‐intensive, yet size fractionation of EVs is essential, as their dimensions influence intercellular signalling, disease progression, and cellular responses to therapies.

Methods : To address these challenges, we developed a platform that simplifies the isolation, enrichment, and size fractionation of EVs from various biological samples. This system can fractionate EVs into size categories, including 100–200 nm, 50–100 nm, and 30–50 nm, all achieved with a simple button push. We isolated EVs from in vitro cancer culture models: benign (BPH‐1), non‐metastatic (22Rv1), and metastatic cell lines (LNCaP, DU145, and PC3), and performed proteomic and transcriptomic analyses to study their roles in prostate cancer progression. Markers identified in vitro were validated in relevant clinical samples (IRB‐approved and obtained informed consent) using Western blot and PCR. Size subpopulations were characterized using NTA and cryo‐EM.

Results : We correlated the size and cargo of EVs with the metastatic potential and androgen receptor (AR) expression of the cancer cell lines, providing insights into disease stage. Our analysis revealed that different EV‐size subpopulations carry different cargos, highlighting the importance of size and metastatic characteristics. Unique proteins were identified for each cell line within each EV size subpopulation. Overlaps among the metastatic lines may indicate pro‐metastatic proteins, while few to no overlaps were found with the non‐metastatic lines. In metastatic cell lines, proteins in AR‐negative cell lines—compared to the AR‐positive one—were predominantly found in smaller EVs (30‐50 nm). Furthermore, different RNA types were distributed in different EV size subpopulations.

Summary/Conclusion : Our innovative approach not only streamLines the isolation, enrichment, and size fractionation of EVs but also generates valuable insights into EV biology and their size heterogeneity. We believe these findings could contribute to the field of EV research and provide a new perspective on their utility in personalized medicine.

* Information Disclosure : Due to a pending IP application, information disclosure is limited.

Funding : Stanford Bio‐X Interdisciplinary Initiatives Program Seed Grant.

Concentrating and purifying extracellular vesicles by superabsorbent polymers and size exclusion chromatography

Markus Bergqvist 1 , Cecilia Lässer 1 , Rossella Crescitelli 2 , Kyong‐Su Park 1 , Jan Lötvall 1

1 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 2 Department of Surgery, Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Introduction : Extracellular vesicles (EVs) are enriched from in vitro cell culture supernatants and various biofluids by multiple types of methods, including ultracentrifugation, which, however, has significant limitations, especially from the perspective of scalability. We have aimed to develop a non‐centrifugation‐based technique, removing non‐EV components of harvested cell culture medium using superabsorbent polymer (SAP), followed by size exclusion chromatography (SEC), with the goal to achieve similar yield and purity as ultracentrifugation (UC) followed by iodixanol density cushion (IDC).

Methods : HEK293 cells expressing CD63 coupled with Thermo Luciferase (T‐Luc) were used as a starting material to help quantify the yield and purity of EVs. After removal of cells and large debris from a 0.5 L cell culture by slow centrifugation, the cell culture supernatant was concentrated by pressure ultrafiltration as a primary step (Sartorius Vivaflow 200). EVs were then concentrated by either UC, centrifugal filter units (MERCK 10 kDa cutoff, CFU) or SAP within a dialysis membrane with a 3.5 kDa cutoff. After concentration of the EVs, further purification by either IDC or SEC using either Sepharose CL‐2B or Sepharose 6B, in either 10 or 20 mL columns (2B‐10, 2B‐20, 6B‐10 or 6B‐20).

Results : The pressure ultrafiltration concentrated the supernatant from the cell cultures from 500 mL down to 20–30 mL. All the subsequent concentration methods, including UC, SAP or CFU, resulted in a final volume of approximately 1 mL. The highest yield was with UC (70%–80%), followed by SAP (60%–70%) and CFU (50%–60%). For evaluation of the four SEC methods and the IDC, the yield was 60%, 50%, 50%, 40% and 60%, respectively, for 2B‐10, 2B‐20, 6B‐10 or 6B‐20 and IDC. Purity was evaluated as the relationship between luciferase signal vs protein concentration measured by BCA. Purity was 26, 25, 15, 15 and 38, respectively, for 2B‐10, 2B‐20, 6B‐10 or 6B‐20 and IDC (arbitrary numbers).

Summary/Conclusion : In summary, UC combined with IDC resulted in the highest purity and highest yield. An alternative approach also achieving high yield and purity is SAP combined with 2B‐10 or 2B‐20. Thus, utilization of SAP followed by SEC is an alternative efficient way to enrich EVs from cell culture.

Different tetraspanin profiles according to biofluid and kidney disease

Ana Flores‐Chova 1 , Olga Martinez‐Arroyo 1 , Sarah Tassinari 2 , Marta Méndez 1 , Laia Garcia‐Ferran 1 , Josep Redón 1,3 , María José Forner 1,4,5 , Benedetta Bussolati 2 , Ana Ortega 1,6 , Raquel Cortes 1

1 Cardiometabolic and Renal Risk Research Group, INCLIVA Biomedical Research Institute, Valencia, Spain; 2 Department of Molecular Biotechnology and Health Science, University of Torino, Torino, Italy; 3 CIBER of Physiopathology of Obesity and Nutrition (CIBEROBN), Institute of Health Carlos III, Minister of Health, Madrid, Spain; 4 Internal Medicine, Hospital Clínico Universitario, Valencia, Spain; 5 Faculty of Medicine, Department of Medicine, University of Valencia, Valencia, Spain; 6 CIBER of Cardiovascular Diseases (CIBERCV), Institute of Health Carlos III, Minister of Health, Madrid, Spain Ana Flores‐Chova and Olga Martinez‐Arroyo are co‐first authors. Ana Ortega and Raquel Cortes are co‐senior authors .

Introduction : The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, the effect of biofluid or disease on their profile remains under‐investigated. This study aimed to characterize the tetraspanin profile in several human biofluids and the effect of kidney diseases.

Methods : We characterized this tetraspanin profile in plasma and urine EVs from 76 lupus patients (22 with lupus nephritis, LN), 18 patients with diabetic nephropathy (DN), and 20 healthy controls using flow cytometry (MACSPlex Kit). Additionally, single EV surface protein characterization was performed using SP‐IRIS (Exoview platform). Written informed consent was obtained, and the study was approved by the Clinical Research Committee of the University Clinic Hospital of Valencia (ref: 2021/062).

Results : In plasma, CD9 showed the highest mean fluorescence intensity (MFI) across all groups; however, CD63 and CD9 had the highest MFI in urine. According to kidney disease, LN plasma samples showed increased CD81 and decreased CD9, while DN plasma had elevated CD9. In urine, LN patients had increased CD9 and CD81 but decreased CD63, whereas DN showed decreased CD9. Single‐vesicle analysis revealed no differences in particle sizes across biofluids or patient groups, but distinct tetraspanin colocalization patterns were noted. In urine, > 65% of CD63+ vesicles in LN were captured by CD63; in plasma, CD81+ and CD63+ vesicles were largely captured by CD81, especially in kidney disease groups. CD81 captured the highest proportions of triple‐ and double‐positive vesicles in both urine and plasma, with the highest levels in LN patients.

Summary/Conclusion : Biofluid and kidney disease have an effect on the EV‐tetraspanin profile. This finding establishes the importance of the tetraspanin characterization before EVs isolation methodologies based on these membrane markers. In addition, further studies are necessary to analyse in more depth what origin‐cell condition tetraspanin profile in kidney disease.

Funding : Carlos III Health Institute: PI18/01405 and PI21/00249 and PI23/01179, I+D+I Projects; FI20/00096 and FI22/00032, PFIS grants and Miguel Servet Contract CP22/00069; CNS2022‐136175, Ayudas consolidación Investigadora, funded by MCIN/AEI/10.13039/501100011033 and Unión Europea «Next Generation EU»/PRTR». IJC2020‐045308‐I, Juan de la Cierva Incorporación, funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future” and co‐funded with European Funds for Regional Development (FEDER).

Engineering extracellular vesicles to transiently permeabilize the blood‐brain barrier

Presenter: Lino Ferreira

University of Coimbra, Coimbra, Portugal

Introduction : Drug delivery to the brain is challenging due to the restricted permeability of the blood brain barrier (BBB). Recent studies indicate that BBB permeability increases over time during physiological ageing likely due to factors (including small extracellular vesicles (sEVs)) that exist in the bloodstream. Here we have investigated in vitro the human BBB opening capacity of sEVs enriched with miRNAs that are overexpressed in sEVs collected from the plasma of aged human individuals.

Methods : To enrich the sEVs with the miRNAs we have used a protocol that has been recently reported by us that makes use of a chemical reagent to transfect the sEVs with miRNAs. We have evaluated the BBB opening properties of 4 miRNA‐enriched sEVs formulations in a static and then one of those in a dynamic (using microfluidics) human in vitro BBB model.

Results : First, we investigated whether some miRNAs identified by us from previous RNASeq data were overexpressed in sEVs collected from plasma of aged individuals. Next, we loaded each of the miRNAs in sEVs collected from human cord blood plasma and evaluated the opening properties of the miRNA‐enriched sEVs in a static or dynamic (under flow) human in vitro BBB model. Our results showed that one of the miRNA‐enriched sEVs significantly increased BBB permeability in a reversible manner by decreasing the expression of claudin 5, an important tight junction protein of brain endothelial cells of the BBB, mediated in part by the knockdown of activating transcription factor 4. To the best of our knowledge, this study identifies for the first time an sEV‐enriched formulation able to transiently open the BBB inspired by physiological ageing.

Summary/Conclusion : Our findings suggest that engineered sEVs have potential as a strategy for the temporary BBB opening, making it easier for drugs to reach the brain when injected into the bloodstream.

Funding : The authors would like to acknowledge the funding from the European Commission through the Marie Skłodowska‐Curie Innovative Training Network “NANOSTEM” (ref. 764958) and Twinning RESETageing project (ref. 952266); the Portuguese funding research institution (FCT) for the funding through the projects PTDC/BTM‐SAL/5174/2020, 2022.07615.PTDC, 2022.02803.PTDC, and the PRR project HfPT‐ Health from Portugal (ref. 02/C05‐i01.01/2022.PC644937233‐00000047).

Optimising immunocapture of extracellular vesicles from cerebrospinal fluid for biomarker discovery in neurodegenerative diseases

Elizabeth R. Dellar 1 , Iolanda Vendrell 2 , Roman Fischer 2,3 , Alexander G. Thompson 1 .

1 Nuffield Department of Clinical Neurosciences, University of Oxford, London, UK; 2 Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, London, UK; 3 Nuffield Department of Medicine, Chinese Academy of Medical Science Oxford Institute, University of Oxford, London, UK

Introduction : Cells of the central nervous system (CNS) secrete EVs into cerebrospinal fluid (CSF) and thus hold potential as a source of new biomarkers for neurodegenerative diseases. Current methods for purification, such as ultracentrifugation and size exclusion chromatography (SEC), require large starting volumes, but immunocapture methods may help to reduce volume and separate EVs by cellular origin. Our aim is to optimise methods for capture and proteomic profiling of CNS‐EVs from CSF for biomarker discovery in neurodegenerative diseases.

Methods : EVs were purified from 200 to 1000 µL CSF by immunocapture with M‐270 Epoxy Dynabeads conjugated to a cocktail of tetraspanin antibodies (CD81, CD63, and CD9), or by SEC. Bound vesicles were lysed with 5% SDS, for subsequent Lys‐C/trypsin digestion with the suspension trapping method (Protifi). Liquid chromatography tandem mass spectrometry with library‐free data‐independent acquisition was used to compare the proteome (1 h gradient on Orbitrap Ascend).

Results : Here we demonstrate that the immunocapture methodology is robust at CSF volumes down to 200 µL, with detection of core EV marker proteins CD9, CD81, TSG101, Syntenin‐1, ALIX, and FLOT1 in all samples, and depletion of non‐vesicular proteins such as Apolipoprotein B and Lectin galactoside‐binding soluble 3 binding protein (LGALS3BP) relative to SEC. We show high proteomic depth with 811 ± 14 proteins using immunocapture from 200 µL CSF, increasing to 1285 ± 224 or 1266 ± 18 in 500 µL or 1000 µL, respectively, compared to 812 ± 66 in SEC‐isolated samples from 500 µL. In the total dataset, detected proteins were significantly enriched for choroid plexus and astrocytic markers, but with no significant enrichment for neuronee markers. We identified proposed neuronal‐specific EV capture target proteins L1CAM, NCAM1, ATP1A3 and GAP‐43 in the data, alongside some additional transmembrane neuronal‐enriched proteins. Precursor peptide mapping showed the presence of cytoplasmic regions for NCAM1, ATP1A3 and some novel proteins, providing some evidence for membrane‐bound over soluble protein forms. No known cytoplasmic peptides were detected for L1CAM or GAP‐43.

Summary/Conclusion : This work demonstrates that the immunocapture methodology allows for robust EV purification from small volumes of CSF using generic EV markers. This approach can be used to test alternative neuronal, microglial and astroglial EV capture targets, which we have identified in the dataset.

Brain targeting using novel extracellular vesicles (EVs)

Presenter: Paniz Shirmast

Griffith University, Gold Coast, Australia

Introduction : Brain infections and neurological disorders are difficult to treat due to the blood‐brain barrier (BBB), which excludes most drug delivery systems, including lipid nanoparticles (LNPs). Extracellular vesicles (EVs), nanometre‐sized lipid bilayers, offer protection to their cargo and have shown promise in delivering therapeutics safely and efficiently. In this study, we aimed to develop a new class of EV‐based nanoparticles capable of achieving brain biodistribution, with the goal of delivering therapeutic molecules for potential treatment of neurodegenerative diseases.

Methods : EVs were collected from HEK293, NSC, and B.END3 cell lines and purified using tangential flow filtration (TFF) with 500 kDa molecular weight cutoff (MWCO) pores. They were further concentrated with Amicon Ultra centrifugal filter tubes (10 kDa MWCO). EVs were characterized following MISEV guidelines using western blot, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). Lipids were extracted from the EVs for lipidomics analysis, and new EVs were formed using the lipids. A GFP‐expressing DNA plasmid was encapsulated using microfluidics. The newly formed EVs were characterized by DLS, transmission electron microscopy (TEM), western blot, and NTA. Packageing efficiency was assessed using PicoGreen and protection assays. Transfection efficiency was measured using flow cytometry, while confocal microscopy tracked nanoparticle entry into cells.

Results : Western blot confirmed the presence of EV markers CD63, CD9, and CD81. DLS showed that HEK293, NSC, and B.END3 EVs had sizes of 30.82, 36.23, and 33.26 nm, respectively. After packageing GFP, sizes increased to 72.45, 67.28, and 79.57 nm, respectively. DLS and NTA results were consistent. Western blot revealed changes in the EVs' protein corona, with surface markers removed during lipid‐based EV formation. The PicoGreen assay showed 94.7% packageing efficiency after adding helper lipids. Intravenous injection of the new EVs demonstrated successful brain biodistribution despite changes to the protein corona.

Summary/Conclusion : Our results show that the newly formed packaged EVs adhere to MISEV guidelines, effectively preserving their cargo and successfully transfecting it into cells. Additionally, our findings suggest that the EVs can reach the brain, with their biodistribution likely influenced by their lipid profile rather than their protein corona. These EVs hold great potential for the treatment of brain‐related diseases.

Funding : Griffith University.

Enhancing extracellular vesicle characterization through high‐resolution mass spectrometry: development of an R package and testing with public plasma datasets

Presenter: Rune Matthiesen

NOVA Medical School, Lisbon, Portugal

Introduction : Quality control and standardization are crucial for advancing extracellular vesicle (EV) research. Key recommendations include using protein‐based characterization methods, such as western blot or ELISA, to evaluate specific EV marker proteins. Non‐EV markers should also be analysed with western blot techniques tailored to the EV sample type. While immunoassays provide high specificity, they face scalability issues due to the substantial amounts of EV samples needed per marker. In contrast, mass spectrometry (MS)‐based proteomics has become a mainstream method, capable of identifying a wide array of EV and non‐EV markers while requiring only small amounts of protein, allowing for the profiling of thousands of proteins simultaneously.

Methods : LC‐MS data from previous studies on human EV samples were downloaded from the ProteomeXchange Consortium and curated to focus on label‐free quantification datasets with relevant metadata for reanalysis using MaxQuant version 2.1. These iBAQ values were normalized to the total iBAQ intensity per sample before being input into the EVqualityMS R package.

Results : The R package “EVqualityMS” provides a simple and efficient way to assess the protein‐based quality of EV preparations by calculating a metric that summarizes the presence of a selected set of non‐EV and EV markers. We demonstrate that this tool is very useful to detect EV preparations that show a different distribution in EV markers from the remaining cohort or study group in large data sets.

Summary/Conclusion : This study provides an R package to facilitate the assessment of quality and other features of the EV preparations based on MS‐based proteomics data in a quantitative, sensitive and high‐throughput approach.

Efficient isolation of extracellular vesicles from human plasma using droplet microfluidics

Beatrice Crestani 1 , Maria Giovanna Lupo 2 , Noemi Torriero 1 , Chiara Maceri 1 , Valentina Moccia 1,3 , Alessio Meggiolaro 1 , Sara Mathlouthi 4 , Mariangela Garofalo 4 , Valentina Zappulli 3 , Nicola Ferri 2,5 , Davide Ferraro 1

1 Department of Physics and Astronomy, University of Padova, Padova, Italy; 2 Department of Medicine, University of Padova, Padova, Italy; 3 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy; 4 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy; 5 Veneto Institute of Molecular Medicine, Padova, Italy

Introduction : Extracellular vesicles (EVs) are considered cargo transporting genetic information of the parental cell in body fluids, being perfectly suitable for liquid biopsy. However, the current use of EVs in clinical practice is still limited. Among the limiting factors, one of the most critical is their isolation. Conventional approaches are characterized by low purity or throughput, or scarce reproducibility. To overcome these limitations, we propose a droplet microfluidic platform for EV isolation by affinity capture using magnetic beads.

Methods : A novel microfluidic device is microfabricated by soft lithography to generate confined droplets devoted to efficiently mixing magnetic beads coated with anti‐human CD9 and CD81 antibodies and the starting sample. Cell culture media from breast cancer cell lines (MDA‐MB‐231) and human plasma from healthy donors (N&#3f3) are used as the starting sample. To evaluate the isolation efficiency capability of the microfluidic device, parallel experiments are performed also in‐batch and by ultracentrifugation (UC). According to the MISEV‐2023, isolated EVs are characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), bicinchoninic acid (BCA) assay, and western blot (WB).

Results : The microfluidic device allows processing up to 2 mL in a faster manner (4 h) than the commercial in‐batch approach (overnight incubation). NTA, BCA and WB show a greater capture efficiency starting from cell culture (2.5‐fold) and, more importantly, a much higher EV enrichment in the case of plasma (more than 10‐fold). These results are due to the microfluidic configuration, which promotes the meeting probability between beads and EVs being co‐encapsulated within the same droplets.

Summary/Conclusion : The device allows an efficient isolation of EVs, which is particularly enhanced in the case of plasma samples, proving how droplet microfluidics represents a promising technology for EV isolation. Currently, experiments devoted to EV elution and analysis within microfluidic devices are under investigation.

Funding : National Recovery and Resilience Plan (NRRP), Mission4, Component2, Investment1.1, calls No.104 (2.2.2022) and No.1409 (14.9.2022) by the Italian Ministry of University and Research (MUR), funded by the European Union–NextGenerationEU–for the Projects OCEAN (2022YYLSJ2) and EXTREME (P2022J2ZC2).

Towards analysis of exogenous siRNA loading homogeneity of bovine milk extracellular vesicles

Maximilian Kieckhöfer 1 , Caroline Schmidt 1 , Franziska Mitrach 1 , Christian Preußer 2 , Michaela Schulz‐Siegmund 1

1 Leipzig University, Leipzig, Germany; 2 Philips University Marburg, Marburg, Germany

Introduction : In a prior study, we established an siRNA loading strategy yielding an average of 150 siRNA copies per milk extracellular vesicle (mEV) utilizing calcium phosphate nanoparticles (CaP‐NP) and dual centrifugation (DC). However, we believe this siRNA distribution is unevenly distributed across the mEV population. A homogenous siRNA loading is crucial for reliable siRNA dosing and development of new delivery systems. To distinguish between loaded and non‐loaded vesicles, we systematically investigated and compared nanoparticle tracking analysis (NTA), flow cytometry (FCM) and nano flow cytometry (nFCM).

Methods : After enrichment, deploying differential centrifugation and size exclusion chromatography followed by ultracentrifugation, the mEV pellet was loaded by DC with fluorescently labelled siRNA bound to CaP‐NP. As a control, mEV‐mimicking liposomes (EVmL) were manufactured by a solvent evaporation protocol and loaded with CaP‐NP or just with siRNA. For fluorescence detection on single vesicles, we used NTA, FCM and nFCM. The loading efficiency was measured by total fluorescence with a plate reader after particle lysis.

Results : NTA analysis showed big differences in fluorescence between CaP‐NP‐loaded mEVs and EVmL loaded without CaP‐NP. We detected much more siRNA‐loaded EVmL than mEVs. The fraction of fluorescent mEVs and EVmL, presumably carrying CaP‐NP, showed a higher particles size than average. With flow cytometry we confirmed a small mEV population with large particles, loaded with fluorescent siRNA. We also stained CaP‐NP with calcein to circumvent low fluorescence intensity when using siRNA. Interestingly, when measuring total siRNA loading in a plate reader after lysis, we detected much higher fluorescence in mEV and EVmL than expected by the small fraction of positive vesicles.

Summary/Conclusion : Our findings show that direct measurement of siRNA loading inside single mEVs is possible but may be limited by a minimal fluorescence yield per vesicle, depending on the instrument employed. The finding that fluorescence was mainly found in large vesicles may result from the concept of encapsulating CaP‐NP in mEVs or just by the minimum fluorescence intensity of a vesicle. Finally, there is a need for higher sensitivity instruments for assessing mEV loading in future research and higher loading concentration to increase the fluorescence intensity per vesicle.

The importance of established and optimized analytical capabilities to develop end‐to‐end and scalable therapeutic extracellular vesicle processing

Presenter: Aslan (Mehdi) Dehghani

Sartorius Stedim North America, Danbury, Connecticut, USA

Introduction : Reliable analytical capabilities are critical for the development of extracellular vesicle (EV)‐based therapies. We have established an analytical toolbox consisting of multiple orthogonal techniques to comprehensively analyse EV samples. This analytical capability was then used to develop an end‐to‐end and scalable process for EVs derived from mesenchymal stem cells (MSCs). The downstream processing was first optimized and then validated and repeated 4 times at 500 mL scale. The process was then scaled up to 2 and 10 L MSC‐derived EVs.

Methods : The analytical toolbox consists of nanoparticle tracking analysis (NTA), flow cytometry (FC) and analytical high‐performance liquid chromatography (HPLC). The assays are established according to MISEV and MIFlowCyt‐EV guidelines. The MSC‐EVs process was optimized and established in a bioreactor using microcarriers. The collected conditioned media was then processed through clarification, tangential flow filtration, anion exchange chromatography and sterile filtration steps. Each purification was then optimized to achieve a full process with high EV recovery and high impurity removal.

Results : Utilizing the optimized analytical capabilities, the EV concentrations, free protein concentrations, free DNA concentrations, and EV associated proteins (corona) were monitored throughout the downstream processing. We achieved over 95% free protein and DNA reduction while maintaining over 50% of the EVs. The recovery and purity results were found reproducible after conducting the downstream process 4 times at 500 mL. The process was also demonstrated to be scalable by repeating at 2 and 10 L scales. Purified MSC‐EVs improved the wound recovery in a dose‐dependent manner, and the EV uptake was shown using live cell imaging. Proteomic analysis showed the common EV markers in agreement with western blot but also showed that 50% of the identified proteins are linked to diseases, suggesting potential targets for therapeutic applications.

Summary/Conclusion : Established and optimized analytical assays are critical to develop therapeutic EVs by systematic evaluation and monitoring of the process. Using optimized and established analytics, we then optimized our purification (downstream process). The optimal process was shown to be reproducible and scalable. We have successfully established the tools for systematic evaluation of EV biomanufacturing platforms to satisfy the therapeutic needs.

Acidic pH‐based elution of extracellular vesicles from magnetic beads

Noemi Torriero 1 , Chiara Maceri 1 , Beatrice Crestani 1 , Valentina Moccia 1,2 , Guillaume Gilliard 3 , Valentina Zappulli 2 , Pietro Parisse 4 , Valeria Rondelli 3 , Davide Ferraro 1

1 Department of Physics and Astronomy, University of Padova, Padova, Italy

2 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy; 3 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy; 4 Istituto Officina dei Materiali (CNR‐IOM), Consiglio Nazionale delle Ricerche, Trieste, Italy

Introduction : Compared to other known methods used for extracellular vesicles (EVs) isolation, immunoaffinity with magnetic beads allows us to obtain EVs with a lower yield but much higher purity. However, one of the main issues related to this technique is the detachment of the EVs from the beads, which often results in the alteration of EVs structural integrity and the dissolution of surface markers, making them unsuitable for functional assays. Here we propose a pH‐based method for the elution of the EVs following CD9‐coated magnetic bead‐mediated immunoaffinity.

Methods : EVs from the MDA‐MB‐231 cell line were previously isolated with ultrafiltration (UF) and characterized with nanoparticle tracking analysis (NTA), bicinchoninic acid (BCA) assay and western blot (WB). EVs were then incubated with beads functionalized with CD9 antibody for 3 h at room temperature in shaking conditions. To elute the EVs from the beads, an acidic solution of glycine‐HCl (pH 3) was added to the beads for 30 min on ice, followed by a basic tris‐HCl solution (pH 8). The beads were then extracted with a magnet, and the supernatant containing the EVs was evaluated with NTA, BCA, WB, transmission electron microscopy (TEM), atomic force microscopy (AFM), small angle X‐ray scattering (SAXS), and infrared spectroscopy (FT‐IR).

Results : In spite of a lower yield of EVs compared to the pre‐enriched UF sample, analyses confirmed the elution from beads and recovery of intact EVs. Pre‐enriched UF‐derived samples had an initial concentration of 10 10 EVs/mL, whereas a final recovery of 10 9 EVs/mL followed detachment from the beads after the acidic treatment. Both TEM and AFM analyses confirmed the higher purity of the eluted EVs, in contrast with UF‐derived EVs, rich in co‐isolated proteins.

Summary/Conclusion : This protocol allows us to detach EVs from magnetic beads following immunoaffinity isolation. Given the potentialities of EVs in several fields, this method represents a promising solution to isolate intact EVs and exploit them for subsequent functional assays. This protocol is also being applied in our Laboratory for the isolation of EVs from raw plasma in microfluidic devices, aiming at obtaining pure EVs for diagnostic purposes.

Funding : Next Generation EU Mission4 Component1, for projects no. P2022J2ZC2 and 2022YYLSJ2.

A microdevice for isolation and quantitation of cardiomyocyte‐secreted extracellular vesicles and monitoring their secretion profile in cardiac differentiation

Ehsanollah Moradi, Saana Pohjavaara, Päivi Järvinen, Markus Haapala, Virpi Talman, Tiina Sikanen

University of Helsinki, Helsinki, Finland

Introduction : Cell type‐selective isolation and detection of extracellular vesicles (EV) is a challenging task, requiring many purification and enrichment steps. Recently, magnetic bead‐based assays and microfabricated electrochemical sensors have been introduced for EV isolation via immunoisolation. Here, we present a microchip, based on electrochemical impedance spectroscopy (EIS), capable of cardiomyocyte‐selective isolation and semiquantitative analysis of EVs in cardiac differentiation.

Methods : The microdevice is made from off‐stoichiometric thiol‐enes and incorporates integrated gold sensors, functionalized with CD172a antibody, facilitating semiquantitative EV analysis based on the resistive EIS component change (deltaR/RA) before and after EV application. We used the chip to monitor the cardiac differentiation of human induced pluripotent stem cells (hiPSCs) by EV release tracking. hiPSCs were differentiated into cardiomyocytes (CM) using sequential Wnt activation and inhibition, and samples were collected on Days 0, 5, 7, 15, and 30. Prior to characterization, the CM‐EVs were pre‐purified and enriched 5‐fold using the ExoEasy kit and analysed for total particle count (nanoparticle tracking analysis, NTA) and total protein content. The selectivity of the CD172a antibody toward cardiac EVs was evaluated by hepatocyte‐secreted EVs and plasma EVs.

Results : Based on the NTA, the small particle (< 300 nm) count increased on days 7 and 9 of the culture, while on day 15, it dropped back to the baseline. Similarly, the total protein amount exhibited a steady rise in the first 2 weeks of the culture, while declining markedly after. Based on the EIS data, the concentration of CD172a(+)‐EVs increased in the differentiation phase (Day 5 to 15), whereas it decreased in the maturation phase (Day 15 to 30). The immunofluorescence staining of the hiPSC‐CMs (Day 30) for quantification of cardiac troponin T‐positive cells confirmed that the differentiation culture produced cardiomyocytes with high (> 90%) purity, which is well in line with the CD172a(+)‐EVs release profile.

Summary/Conclusion : Here, we demonstrate how the EV release profile can potentially be used for predicting hiPSC‐CM differentiation noninvasively with the help of cardiomyocyte‐selective EV isolation on CD172a‐coated sensors, followed by semiquantitative analysis of EV concentration through in situ EIS.

Funding : This work has been funded by the Research Council of Finland, the European Research Council, and the Finnish Cultural Foundation.

Nanoscale detection of EpCAM‐positive extracellular vesicles using magnetic beads and interferometric scattering microscopy

Sena Yaman 1 , Ugur Aygun 2 , Ugur Parlatan 2 , and Gozde Durmus 1

1 Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California, USA; 2 Department of Radiology, Canary Center at Stanford, Stanford University, Stanford, California, USA

Introduction : Extracellular vesicles (EVs) positive for epithelial cell adhesion molecule (EpCAM) hold great potential as predictive and prognostic tools for epithelium‐derived tumours. Here, we captured EpCAM+ EVs on nanometre‐sized magnetic beads and imaged them with single‐particle sensitivity using custom‐built Interferometric Scattering Microscopy (iSCAT). This approach requires < 1 µL of sample, making it highly suitable for applications with limited sample availability.

Methods : EVs were isolated from MC38 Y01 (a hybrid of a murine colon adenocarcinoma) cell culture media. After 48 h of incubation in DMEM supplemented with 10% exosome‐depleted FBS, 4 mL of the culture media was collected, and EVs ranging from 30 to 200 nm were isolated using a microfluidic filtration chip. First, 50 nm streptavidin magnetic beads (Miltenyi Biotec) were functionalized with biotinylated mouse CD326 (EpCAM) monoclonal antibody (Invitrogen). Second, the antibody‐coated beads were incubated with 50 µL of isolated EVs for 1 h. For imaging, we designed a custom iSCAT setup that uses a dielectric thin film on the substrate to enhance contrast by providing a background reference field. This setup enables single‐particle visualization by capturing interference between scattered light from particles and the reference field. The resulting interferometric images reveal particle volume and shape, allowing real‐time tracking of particle dynamics. We performed interferometric imaging and nanoparticle tracking analysis (NTA) for magnetic beads, antibody‐coated magnetic beads, and magnetic bead‐antibody‐EV complexes.

Results : Our setup enabled label‐free imaging of EpCAM+ EVs on magnetic beads, differentiating between bare magnetic beads, antibody‐functionalized beads, and bead‐EVEpCAM complexes. Interferometric images provided detailed size and morphology information, with quantitative analysis showing size increases and shape circularity changes upon EV binding. NTA yielded mean diameters of 58.9 nm for magnetic beads, 63.3 nm for antibody‐coated magnetic beads, and 107.7 nm for bead‐EV complexes. In the case of the bead‐EV complexes, three peaks were observed, confirming the conjugation and elongation of the particles compared to antibody‐coated magnetic beads.

Summary/Conclusion : Here, we developed a rapid, cost‐effective, and easy‐to‐use method to capture, detect, and quantitatively analyse EpCAM+ EVs released from a cell line using a low volume of sample. With additional modifications, this protocol could be adapted for detecting other EV subtypes directly from plasma.

Funding : UA, MSCA‐101066038.

Comparative extracellular vesicle purity assessment of independent isolation approaches

Kekoolani S. Visan 1 , Yue Su 1 , Sarah Voss 1 & Andreas Möller 1,2

1 Chinese University of Hong Kong, Hong Kong SAR, China; 2 QIMR Berghofer Medical Research Institute, Brisbane, Australia

Introduction : Extracellular vesicles (EVs) play a crucial role in intercellular communication and possess the potential to act as liquid biopsy biomarkers. Hence there is great interest regarding their contribution to biological processes. As a result, researchers continue to pursue optimal methods for the isolation of ‘pure’ EVs. EV purity remains an important topic of discussion, as the definition of ‘purity’ depends on the source and intended use of EVs. Different isolation methods can modify EV cargo and the biomolecular corona, impacting functional EV‐induced effects and analysis of EV contents. Ensuring appropriate purity is imperative so that any result produced is truly representative of EVs and not of co‐isolated materials.

Methods : Cell culture media (CCM) and blood are the most utilised sources for EV studies; hence, we assessed the purity of EVs isolated from both serum‐containing CCM (SK‐MES‐1 cell line) and plasma (healthy individuals). Traditional isolation methods were utilized including ultracentrifugation (CCM), tangential flow filtration (CCM) and size exclusion chromatography (plasma), as well as a novel device called the exosome detection method via the ultrafast‐isolation system (EXODUS) (CCM and plasma). Evaluation of EV purity was performed by particle (nanoparticle tracking analysis) and protein (Bradford assay) quantification, morphology visualisation (transmission electron microscopy), and assessment of protein absence or presence (traditional and capillary‐based western blot analyses, ELISA and mass spectrometry).

Results : The isolation methods employed identified significant differences in the abundance of EV and non‐EV materials. Size exclusion chromatography isolated plasma EVs with a high abundance of EV transmembrane and cytosolic markers, as well as corona‐related complement, fibrinogen, and extracellular matrix proteins. However, this was accompanied by high quantities of non‐EV immunoglobulins. EXODUS isolated plasma EVs with a high abundance of EV integrins and corona‐related complement proteins, albeit with high lipoprotein contamination. Ultracentrifugation, tangential flow filtration and EXODUS produced great variability in particle and protein yields from serum‐containing CCM, resulting in inconsistent detection of EV markers, but consistent ineffective removal of bovine albumin.

Summary/Conclusion : Each isolation approach produces EVs with different biomolecular properties, which provides specific advantages and disadvantages, depending on the intended use of EVs. Therefore, isolation methods need to be selected carefully for downstream applications.

ATR‐FTIR as a tool for process control during EV isolation

Alexander Popoff 1 , Cristian‐Tudor Matea 2,3 , Patrick Zimmerebner 2,3 , Andreas Marl 2,3 , Mario Gimona 3,4,5 , Eva Rohde 3,5,6 , Nicole Meisner‐Kober 2,3 , Daniela Schuster 1

1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Paracelsus Medical University, Salzburg, Austria; 2 Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria; 3 Ludwig Boltzmann Institute for Nanovesicular Precision Medicine, Salzburg, Austria; 4 Research Program “Nanovesicular Therapies,” Paracelsus Medical University, Salzburg, Austria; 5 GMP Unit, Paracelsus Medical University, Salzburg, Austria; 6 Department of Transfusion Medicine, University Hospital, Salzburger Landeskliniken GesmbH (SALK) and Paracelsus Medical University, Salzburg, Austria

Introduction : Extracellular vesicles (EVs) harbour great therapeutic potential either by their intrinsic properties or as vehicles for drug delivery. Therefore, isolation of EVs from their biological sources is a crucial aspect. Among commonly known isolation techniques like ultracentrifugation and preparative size exclusion chromatography (SEC), tangential flow filtration (TFF) offers great potential for upscaling but is time‐consuming and requires a process control like analytical SEC. Here we show that attenuated total reflection Fourier transform infrared spectroscopy (ATR‐FTIR) can serve as a simple and fast method for the quality control of EVs.

Methods : For the analysis we collected different milk‐derived EV samples during various TFF stages and determined the respective purity by analytical SEC, followed by acquisition of ATR‐FTIR spectra. This sample set we divided into a training and into a test set and created multivariate analysis models to predict the SEC purity. Accuracy and precision of the models were analysed with the test set.

Results : While the training sets showed coefficients of determination (R 2 ) above 0.95, the test sets had R 2 values greater than 0.75 and errors of prediction below 10%. In addition, weighting of spectral variables indicated that wavenumbers of lipid, phosphate and carbohydrate stretching are associated with higher SEC purity. In contrast, protein‐associated wavenumbers, like for instance the Amide I and Amide II bands at 1660 cm −1 and 1550 cm −1 are clear indicators of lower SEC purity.

Summary/Conclusion : Here we demonstrate that ATR‐FTIR can serve as process control during the TFF process, for example, which requires several buffer exchange steps. Due to its simple procedure, the low sample amount required and time efficiency, this technique can be a promising supplement for industrial scale EV isolation. In addition, multivariate analysis revealed spectral areas that can indicate molecule classes that can further lead to the discovery of purity markers.

Funding : The project was partly funded as part of EV‐TT (WISS 2025 and EFRE/IWB 20102‐F1900731‐KZP).

Multiplexed immunomagnetic microfluidic chip for isolation and detection of HER2‐positive exosomes

Presenter: Byeonggeol Mun

Yonsei University, Seoul, Republic of Korea

Introduction : Exosomes are useful for cancer diagnosis and monitoring. However, clinical samples contain impurities that complicate direct analyses of cancer‐derived exosomes. Therefore, a microfluidic chip‐based magnetically labelled exosome isolation system (MEIS‐chip) was developed as a lab‐on‐a‐chip platform for human epidermal growth factor receptor 2 (HER2)‐positive cancer diagnosis and monitoring.

Methods : Various magnetic nanoclusters (MNCs) were synthesized with different degrees of magnetization, and antibodies were introduced to capture HER2‐overexpressing and common exosomes using immunoaffinity. MNC‐bonded exosomes were separated into different exits according to their magnetization degrees.

Results : The MEIS‐chip efficiently separated HER2‐overexpressing exosomes from common exosomes that did not contain disease‐related information. The simultaneous separation of HER2‐ and non‐HER2‐overexpressing exosomes provided a means of analysing high‐purity HER2‐overexpressing exosomes while minimizing the contribution of non‐target exosomes, reducing misdiagnosis risk. Notably, common exosomes served as a negative control for monitoring real‐time changes in HER2 expression.

Summary/Conclusion : These findings support the application of MEIS‐chip for cancer diagnosis and treatment monitoring via effective exosome isolation.

Funding : This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number RS‐2024‐00448784).

Pre‐analytical considerations to augment signal‐to‐noise ratio in EV flow cytometry

Yohan Kim 1 , Chisom Uzendu 2 , Mohamed Ahmed 1 , Jack Andrews 3 , Ree Erickson 1 , Edwin Van Der Pol 4 , Fabrice Lucien 1,5

1 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 2 Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA; 3 Department of Urology, Mayo Clinic, Scottsdale, Arizona, USA; 4 Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands; 5 Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA

Introduction : Nanoscale flow cytometry (nFCM) is a suitable platform for high‐throughput, single extracellular vesicle (EV) enumeration and characterization from biofluids with minimal handling and processing. However, the small size of EVs and low number of surface antigens make detection above the background challenging. There is a need to develop strategies to augment the signal‐to‐noise ratio for the sensitive detection of rare EV populations. Therefore, we conducted a systematic evaluation of pre‐analytical labelling conditions and their impact on the quantification of different EV subsets from clinical samples.

Methods : The Apogee A60‐Micro Plus nFCM was calibrated and standardized using Exometry and Bangs Labouratories fluorescence beads according to the MIFlowCyt‐EV guidelines. We tested the impact of the following conditions on the limit of detection (LoD) and fluorescence quantification of EVs from patient platelet‐poor plasma samples and recombinant PSMA+ EV spiked samples: direct and indirect EV labelling, pre‐ and post‐labelling size exclusion chromatography (SEC). Lastly, we assessed the impact of multiplexing on the quantification of EVs from blood samples collected from localized prostate cancer patients ( n = 134) and healthy donors ( n = 7).

Results : Direct labelling of PSMA+EVs with a fluorescent primary antibody increased the percentage of PSMA+ EVs detected compared to indirect labelling with a fluorescent secondary antibody (45% vs. 35%, p = 0.0005) due to a reduction in LoD. Both pre‐ and post‐labelling SEC successfully removed ApoB+ lipoproteins (6‐fold decrease, p = 0.008). Although both pre‐ and post‐labelling SEC reduce LoD (2.5‐fold, p = 0.0156), only post‐labelling SEC leads to higher PSMA+ EV concentrations measured (2.0‐fold, p = 0.03). In the multiplex assay, using PSMA and STEAP1 antibodies, we observed increased concentrations of both single‐positive and double‐positive PSMA+ and STEAP1+ in samples from localized prostate cancer patient samples compared to healthy individuals ( p < 0.0001).

Summary/Conclusion : SEC is beneficial to improve the signal‐to‐noise ratio for the sensitive detection and quantification of plasma‐derived EVs. Careful selection of fluorophore and optimized fluorophore antibody conjugation improves fluorescence‐based quantification of EVs. This work serves as a framework for reproducible EV analysis by flow cytometry, offering new opportunities for the development of non‐invasive diagnostic tests.

Size‐exclusion chromatography and centrifugation protocols to enrich human plasma EVs: protocol optimization ahead of human clinical sample analyses

Hedayatullah Hayat, Dianne Cooper, Lucy V. Norling, Mauro Perretti

Queen Mary University of London, London, UK

Introduction : We compared centrifugation‐based with size exclusion chromatography (SEC) protocols, taking advantage of NanoFCM and other EV analytical approaches.

Methods : Heparinized blood ( n = 3 donors; Queen Mary Ethics Research Committee approval 0391) was centrifuged at 200 × g 20 min to prepare plasma. Then, two protocols were followed. (A) Plasma (500 µL) underwent three centrifugation steps: (i) 4400 × g for 15 min at 4°C; (ii) 13,000 × g for 2.5 min at 4°C; (iii) 13,000 × g for 2.5 min at 4°C. Supernatants stored at −80°C for further use. (B) Chromatography columns (BioRad Econo‐Pac, cat#7321010) were loaded with 14 mL Sepharose CL‐2B (cat#10217754) followed by 10 mL EV‐free, double filtered PBS (buffer) overnight at 4°C. After three washes, plasma (500–250–100 µL) was loaded. Elution was done with 10 mL buffer + 0.03% Tween and fifteen 0.5 mL eluted fractions were collected. In all cases, EVs were stained with CellMaskGreen (CMG) and analysed with NanoFCM, calibrated using dual‐laser quality control beads and Silica Nanospheres Cocktail #1 (65–155 nm). Quality control beads, sizing beads and fluorescently labelled EVs were all acquired with a sampling pressure of 1.0 kPa, and events were recorded for 1 min. The proportion of total events and CMG+ events was calculated. NanoSight NS300 (Malvern Instruments Ltd., Malvern, UK) analyses were also conducted. In some cases, the Oxford Nanoimager (ONI) dSTORM microscope (Nanoimager S, Oxford Nanoimaging) and Western blotting were run. Proteins were quantified with the Bradford Assay (Sigma).

Results : Centrifuged plasma EVs gave values of 30% to 40% CMG+ events. SEC fractionation of plasma yielded high EV numbers in fractions 5 to 10. Protein concentration was elevated from fraction 10 to 15. NanoFCM analyses indicated 70% ± 15% CMG+ EVs out of total events, with 500 µL plasma starting volume being optimal. NanoSight analyses calculated a mode of 157 ± 12 nm for SEC samples, against 120 ± 12 nm for centrifuged plasma samples ( N = 3 donors). Cell‐marker staining is ongoing with ONI microscopy and Western blotting analyses.

Summary/Conclusion : Reliable characterization is of paramount importance for diagnostic and prognostic use of EV numbers and types. We perfected a protocol for EV separation for plasma based on SEC and subsequent quantification of EVs, to be applied for clinical investigations, including an RCT for myocardial infarction ( NCT06465303 ).

Funding : Barts Charity Grant MGU0509.

Comparative analysis of methods for the purification of extracellular vesicle suspensions from human mesenchymal stem cell culture

Presenter: Yuliia Khmelnytska

Medical Center ‘M.T.K.,’ BU “Reocell,” Kyiv, Ukraine

Introduction : The extracellular vesicles (EVs) secreted by cell cultures have different compositions and may contain protein impurities. Existing extraction methods lead to the ingress of impurities into the target product. It is important to investigate the fractional composition of EV suspensions and their changes during concentration using tangential filtration (TF). The study aimed to analyse changes in the fractional composition of EV when using TF.

Methods : Mesenchymal stem cells from human adipose tissue (obtained with informed consent) were cultured in serum‐free DMEM/F12 medium (Sigma Aldrich) and in foetal bovine serum‐free DMEM/F12 medium (Sigma Aldrich) for 7 days without medium change. EVs were isolated by ultracentrifugation and TTF with 100 and 300 kDa cartridges, followed by ultracentrifugation (100,000 × g , 60 min). Total protein was measured fluorometrically (Invitrogen Qubit 4), EV concentration and size were measured by a flow cytometer (CytoFLEX Nano Beckman Coulter), and images were obtained by TEM (CEO).

Results : In conventional ultracentrifugation, the serum‐free medium has a predominantly 20‐50 nm fraction (77%) and a protein of 0.2 mg/mL, whereas in serum medium the 20–50 nm fraction is reduced to 66% and the protein increases to 4 mg/mL. TF with ultracentrifugation on 100 and 300 kDa cartridges significantly increases the protein content: for the 30 kDa cartridge in serum medium, up to 54 mg/mL with a 20–50 nm fraction (85%). The concentration of extracellular vesicles in the > 300 kDa fraction was 1 × 10⁹ EV/mL in serum medium and 6.4 × 10⁸ EV/mL in serum‐free medium. In contrast, in the < 100 kDa fraction the concentration was the same in both media – 1.5 × 10⁷ EV/mL.

Summary/Conclusion : Cultivation of MSCs in serum‐free medium reduced the protein in the culture by 4 times compared to serum medium. FBS increases the yield of EV because it contains its vesicles. TF reduces serum protein levels but does not remove EV from the serum. The purity of the final product depends on the choice of cartridge and media: tangential filtration with ultracentrifugation provides the content of the 20–50 nm fraction of 54%–85%.

Development of a reporter protein for dynamic labelling of cell type‐specific extracellular vesicles

Presenter: Jannik Hjortshøj Larsen

University of Southern Denmark, Odense, Denmark

Introduction : Emerging evidence suggests that metabolism is modified by organ system crosstalk using extracellular vesicles (EVs). We have gained significant knowledge of the EV's RNA and protein cargo. Yet, the mechanisms controlling the sender cell's EV secretion rate and the receiving cells’ uptake are largely unknown in living animals. We hypothesized that a reporter protein enabling the tagging of cell type‐specific EV could be used to gain insights into the in vivo dynamics of intercellular EV transfer.

Methods : We designed two different EV reporter proteins based on CD9. The first reporter protein, CD9‐truncated‐APEX2, was a truncated version of CD9, replacing CD9's large extracellular domain and final transmembrane domain with the peroxidase APEX2 and an ALFA tag. The second reporter protein, termed CD9‐APEX2, had a replacement of the CD9's extracellular domain with APEX2 and the ALFA tag but still contained CD9's last transmembrane domains. We characterized the constructs by stable transfection of HeLa cells. The labelling of cells and EVs by the APEX2 activity was tested by a 15‐min treatment with tyramide‐biotin. EVs were isolated by polyethylene glycol 6000 (PEG) precipitation and fractionated using size exclusion chromatography (SEC) followed by western blotting. Immunoprecipitation (IP) was performed using ALFA Selector ST magnetic beads or recombinant anti‐ALFA conjugated magnetic dynabeads.

Results : Our result shows the potential of both CD9‐APEX2 constructs, as in vitro EVs compose the fusion protein within the membrane as both constructs led to biotin tagging in SEC fractions positive for CD63. Furthermore, we successfully pulled down EVs from both constructs with IP targeting the ALFA tag, though CD9‐APEX2, containing the last domain, showed increased promise, as a vast greater amount were precipitated upon investigating CD63 expression. Lastly, this indicates the APEX2 resides on the outside of the EV membrane.

Summary/Conclusion : Our results indicate that CD9‐APEX2 allows the labelling and affinity isolation of cell type‐specific EVs. When expressed in mice, this reporter protein has the potential to label secreted EVs, enabling a deeper insight into the dynamics of EVs in living animals.

Funding : Funding provided by Independent Research Fund Denmark.

Filter‐aided extracellular vesicle enrichment (FAEVEr) using 300 kDa MWCO 96‐well plates for high‐throughput proteomics

Presenter: Jarne Pauwels

VIB‐Ugent, Ghent, Belgium

Introduction : Cells secrete small extracellular vesicles (sEVs) with a diameter < 200 nm, which are critical mediators of intercellular communication and hold significant potential for disease diagnostics and therapeutics. Traditional methods for sEV isolation, such as ultracentrifugation (UC), are often not scalable and may result in variable yields and purities, especially for proteome studies. This study investigates a streamLined 96‐well format for EV enrichment for mass spectrometry‐based proteome analysis.

Methods : We present a high‐throughput filter‐aided EV enrichment (FAEVEr) workflow for the enrichment of sEV from conditioned medium (CM) and biofluids with limited sample input (200–1000 µL). In short, CM is pre‐cleared using 96‐well 0.2 µm filtration plates, followed by sEV enrichment using ultrafiltration (UF, 300 kDa MWCO 96‐well plates) at low centrifugation speed (1000 × g for 10 min). Retained particles are purified by using relatively high percentages of the detergent TWEEN‐20 for consistent and near‐complete removal of non‐EV proteins. Purified sEVs are immediately lysed for optimal protein recovery and prepared for LC‐MS/MS analysis on a 96‐well S‐Trap plate.

Results : Our results indicate that FAEVEr 96well effectively and consistently retains sEV particles and show that including TWEEN‐20 during wash steps results in consistent high sEV purity without compromising sEV integrity. Mass spectrometry analysis revealed high reproducibility of protein identifications with few missing values compared to UC, and that increasing the percentage of TWEEN‐20 in the wash buffer translates to a more efficient removal of non‐EV proteins and a higher abundance of membrane proteins and EV biogenesis‐related proteins.

Summary/Conclusion : The FAEVEr 96‐well strategy enables the simultaneous processing of multiple samples, significantly reducing processing time yet improving the reproducibility, consistency and performance between replicates with overall fewer missing values, crucial for ensuring the validity and comparability between experimental results.

Use of advanced aptamer technology in affinity purification of EV from biological fluids

Rajindra Napit 1 , Satendra Jaysawal 1 , Jasmine Catague 1 , Haben Melke 1 , Rocky Chowdhury 1 , Alin Rai 2 , David Greening 2 , Lingxue Kong 1 , Wei Duan 1

1 School of Medicine, Deakin University, and IMPACT Strategic Research Centre, Waurn Ponds, Victoria, Australia; 2 Molecular Proteomics and Helen Amelia Hains Fellow at Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia

Introduction : Extracellular vesicles (EVs) play a vital role in intercellular communication by transporting genetic material and proteins. Despite their significance, current purification methods such as ultracentrifugation and chromatography are time‐consuming and can damage EVs, hindering their potential clinical applications. Given their importance in diagnostics and therapeutics, there's a pressing need for alternative purification techniques. This project aims to address this need by exploring new methods to isolate and enrich EVs effectively.

Methods : Methodology—the method involves utilizing predictive tools to aid in optimizing aptamer design, ensuring spacer arm addition minimally impacts aptamer affinity. Followed by the use of denaturing agents to disrupt the 3D structure of aptamers, enabling effective capture of extracellular vesicles (EVs) without the need for harsh reagents like detergent, high salt, or heat. Aptamers targeting universal EV biomarkers are employed for small EV capture. Streptavidin‐biotin interaction was exploited with streptavidin magnetic beads and biotin‐aptamer to capture and release the EV from cell culture media (CCM) and biological fluid plasma. Protein profiling of the purified EV fraction was performed in collabouration with the Baker Heart and Diabetes Institute.

Results : The aptamer affinity was then tested on antibody captured EVs to assess the binding capacity of aptamer. The result showed the modified aptamer is capable of binding to EVs in PBS buffer. The aptamer has been successfully used to capture and release the EV from both CCM and plasma. Furthermore, proteomics analysis revealed about ∼861 proteins on EV purified from CCM and ∼632 proteins from plasma purified EV. The proteome profile revealed that aptamer purified EV does contain the EV markers along with other proteins.

Summary/Conclusion : By leverageing predictive tools for aptamer design and employing denaturing agents, this research demonstrates a promising approach for efficient EV capture under mild or physiological conditions. The successful modification of the DNA aptamer against EV marker and its demonstrated EV purification capability could pave the way for further development of novel development in EV research along with potential clinical applications.

Targeted proteome analysis of extracellular vesicles from gastric cancer cell lines

Presenter: Tanay Kumar Sinha

Uppsala University, Uppsala, Sweden

Introduction : Early detection of gastric cancer (GC) is challenging due to cost and invasiveness of current screening methods. Extracellular vesicles (EVs) may be a promising alternative due to their protein composition. This study aims to explore the use of small‐EVs (sEVs) as biomarkers for early detection of gastric cancer by analysing 430 proteins using multiplex Proximity Extension Assay (PEA) in sEVs purified from gastric cancer cell lines and comparing them to those in the original cell lines and prostasomes from healthy individuals.

Methods : sEVs from four GC cell lines (AGS, MKN7, MKN45, and KaToIII) and prostasomes from healthy individuals were purified using differential ultracentrifugation and characterized per MISEV guidelines with TEM, NTA, and Western Blot. Protein levels in sEV and prostasome samples were measured using multiplex PEA with two commercial panels (oncology and inflammation) and three experimental panels (cancer, cellular pathway, and neurology). High‐expression proteins were validated using in‐house affinity‐based proximity methods, including solid phase proximity ligation assay (sp‐PLA) and extracellular vesicle‐PLA (EV‐PLA), followed by STRING analysis to understand protein‐protein interactions in GC sEVs.

Results : A total of 208 proteins were detected in at least one sEV sample, with 21 proteins present in all GC sEV samples. Principal component analysis (PCA) of protein expression profiles from GC cell lines, their sEVs, and prostasomes facilitated the differentiation of GC cells from their related sEVs, primarily through PC1, and revealed a distinct pattern for sEVs from the gastric carcinoma KaToIII cell line compared to those from three adenocarcinoma GC cell lines. 39 proteins showed significantly higher expression levels in GC sEVs compared to prostasomes. EphA2 and cMet were identified as the markers with the highest expression levels in GC sEVs, and their presence on the GC sEV surface was validated using SP‐PLA, EV‐PLA, and WB.

Summary/Conclusion : Using PEA, we identified highly expressed proteins, including EphA2, cMet, SOX‐2, IL‐6, and PTK‐2, in sEVs from GC cell lines. PEA's sensitivity enabled detection of less abundant sEV proteins, and we used STRING to predict the proteins' involvement in pathways promoting disease proliferation and invasion. These findings support further investigation and optimization of PEA for large‐scale screenings in larger patient cohorts and various body fluids. In conclusion, we identified a protein panel as potential GC‐specific biomarkers.

Fully‐automated cultivation of iMSCs and purification of iMSC‐EVs using the AutoCRAT system

Anna‐Lena Felser 1 , Tanja Jasmin Kutzner 2 , Moritz Meyer 3 , Kai Janning 1 , Yasemin van Heuvel 3 , Tim Milfeit 2 , Marco Carvalho Oliveira 3 , Bernd Giebel 2 , Patrick Bongartz 3 , Laura Herbst 1 , Bastian Nießing 1 , Robert H. Schmitt 1,4

1 Fraunhofer Institute for Production Technology IPT, Aachen, Germany; 2 University of Duisburg‐Essen, Essen, Germany; 3 BioThrust GmbH, Aachen, Germany; 4 RWTH Aachen University, Aachen, Germany

Introduction : Despite significant advancements in regenerative medicine, osteoarthritis (OA) remains a prevalent illness. Mesenchymal stromal cells (MSCs) are proposed as a therapeutic treatment for OA. The clinical efficacy of MSCs has been inconsistent; while several successful phase I and II trials have been conducted, phase III trials have raised doubts about their effectiveness, and no therapy has been approved for patients outside of these trials. These conflicting findings are partly due to significant biological variability and the lack of standardization in the MSC manufacturing process. Since MSCs exert their therapeutic functions mainly through their secretome, particularly via extracellular vesicles (EVs), MSC‐EVs have gained attention as a new drug product for various diseases, including OA. A major challenge in this field is to define robust and scalable production strategies. To this end, automation is a key technology to increase throughput and improve iMSC manufacturing efficiency in scalable EV production.

Methods : Within the EU project AutoCRAT, we have developed a fully automated Laboratory platform for cultivating induced pluripotent stem cells (iPSCs), differentiating them into iMSCs, and bioreactor‐based expansion of iMSCs to generate iMSC‐EVs.

Results : We incorporated bioreactors designed to accommodate three distinct vessels for maximum scalability. Liquid handling is performed by a dedicated pumping station. Samples can be taken automatically and analysed for cell count and viability at‐line, while temperature, pH, dissolved oxygen, glucose, and lactate are monitored online. An FPLC with an automated fraction collector is integrated into the EV module for downstream processing of the EV‐containing supernatant. We constructed a housing for the fraction collector with openings on both sides to allow for sterile purification of the EV‐containing supernatant. In our ongoing project, we confirmed that the platform enables the manufacturing of bona fide MSC‐EV products.

Summary/Conclusion : The platform establishes the groundwork for the automated production of MSC‐EV products, including scalability and enhanced manufacturing efficiency. Coupled with the potential for automated, repeated quality control evaluations, measures can be implemented to regulate the process for optimal production of highly abundant, high‐quality EVs.

Funding : This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 874671.

Development of a novel EV purification and cargo transfection method for efficient gene delivery

Presenter: Vinh Lam

System Biosciences, Palo Alto, California, USA

Introduction : Extracellular vesicles (EVs) are membrane‐bound particles that mediate intercellular communication, participating in various physiological and disease process. Therefore, EVs hold immense potential for delivering therapeutic genetic materials, such as mRNA and plasmid DNA, into target cells. However, current EV purification and cargo‐loading techniques often face challenges in efficiency, yield, and bioactivity retention. Here, we present a novel, streamLined approach that combines optimized exosome purification with an efficient cargo transfection method.

Methods : Our novel, two‐step protocol utilizes a combined polymer‐based precipitation and mixed‐mode chromatography isolation method for exosome purification that enhances yield and reduces contaminants. For cargo loading, we designed specific transfection reagents for plasmid DNA and mRNA, respectively, and optimized the protocol for efficient genetic material loading into EVs.

Results : The new purification method significantly improved exosome purity and yield compared to traditional techniques such as ultracentrifugation and conventional SEC, as confirmed by nanoparticle tracking analysis (NTA) and western blotting. The new method was applied to various biological samples such as serum, plasma and cell culture medium, and consistently achieved high yield and cleaner EVs. In an in vitro assay, our cargo transfection method demonstrated that EVs loaded with cargo successfully delivered and expressed the genetic material in target cells. In addition, the incorporation efficiency of mRNA or plasmid DNA using our EV purification method is higher than that achieved with traditional methods.

Summary/Conclusion : This study presents an efficient, scalable method for EV purification and cargo transfection, with improved purity and loading efficiency, offering a promising strategy for EV‐based gene therapy.

Physicochemical characterization and uptake of EVs isolated via tangential flow filtration, anion exchange and size exclusion chromatography

Presenter: Naomie Zhou

University Medical Centre Utrecht, Utrecht, The Netherlands

Introduction : Cells secrete extracellular vesicles (EVs) into their environment, where they participate in various physiological and pathological processes. For research purposes and therapeutic applications, EVs need to be separated from contaminants present in complex media or biofluids. However, in pursuit of effective EV isolation methods, balancing EV purity, yield, and integrity remains challenging. This study evaluates anion exchange chromatography (AEX) against routine EV enrichment methods—tangential flow filtration (TFF) and size exclusion chromatography (SEC). By comparing particle characteristics and uptake efficiency, we highlight how the isolation method impacts downstream applications.

Methods : Conditioned medium from MDA‐MB‐231 cells expressing HiBiT‐tagged CD63 was concentrated using TFF and ultrafiltration. Following storage (TFF) or further purification (AEX and SEC), EV‐containing fractions were buffer‐exchanged and concentrated by ultrafiltration. Particle size and yield were assessed via nanoparticle tracking analysis, and protein quantity and profiles were analysed by micro BCA and western blot. After EV treatment, HiBiT‐tagged EV uptake was measured via luminescence in recipient HeLa cells after removing bound EVs.

Results : EVs eluted from AEX in two distinct peaks, named AEX1 and AEX2. AEX1 contained the smallest particles (D10 = 76.4 to D90 = 163.8 nm, modal size at 89.6 nm), followed by SEC (84–160.7 nm, modal size = 104.7 nm), TFF (85.5–181.7 nm, modal size = 103.5 nm) and AEX2 (81.5–170 nm, modal size = 116.8 nm). Combined AEX1 and AEX2 particle yields, with most EVs present in AEX1, were similar to yields obtained by TFF. SEC caused significant particle loss, reducing yield by 46% compared to TFF. AEX1‐ and TFF‐EVs had higher protein contamination, whereas SEC provided the lowest protein‐to‐particle ratio. Interestingly, AEX1‐ and TFF‐EVs were internalized significantly more efficiently than SEC‐ and AEX2‐EVs.

Summary/Conclusion : TFF delivers the highest yield but with notable protein contamination. SEC achieves the best protein‐to‐particle ratio, although with reduced yields. AEX offers a middle ground; AEX1‐EVs show increased uptake alongside higher protein contamination and yield, while AEX2‐EVs demonstrate an improved protein‐to‐particle ratio but reduced uptake and yield. Results indicate a positive correlation between the presence of protein in EV preparations and EV uptake. In future studies, we will explore how differences in EV purity and uptake efficiency impact their functional effects.

Subtopic EV detection 1

Leverageing an antiviral peptide for the targeted disruption of tumour‐derived exosomes to enhance cancer immunotherapy

Presenter: Sol Shin

Sungkyunkwan University, Seoul, Republic of Korea

Introduction : Immune checkpoint blockade therapy (ICB), which targets the PD‐1/PD‐L1 axis, has revolutionized cancer treatment by restoring T cell effector functions. Despite the inherent advantages and high therapeutic efficacy of ICB, a significant unresolved challenge remains, namely that the majority of patients (70%–80%) do not benefit from ICB due to primary and acquired tolerance. Tumour‐derived exosomes (T‐EXOs), carrying immunosuppressive PD‐L1, have been shown to contribute to this resistance, inhibiting T cell activation and reshaping the tumour microenvironment (TME) to favour tumour growth. Effective therapeutic strategies are needed to inhibit T‐EXOs directly rather than targeting complex biogenesis pathways.

Methods : We developed the Lipid Envelope Exosome Disruption (LEED) strategy, utilizing a membrane‐curvature‐sensing antiviral peptide to disrupt the lipid membrane of T‐EXOs. As a first attempt to prove the LEED strategy, we harnessed the antiviral AH‐D peptide capable of recognizing high membrane curvature. The efficacy of the peptide was evaluated on T‐EXOs originating from various cancer cell types. Additionally, we investigated the therapeutic potential of combining the T‐EXO‐disrupting peptide with PD‐1 antibody‐based ICB therapy in a tumour‐bearing mouse model.

Results : Peptide‐mediated membrane disruption of T‐EXOs was significantly enhanced under the acidic pH conditions found in tumour microenvironments. This disruption resulted in a reduction in circulating levels of exosomal PD‐L1 and the restoration of CD8+ T cell effector function. The combination treatment of AH‐D peptide and aPD‐1 led to an enhanced therapeutic response, which reconstituted the tumour microenvironment (TME) to support anti‐tumour immunity. Additionally, AH‐D peptide treatment demonstrated an anti‐metastatic effect, preventing the formation of pre‐metastatic niches.

Summary/Conclusion : The LEED strategy demonstrates the potential of peptide‐mediated T‐EXO disruption to overcome ICB resistance, enhance anti‐tumour immunity, and prevent metastasis. This approach represents a promising avenue for improving cancer immunotherapy. Furthermore, our LEED strategy is anticipated to be broadly applicable to various combination treatment regimens, including both cancer immunotherapy and conventional chemotherapy.

A new class of universal reference EVs expressing horseradish peroxidase (HRP)

Daniela Waas 1 , Marc Juraschitz 2 , Yen‐Fu Adam Chen 1 , Harald Waltenberger 2 , Wolfgang Hammerschmidt 3 , Reinhard Zeidler 3,4 , Sonja Molinaro 2 , Kathrin Gärtner 1

1 Eximmium Biotechnologies GmbH, Planegg, Germany; 2 Microcoat Biotechnologie GmbH, Bernried, Germany; 3 Helmholtz Munich, Neuherberg, Germany; 4 LMU University Hospital Munich, Munich, Germany

Introduction : Extracellular vesicles (EVs) hold great promise as circulating biomarkers for diagnostic purposes (‘liquid biopsy’) and therapeutic applications. Thus, many research groups worldwide investigate important aspects of EVs, their biology and medical significance. For this, a large number of different procedures and protocols have been established, making it difficult or almost impossible to compare and replicate results. Consequently, the interpretation of diagnostic tests remains problematic, particularly because the use of reliable reference EVs has not yet gained widespread acceptance.

Methods : We designed and generated a new class of reference EVs derived from a recombinant HEK293 cell line expressing horseradish peroxidase (HRP). We characterized the HRP‐positive EVs and evaluated their performance in spike‐in and recovery experiments using different common EV isolation methods (e.g., SEC, density gradient centrifugation). We further investigated the sensitivity of detection of EV‐associated HRP and its stability in complex biological materials, such as serum.

Results : HRP+ EVs can be quantified easily and with unmatched sensitivity either directly via measuring HRP activity or indirectly via immunodetection of HRP on the EV surface. They exhibit common EV characteristics and the HRP activity remains stable in complex biological matrices and during EV isolation processes.

Summary/Conclusion : In the present study we show that HRP+ EVs derived from an engineered HEK293‐derived cell line can be easily detected and tracked enzymatically and immunologically and used for normalization of endogenous EVs contained in various biological specimens. The detection of these EVs via the enzymatic activity of HRP represents a highly sensitive and robust system, which can be used to (1) trace EVs through various isolation methods, (2) standardize protocols and SOPs, (3) calculate the yield after isolation, (4) back‐calculate the total EV numbers in biological and medical samples and cell culture supernatants and (5) normalize clinical specimens prior to diagnostic downstream analyses. We want to introduce our reference EVs to the EV community to foster standardization and harmonization in the field for both scientific application and routine clinical use.

Development of a disease‐specific analytical reference material for an EV‐based biomarker study

Presenter: Md. Khirul Islam

University of Turku, Turku, Finland

Introduction : Extracellular vesicles (EV) have emerged as promising biomarker carriers for various diseases. However, their clinical translation is hindered by the lack of standardized analytical reference materials necessary for developing reproducible and robust detection assays. While recent advancements have introduced reference materials for EV research, these often lack disease‐specific molecular signatures. Existing reference materials, derived from artificial sources or healthy donors, fail to accurately represent the diverse biomolecular content of disease‐associated EVs, thereby limiting their applicability in biomarker detection. To address these challenges, we have developed and characterized an analytical reference material derived from Du145 prostate cancer cell line EVs, aimed at standardizing EV‐based biomarker research.

Methods : EVs were separated from Du145 cancer cell line using ultracentrifugation (UC) and size exclusion chromatography (SEC), resulting in ucEV and secEV fractions. These isolates were then compared with commercially available recombinant EV (rEV) standard reference material. These ucEV, secEV and rEV were then characterized by tetraspanin expression profiling, nanoparticles tracking analysis (NTA), and transmission electron microscopy (TEM). To assess their potential as reference material, Du145‐EV was used to standardize a time‐resolved‐fluorometry‐immunoassay (TRFIA). Then the TRFIA platform was employed to detect surface proteins and glycans on ucEV, secEV and rEV using various antibodies and lectin panels.

Results : The TRFIA assay demonstrated high sensitivity and linearity for Du145‐EV detection compared to commercial rEV. Notably, ucEV and secEV showed higher expressions of disease‐specific proteins and glycans, which were absent in commercially available rEV. These findings reveal the superior expressions of molecular content on Du145‐EV, which could serve as analytical standards for EV‐based biomarker studies.

Summary/Conclusion : We introduce a Du145‐EV reference material, enriched with disease‐specific proteins and altered surface glycans, offering a biologically relevant standard for EV research and diagnostics. This material can serve as a reference for method development, data normalization, and assessment of analytical variables in EV studies. Next, we aim to validate its clinical utility and expand its application in EV‐detection methodologies, including lateral flow immunoassay (TRFIA) and TRFIA, within our lab setting.

Funding : University of Turku, Finland.

Development of a novel reference material for the flow cytometry analysis of extracellular vesicles

Presenter: Zoltán Varga

HUN‐REN Research Centre for Natural Sciences, Budapest, Hungary

Introduction : Hollow organosilica beads (HOBs) have previously been established as reference materials (RMs) for calibrating extracellular vesicle (EV) size gates in flow cytometry (FCM). However, established synthesis methods for HOBs have proven challenging when scaling to larger particle sizes (above 400 nm), thereby necessitating a more reproducible approach. In this study, we introduce a novel synthesis route using positively charged polystyrene (PS+) core templates, combined with fluorescent labelling to generate dim hollow particles with MESF values between 10 and 1000 to enhance reproducibility in EV measurements by FCM.

Methods : PS+ template particles were synthesized following a published protocol involving the controlled polymerization of styrene in water. These templates were then utilized in a modified sol‐gel process with TEOS and L‐arginine as catalysts to produce core‐shell particles, which were then calcined to yield hollow structures. The resulting particles were characterized for size distribution, concentration, and light scattering properties. A subsequent fluorescent labelling protocol was applied to achieve the targeted MESF range, enabling the generation of dim particles suitable for FCM calibration.

Results : The novel synthesis route produced RMs with uniform, reproducible hollow structures that overcome the size limitations of previous preparation methods. The resulting particles closely mimic EV characteristics regarding shell thickness and refractive index. Fluorescent labelling successfully yielded particles with MESF values between 10 and 1000, as confirmed by flow cytometry, ensuring improved calibration capability for EV analysis in FCM.

Summary/Conclusion : We have developed an innovative and reproducible method for synthesizing hollow particles using PS+ core templates, addressing the challenges of producing larger particles. We combined this with precise fluorescent labelling to generate particles with controlled dim fluorescence. These RMs exhibit EV‐like physical properties and provide robust calibration standards for FCM, promising enhanced accuracy and reproducibility in EV quantification for clinical diagnostics and research applications.

Funding : This work was funded by the National Research, Development, and Innovation Office under the grants ADVANCED‐150077, STARTING‐150629, TKP2021‐EGA‐31, and 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021.

Customizable liposomes as standardized fluorescent calibrators for submicron extracellular vesicle analysis by flow cytometry

Isabella Pesce 1 , Marinella Pinelli 2 , Lorenzo Lunelli 3,4 , Cristina Potrich 3,4 , Martin M. Hanczyc 2

1,2 University of Trento, Trento, Italy; 3 Fondazione Bruno Kessler‐Trento, Trento, Italy; 4 Italian National Research Council‐Trento, Trento, Italy

Introduction : Characterization of extracellular vesicles (EVs) by optical methods is challenging due to their small size, high polydispersity and low refractive index. Their limited abundance in biological fluids and low antigen density make their analysis by flow cytometry even more complex. Flow cytometers with improved sensitivity and detection limits and suitable standardized methods can greatly contribute to EV characterization. By taking advantage of both the ImageStreamX MkII imaging flow cytometer and the BD‐FACSymphony A1 flow cytometer equipped with the small‐particle detector, we aim at setting up suitable reference nanoparticles based on biotinylated liposomes for small particle analysis. Biotin is exploited for the binding of fluorophores conjugated with streptavidin, obtaining useful and flexible tools for the optimization of flow cytometer settings and protocols.

Methods : Biotinylated liposomes of different lipid compositions are prepared both as giant unilamellar vesicles obtained by droplet transfer followed by extrusion and as large unilamellar vesicles obtained by direct extrusion of multilamellar vesicles. Liposomes are characterized for size and concentration with NanoSight NS300. Monovalent and tetravalent streptavidins conjugated with various fluorophores are used to label biotinylated liposomes, and streptavidin fluorescence is quantified by using commercially available molecules of equivalent soluble fluorochrome (MESF) calibration beads.

Results : Biotinylated liposomes were prepared by two standard methods and then varied by diameter, lipid composition, concentration and amount of added fluorescent streptavidin to establish the best protocol for the preparation of standard fluorescent calibrators for flow cytometry. The stability of biotinylated liposomes as size and concentration was evaluated over a 2‐month period, resulting in small variations. Biotinylated liposomes stained with different combinations of fluorescent streptavidin gave efficient binding and fluorescent signals after 30 min, showing the versatility of this tool for the different applications. Preliminary tests were performed to assess the potential of biotinylated liposomes as calibration nanoparticles for EV analysis.

Summary/Conclusion : We successfully developed new versatile nanoparticles as fluorescent calibration nanoparticles for small particle analysis by flow cytometry. The proposed biotinylated liposomes have the huge advantage of being more similar to natural EVs with respect to standard polymeric calibration beads.

EVCA—Advancing excellence in multidisciplinary research and fostering long‐lasting collabourations in extracellular vesicles leading institutions

Presenter: Rúben Silva

University of Lisbon, Lisbon, Portugal

Introduction : EVCA stands for Extracellular Vesicles in Clinical Applications and is an acronym of the project titled ‘Centre for Excellence in Diagnostic and Advanced Therapeutics based on Extracellular Vesicles.’ EVCA focuses on methods and standard operating procedures for EVs and advanced therapeutics and diagnostics based on EVs. By partnering with internationally leading institutions—Institut Curie, France, and CIC bioGUNE, Spain—EVCA will leverage the existing multidisciplinary knowledge, experience, research and training best practices, widen collabourative networks, and step up the excellence of the researchers and research management at NOVA Medical School in order for it to become an internationally‐recognized Centre of EVs in clinical applications, research and innovation.

Methods : Since the start of the project, excellent dissemination, outreach, training, and mentoring activities have been implemented, supported by many initiatives, including Think Tank sessions, winter retreat, summer school, training workshops, and staff exchanges. The essential information about the project is disseminated through a well‐structured website (http://evca‐twinning.eu), carefully curated mailing lists and active social media channels, including LinkedIn and X.

Results : In the first reporting period of the EVCA, 4 high‐quality papers were published on (i) gut‐diet EV interaction in obesity, (ii) the role of EVs in skin intercellular communication, pigmentation and skin cancer, (iii) EV‐based bladder cancer diagnosis, and (iv) urinary EV‐based biomarkers for prostate cancer. Additionally, EVCA members were selected for 16 oral presentations and 13 poster presentations. Moreover, the previously mentioned organized events had more than 100 participants, and the project also supported the attendance of 50+ researchers in other initiatives and conferences, including MOVE, ISEV, PNEV23 and SINAL24 among others. Finally, EVCA was present in outreach events such as the European Researcher Night and reached 900+ followers across the social media platforms.

Summary/Conclusion : During this time progress was made towards the goals of the project. One of the main impacts was the consolidation of the collabourations between partners to make them long‐lasting—common participation in different initiatives and common scientific publications. Remarkably, the EVCA project had a profound impact on the institutional scientific landscape of NMS, fostering over 200% growth in the number of research groups studying EVs.

The dynamin inhibitor, dynasore, modulates longitudinal bone growth in a two‐phased dose‐dependent manner

Presenter: Jose G. Marchan‐Alvarez

Karolinska Institutet, Stockholm, Sweden

Introduction : Growing bones increase in length by the activity of chondrocytes in the epiphyseal cartilage. Chondrocytes regulate the sequential differentiation, proliferation, and mineralization process by releasing and uptaking extracellular vesicles (EVs). The internalisation of such EVs predominantly occurs via clathrin‐ and caveolin‐dependent endocytosis, in which dynamin is recruited to nascent vesicles in the inner leaflet of the plasma membrane, promoting vesicle scission. Inhibition of dynamin using selective inhibitors, such as dynasore, nearly abolishes EV internalization. Yet, dynasore may also induce non‐specific effects, including disruption in lipid trafficking and recycling. Since the role of EVs in growing cartilage remains to be fully elucidated, we sought to dissect the general effects of dynamin inhibition on bone growth.

Methods : The three central metatarsals were micro‐dissected from both hind paws of three‐day‐old C57BL/6 mice. Metatarsals were cultured for six days. On Days 0, 2, and 4, bones were treated with dynasore at concentrations of 40, 80, 160, and 220 µM. Bafilomycin, a well‐established compound known to induce bone overgrowth, was used as a positive control. Histological characteristics were evaluated using Fast Green/Safranin‐O staining, whereas phosphorylated ribosomal protein S6 (phospho‐RPS6), and SQSTM1 were studied by immunostaining.

Results : Our findings reveal that dynasore regulates longitudinal bone growth in a dose‐dependent manner, with these effects becoming evident as early as Day 2. At a high concentration (220 µM), dynasore significantly reduced bone length. By contrast, at a lower concentration (40 µM), we observed a marked increase in bone growth, resembling the effects of bafilomycin. This enhanced growth was associated with a gain in the length of growth plate zones such as the resting and proliferating zones. Furthermore, treatment with 40 µM dynasore significantly elevated levels of phospho‐RPS6 and SQSTM1, reflecting activated mTORC1 signalling.

Summary/Conclusion : A low dose of dynasore leads to pronounced bone growth, particularly by stimulating the length of the resting and proliferating zone and targeting hypertrophic chondrocyte activity through phospho‐RPS6 and SQSTM1. Thus, this study highlights an unrecognized role of dynasore in bone growth stimulation by potently activating the mTORC1 pathway in chondrocytes.

Funding : This work was supported financially by the Novo Nordisk Foundation and Karolinska Institutet.

Synthetic extracellular vesicles mimicking natural ones and their applications as reference particles

Presenter: Seong Jun Park

Ewha Womans University Mokdong Hospital, Seoul, Republic of Korea

Introduction : Since the complexity and heterogeneity of natural extracellular vesicles (nEVs) present challenges for their use in diagnostics and therapeutics, we have previously developed synthetic EVs (sEVs) using artificial intelligence (AI) and a microfluidic mass production platform. The basic requirements for referencing the nEVs are comparable size, low polydispersity index (PDI), reliable tropism and biocompatibility. For the validation of our sEVs as reference nanoparticles of nEVs, we have conducted the physicochemical characterization and bioequivalence tests of sEVs in both cellular and animal models, demonstrating the potential of sEVs in standardizing EV‐based biomedical applications.

Methods : The sEVs were characterized following MISEV 2023 guidelines, including particle size, zeta potential and encapsulation efficiency, using nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), flow cytometry, and transmission electron microscopy (TEM). Cellular uptake was assessed in HaCaT cells, and in vivo bioequivalence was evaluated in mice using nEVs from human salivary gland‐derived mesenchymal stem cells for comparison.

Results : The DLS measurements confirmed particle sizes below 150 nm with a PDI under 0.1, indicating precise and uniform production. Encapsulation efficiency exceeded 95%, and zeta potential was around 50 mV. Cellular uptake assays in HaCaT cells showed higher uptake of sEVs than that of nEVs. While sEVs exhibited slightly higher cytotoxicity at low concentrations than nEVs, they were comparable at higher concentrations. In vivo mice studies demonstrated minimal hepatic and renal toxicity, similar to nEVs.

Summary/Conclusion : Our findings suggest that sEVs can serve as the reliable reference particles for the EV research and applications, supporting their role in diagnostics and therapeutics. Their homogeneous mass production and customizable compositions will be able to offer a path toward more standardized and effective EV‐based biomedical solutions.

Funding : This work was supported by the National Research Foundation of Korea (NRF) and the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea Government (Ministry of Science and ICT) (RS‐2024‐00432946), the NRF grant funded by the Korea government (MSIT) (No. 2021R1A2C3011254), and the Technology Development Program funded by the Ministry of SMEs and Startups (MSS, Korea) (RS‐2024‐00506850).

Subtopic EV sizing and counting 1

A nanoscopic ruler to understand differences in concentration measurements across techniques

Mendel Engelaer 1 , Mona Shahsavari 1 , Britta Bettin 1 , Erik Mul 2 , Romy Meier 2 , Wouter W. Woud 3 , Inge Nelissen 4 , Ton G. van Leeuwen 1 , Edwin van der Pol 1

1 Amsterdam UMC, Amsterdam, The Netherlands; 2 Sanquin, Amsterdam, The Netherlands; 3 ExoVectory, Nijmegen, The Netherlands, 4 VITO, Mol, Belgium

Mendel Engelaer and Mona Shahsavari are co‐authors .

Introduction : Size distributions of extracellular vesicles (EVs) are commonly characterized using techniques such as conventional and imaging flow cytometry, microfluidic resistive pulse sensing (MRPS), and nanoparticle tracking analysis (NTA). These techniques differ in sensitivity and thus in their ability to detect small particles, such as EVs, which directly affect measured particle concentrations. As EV concentrations increase with decreasing diameter, slight differences in the diameter of the smallest detectable particle can significantly impact measured concentrations. Therefore, determining and reporting the diameter of the smallest detectable particle is essential for comparability and reproducibility. Currently, no method exists to determine this diameter across instruments. Here, we present a practical method for determining the diameter of the smallest particle detectable across instruments.

Methods : A bead mixture with a flat size distribution containing polystyrene beads between 30 and 100 nm was prepared and measured with conventional flow cytometers (FCMs, Cytek Biosciences Northern Lights, Apogee A60‐micro), an imaging FCM (Cytek Biosciences, ImageStreamX Mk II), NTA (Particle Metrix, ZetaView), and MRPS (Spectradyne, nCS1). The bead mixture was used to (1) determine the diameter of the smallest detectable particle with each instrument and (2) correct the measured concentration in a urine sample for differences in the diameter of the smallest detectable particle between instruments.

Results : The smallest polystyrene bead detectable by the different instruments ranged from 52 nm to 76 nm. The measured EV concentration before correction differed 31‐fold between instruments, ranging from 1.3E7 to 3.9E8 mL −1 . After correction for the smallest detectable size determined with the bead mixture, concentration differences were reduced to 11‐fold, ranging from 3.8E6 to 4.0E7 mL −1 .

Summary/Conclusion : Using the first sample with a flat size distribution, we provide a simple and universal method to determine the smallest detectable particle diameter across different instruments. Our results show that differences in the ability to detect small particles can be accounted for when measuring and reporting concentrations, improving reproducibility. In the future, a bead mixture with EV‐like properties, such as a low refractive index, could further enhance implementation and reproducibility in the field.

Funding : NWO: VIDI 19724 & MEDPHOT P18‐26.

Label‐free technology to assess protein loadings on the surface of a biological sample

Presenter: Evgenia Kim

Beckman Coulter Life Sciences, Indianapolis, Indiana, USA

Introduction : The present work relates to characterizing biological samples with different loadings of proteins on their surfaces by flow cytometry. While several methods, such as atomic force microscopy, transmission electron microscopy, surface plasmon resonance, Raman spectroscopy, and so forth, can also be used for such assessment, they are time‐ and labour‐intensive and lack sufficient throughput.

Methods : Flow cytometry, on the other hand, is a powerful high‐throughput method to characterize and enumerate biological particles through single particle detection. CytoFLEX nano flow cytometer is a dedicated flow cytometer for nanoparticle characterization, it is equipped with two violet side scatter channels that provide a large dynamic range of detection, along with three side scatter channels for blue, yellow, and red scatter signals.

Results : In this study, we demonstrate the capabilities of the instrument to detect and analyse the change of refractive index caused by different numbers of antibodies bound to the surface of murine leukaemia virus.

Summary/Conclusion : High scatter sensitivity of CytoFLEX nano Flow Cytometer enables detection of small changes of medium scatter intensities caused by varying amounts of protein loaded on the surface of the biological sample.

Comparative analysis of the CytoFLEX nano flow cytometer and the CytoFLEX LX flow cytometer

Presenter: Evgenia Kim

Beckman Coulter Life Sciences, Indianapolis, Indiana, USA

Introduction : This work aims to demonstrate the performance of the CytoFLEX nano flow cytometer and provide a comparative analysis with the conventional CytoFLEX LX flow cytometer.

Methods : Performing a side‐by‐side comparison analysis between scatter and fluorescence characteristics will provide insightful information about the beneficial features of the CytoFLEX nano flow cytometer as a dedicated instrument designed for nanoparticle characterization.

Results : In this study, we will demonstrate the instrument's performance using commercially available control beads and samples, such as plasma EVs, RBC EVs, platelet EVs, etc. As a part of the post‐processing step, we will demonstrate the capabilities of calibration techniques for size and refractive index based on NIST control beads (nanoViS Nanoscale Sizing Standards) with known sizes and refractive indices.

Summary/Conclusion : Calibrating the arbitrary units of medium scatter intensity (MSI) to absolute units of size and refractive index allows for the comparison of results measured on two different instrument platforms.

Label‐free characterization of biological nanoparticles

Presenter: Evgenia Kim

Beckman Coulter Life Sciences, Indianapolis, Indiana, USA

Introduction : Flow cytometry is a powerful method to analyse heterogeneous particle populations based on single particle detection and is widely used for cellular analysis. However, the sensitivity of most current conventional cytometers has limitations to detect nanoparticles with sizes below 200 nm, creating a challenge for extracellular vesicle analysis.

Methods : This work aims to demonstrate the performance of the CytoFLEX nano Flow Cytometer as a sensitive label‐free tool to characterize nanoparticles by leverageing its capability to collect and analyse scatter parameters at multiple wavelengths.

Results : In this study, we will demonstrate the instrument's performance using commercially available samples, such as platelet EVs, RBC EVs and liposomes.

Summary/Conclusion : As a part of post‐processing step, we will demonstrate capabilities of calibration techniques for size and refractive index based on NIST control beads with known size and refractive indices and biological controls of murine leukaemia virus (MLV), Herpes simplex virus (HSV), adenovirus, and vaccinia virus.

Nanochannel microscopy for single extracellular vesicle analysis

Viktoria de Carvalho, Elin Persson, Aaron Domenzain Del Castillo Cerecer, Alex Lech, Sriram KK, Daniel Midvedt, Giovanni Volpe, Fredrik Westerlund, Elin Esbjörner

Chalmers University of Technology, Gothenburg, Sweden

Introduction : Extracellular vesicles (EVs) are membrane‐enclosed biological nanoparticles that play crucial roles in cell‐to‐cell communication in both health and disease. A major problem in understanding EV biology relates to their vast heterogeneity in terms of both physical and biochemical properties. To address this problem, single‐particle methods are needed. This project uses and further develops a unique single particle analysis platform, combining nanofluidics with fluorescence microscopy, to shed light on EV heterogeneity.

Methods : The nanofluidic device, with nitrogen inlets allowing for pressure‐driven flow through microchannels and nanochannels, is mounted on an inverted fluorescence microscope. Fluorescent particles diffusing in the nanochannels are recorded with single or multicolour fluorescence detection. Using a machine learning algorithm, particle trajectories are retrieved, from which diffusivity and, based on that, size are determined. The nanochannels, with a cross section of 300 × 300 nm 2 , allow tracking of particles in the focal plane, while avoiding particle trajectories from crossing each other. EVs from engineered HEK293t cells overexpressing CD63 fused to mCherry are extracted from cell culture media and isolated via tangential flow filtration (TFF) and ultrafiltration (UF). We also investigate EVs collected from cell media directly in the nanochannels, avoiding sample preparation biases.

Results : Previous results have demonstrated that our method can distinguish between 50 and 100 nm polystyrene bead populations with high specificity in a mixed sample. Here, we show that EVs isolated with TFF and UF can freely diffuse in the nanochannels, their emission intensity can be recorded, and their hydrodynamic size can be determined. We also show that EVs taken directly from cell media can be analysed in the nanochannels.

Summary/Conclusion : The nanofluidic platform can be used to determine the hydrodynamic size of EVs in solution using fluorescence microscopy and pressure‐driven flow. Subsequent projects will use this technique to further investigate EV populations from engineered HEK293t cells with CD69‐mCherry undergoing different treatments, such as oxidative stress, which may affect EV population characteristics.

Funding : Knut and Alice Wallenberg Foundation (KAW).

Size‐based profiling of single small EVs in silicon nanochannels

Ivy Bhattacharya, Marzia Iarossi, Navneet Chandra Verma, Diana Huttner, Amit Meller

Technion—Israel Institute of Technology, Haifa, Israel

Introduction : Extracellular vesicles (EVs) are increasingly being accepted as circulating biomarkers accessible to liquid biopsies. However, the heterogeneity in the size profile of EVs originating from different sources, even when identified as small EVs (sEVs), and the contrasting dearth of evidence directly correlating EV size to its biophysical characteristics and, in turn, to its diagnostic potential, will likely mandate the development of novel, precise and democratic technologies for EV detection and sizing. Recently, the ability to fabricate sub‐wavelength solid‐state nanochannels and load them with biomolecules of interest has opened new avenues to achieve far more sensitive quantification of both concentration and single‐EV trajectory dynamics over time.

Methods : To fabricate our nanochannel device in‐house, we used a combination of chemical dry etching and anodic bonding. To minimize pre‐processing of an HCT‐116 derived EV sample so that we retain accessibility of the device, we first fluorescently labelled the lipid membrane with DiD using the salt‐change method to avoid conflation of dye aggregates and EVs. To further ensure sample purity, we labelled the particles with a dye‐antibody complex specific to CD63 and assured colocalization of the dyes on the particles identified as EVs on our simple widefield optical setup.

Results : We develop a generic 4‐dimensional single EV analysis method that allows not only highly sensitive EV detection but also size recognition based on single particle tracking of individual EVs during their electromigration. Tracking of the EVs provides rich multi‐dimensional information, which includes their migration velocity profiles and their diffusion velocity profiles, as well as their available lipid surface area. This information is then processed by a neural network trained on particles of known radii to output a most probable radius value for a specific EV, with an accuracy up to ±4 nm.

Summary/Conclusion : We build an experimental and analytical framework that raises the limit of detection of EVs in a sample by several orders of magnitude compared to conventional techniques of EV counting and sizing. Importantly, our method does not rely on specific antibodies for EV sensing and is non‐destructive in the sense that EVs retain their biophysical characteristics and membranal integrity.

Single EV charge characterization using nanochannel fluorescence microscopy

Elin Persson 1 , Viktoria de Carvalho 1 , Aaron Domenzain Del Castillo Cerecer 2 , Alex Lech 2 , Sriram KK 1 , Daniel Midtvedt 2 , Giovanni Volpe 2 , Elin Esbjörner 1 , Fredrik Westerlund 1

1 Chalmers University of Technology, Gothenburg, Sweden; 2 University of Gothenburg, Gothenburg, Sweden

Introduction : Extracellular vesicle (EV) heterogeneity is one of the largest obstacles when characterizing EVs into subpopulations. Traditional subgroups include exosomes and microvesicles, which are based on their origin in the cell. While origin is believed to hold important information on the function and specificity of EVs, there are to date no single characteristics which confidently separate EVs into these subgroups, highlighting the need for better characterization methods. One way to tackle the heterogeneity problem is by multi‐parameter characterization of single EVs. We here present a single EV characterization method, based on a nanofluidic device coupled to fluorescence microscopy, where we characterize single EVs based on hydrodynamic radius, protein content and the often‐overlooked characteristic charge, within the same device and in a high‐throughput fashion.

Methods : Synthetic liposomes with increasing negative charge were used as a model system for charge determination. Particles were loaded on the nanofluidic chip, and their electrophoretic mobility in the applied electric field was measured. Dynamic light scattering was used as a reference measurement of the zeta potential. HEK293T cells were cultured, and EVs were harvested and purified by tangential flow filtration followed by ultrafiltration. Isolated EVs were then labelled with fluorescent antibodies against CD63 and CD9. EVs were loaded on chip, and their charge distribution was investigated via a machine learning‐based particle tracking algorithm.

Results : The charge determination was validated using liposomes with known zeta potential. Particle populations with a mean zeta potential difference of 3 mV could be separated as different populations. Preliminary investigations of CD63‐ and CD9‐positive EVs show that we can distinguish charge variation within EV samples that could be traced to cell origin.

Summary/Conclusion : We present a novel characterization method, based on nanofluidics and fluorescence microscopy, to characterize single EVs in a high throughput manner. Using synthetic liposomes, we could distinguish a difference in zeta potential of 3 mV as two separate populations. The ability to investigate the charge of single EVs could hold great potential for revealing new subgroups of EVs as well as revealing fundamentals of EV biogenesis.

Funding : Knut och Alice Wallenbergs Stiftelse.

A comparative analysis of reproducible EV size and concentration measurement using nanoscale flow cytometry and fluorescent microfluidic resistive pulse sensing

Yohan Kim 1 , Ahmad Sabbah 1,2 , Khaled Elshaer 1,2 , Ngoc Do 3 , Emmalee Northrop‐Albrecht 4 , John B. Kisiel 4 , Jean‐Luc Fraikin 3 , Fabrice Lucien 1,5

1 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 2 College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; 3 Spectradyne LLC, Signall Hill, California, USA; 4 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA; 5 Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA

Introduction : Reproducible enumeration of extracellular vesicles (EVs) is crucial for exploring their biological roles and translating them into clinical applications. Nanoscale flow cytometry (nFCM) and fluorescent microfluidic resistive pulse sensing (F‐MRPS) technologies are single‐vesicle analytical methods with immunophenotyping capabilities. As such, they can serve for orthogonal validation of reproducible EV size estimation, as well as corresponding concentration measurements in specific size ranges. In this study, we conducted a comparative analysis of nFCM and F‐MRPS using NIST‐traceable standard beads and human biological samples.

Methods : Apogee Mix and Spectradyne NIST‐traceable calibration polystyrene and silica beads (180 to 1300 nm), platelet‐poor plasma ( n = 5 patients), cell‐free urine ( n = 5 patients), and stool EV preparations ( n = 4 patients) were analysed by nFCM (Apogee A60 Micro Plus) and MRPS (nCS1) instruments. Exometry and Spectradyne calibration beads, along with Bangs Labouratories fluorescence beads, were used for calibration of nFCM. The impact of wetting agent (Poloxamer‐188, Tween‐20) and pre‐analytical size‐exclusion chromatography (SEC) on reproducible EV quantification by MRPS was also tested. PSMA+ EV spiked plasma was analysed by nFCM and fluorescent F‐MRPS (ARC particle analyser) using fluorescently conjugated PSMA antibody.

Results : Both technologies performed compatible size and concentration measurements of NIST‐traceable beads of mixed sizes (median CV = 7.7%). To make the comparison meaningful, the analysis was restricted to the size range between 180 and 900 nm, which nFCM reliably detects by estimating uniform refractive index for all particles. Next, we observed that both nFCM and MRPS demonstrated excellent replicability (CV = 7.6%–10.7% for MRPS and CV = 4.4%–7.5% for nFCM) in total particle measurements across all biological samples. nFCM and MRPS provided concordant particle concentration measurements in SEC‐processed samples diluted in Poloxamer‐188 wetting agent (CV = 12%, 18%, and 19% for plasma, urine, and stool samples, respectively). Lastly, we observed that similar PSMA+ EV concentrations were measured by nFCM and F‐MRPS from spiked plasma samples with and without SEC sample processing ( n = 3, p = 0.77).

Summary/Conclusion : This study highlights that both nFCM and F‐MRPS can provide reproducible and comparable size determination and quantification of EVs over a common range of measurable particle sizes. With appropriate instrument calibration, these techniques can serve as complementary orthogonal methods for characterizing EVs from various biological sources.

Characterisation of biological nanoparticles using dual‐angle interferometric scattering microscopy (DAISY)

Presenter: Fredrik Eklund

Holtra AB, Göteborg, Sweden

Introduction : Multiple challenges remain in characterizing colloidal particles of low optical contrast in their native liquid environment. One important challenge is to distinguish between different types of biological submicron and nanoparticles in a heterogeneous sample. This can be especially difficult when the particles to be characterized are surrounded by a high concentration of smaller nanoparticles or large molecules.

Methods : We present a method and an instrument which enables powerful particle characterization in a broad size range from nanoparticles to microparticles. The method is based on particle tracking and a combination of holographic and interferometric scattering microscopy, which use two opposing illumination angles.

Results : First, we demonstrate characterization of heterogeneous particle dispersions in the size range 50–500 nm, where hydrodynamic size as well as quantitative optical properties of individual particles are determined. The combination of hydrodynamic and optical parameters enables differentiation of particles based on their refractive index, which, in the case of biological particles, is indicative of their density. Further, for particles in the submicron range, the combination of quantitative optical data from opposing illumination angles enables us to estimate the distribution of mass between the centre and periphery of a particle or to estimate particle size from purely optical data. Second, we demonstrate the possibility of using holographic microscopy in combination with gold nanoparticle labelling to detect specific EV or virus. One advantage is that unlabelled biological particles as well as individual gold nanoparticles below the detection limit will not interfere much with the detection of complexes formed between gold nanoparticles and biological nanoparticles, enabling the use of an excess of labelling particles and also specific detection in minimally diluted biological samples. Compared with molecular based methods, this approach therefore has the potential advantage of better avoiding false positive results by directly detecting and counting intact viruses and EVs. Further, compared to using fluorescent labels, comparatively few binding sites are needed for detection.

Summary/Conclusion : Particle tracking using holographic/interferometric microscopy enables powerful optical characterization of biological submicron‐ and nanoparticles.

A comparison of flow cytometry technologies for extracellular vesicle analysis

Georgina Hopkins 1 , Davis Tucis 1 , William Browne 1 , Stella Cochrane 2 , Victoria James 1 , David Onion 1 , Lucy C. Fairclough 1

1 University of Nottingham, Nottingham, UK; 2 SERS Unilever, Wirral, UK

Introduction : Conventionally, extracellular vesicles (EVs) are isolated and then analysed by nanoparticle tracking analysis (NTA), western blotting, and transmission electron microscopy (TEM). It would be advantageous to have a single technology that can rapidly enumerate, size and phenotype EVs, without the absolute need for purification, to enable higher throughput Laboratory and translational studies. The development of flow cytometers (FCs) with high sensitivity has enabled its application to EV analysis, importantly without the need for prior EV isolation. Yet, with increasing FC technologies available, there is a lack of clarity over the suitability of each technology for EV research. Thus, we examined methods for EV quantification, sizing, and tetraspanin characterisation, using a range of FCs, namely conventional, imaging, and nanoscale.

Methods : Supernatants containing EVs from human bronchial epithelial cells (MucilAir, Epithelix Sárl) were stained with 10 µM calcein‐AM in combination with 2.5 µg/mL of fluorescent‐conjugated anti‐CD9, anti‐CD63, and anti‐CD81 for 1hr at 37°C. The supernatants were then analysed using an ImageStreamX MKII (Amnis), CytoFLEX S (Beckman Coulter), and CytoFLEX nano (Beckman Coulter). Size exclusion chromatography was used to isolate EVs from cell supernatants before performing TEM, super‐resolution microscopy, and NTA.

Results : TEM, NTA, and super‐resolution microscopy confirmed the presence, size, and tetraspanin expression of EVs in MucilAir supernatants before analysing by FC technologies. Conventional FC, using 405 nm side scatter, was inadequate in detecting EVs below 126 nm in size, but could accurately size and quantify tetraspanin expression in larger EVs. Imaging and nanoscale FCs were more sensitive in size detection, reaching 40 nm. Tetraspanins were also successfully quantified on small EVs by imaging and nanoscale FC. However, the imaging FC allows for more fluorophore combinations and thus a larger panel for tetraspanin analysis.

Summary/Conclusion : A comparison of available FC technologies highlights that EVs can be detected using side scatter triggering by conventional and nanoscale flow cytometers, but the sensitivity of nanoscale flow cytometry is greater than conventional FC and similar to that of imaging FC.

Funding : This research was funded through a BBSRC‐Unilever PhD studentship.

An optimized flow cytometry method for single extracellular vesicle subtyping, counting, and isolation

Pasquale Simeone 1,2 , Davide Brocco 3 , Francesca D'Ascanio 1,2 , Domenico De Bellis 1,2 , Giulia Colasante 1,2 , Arianna Aquilini‐Mummolo 1,2 , Rosalba Florio 4 , Anna Piro 4 , Serena Pilato 4 , Maria Concetta Cufaro 1,5 , Alice Di Sebastiano 1,4 , Damiana Pieragostino 1,5 , Michela Battistelli 6 , Laura De Lellis 4 , Piero Del Boccio 1,4 , Serena Veschi 4 , Antonella Fontana 4 , Alessandro Cama 4 , Simone Dinarelli 7 , Massimo Bottini 6

1 Center for Advanced Studies and Technology (CAST), Chieti, Italy; 2 Department of Medicine and Ageing Sciences, Chieti, Italy; 3 Department of Medical, Oral and Biotechnological Sciences, Italy 4 Department of Pharmacy, Chieti, Italy; 5 Department of Innovative Technologies in Medicine and Dentistry, Chieti, Italy; 6 Department of Biomolecular Sciences (DiSB), Urbino, Italy; 7 Institute for structure of Matter (ISM), Italy National Research Council (CNR), Rome, Italy

Introduction : Extracellular vesicles (EVs) are released during different physiological processes and are increasingly thought to be new potential biomarkers and/or promising therapeutic tools. Flow cytometry has great potential as a powerful and solid single vesicle technique to study EVs. However, validated flow cytometry protocols for EV subtyping are lacking. Here we undertook the task of developing an optimized flow cytometry procedure for EV analyses, paralleling different protocols and instruments (conventional, high‐sensitivity, spectral, and nano‐flow platforms, as well as imaging flow cytometers).

Methods : Adhering to current guidelines, a simple, single‐vesicle procedure to identify, classify, enumerate, and separate by fluorescence activated cell sorting EVs from different cell origins has been optimized. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. LCD‐positive EVs isolated from cell supernatants and different biofluids (i.e., peripheral blood and urine) by cell sorting, were phenotypically characterized by flow cytometry and western blot. They were also morphologically analysed by atomic force microscopy (AFM), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The dynamic range and the limit of detection of this method were also calculated, using different flow cytometry platforms.

Results : The application of the newly optimized PFC protocol here described allowed the obtainment of highly purified EV samples, expressing EV markers (CD63 and Flotillin‐1), being negative for cytochrome C and other typical EV preparation contaminants (i.e., apolipoproteins). LCD positive EV characterization revealed that they displayed a globular shape and typical EV sizes. We also obtained repeatable EVs counts for different EV subtypes, while the proteomic analysis of their cargoes revealed that they carry binding proteins and bioactive molecules.

Summary/Conclusion : The use of the optimized protocol here proposed, taking advantage of the LCD staining, allowed EV subtyping, repeatable counts and sorting of EVs from different sample types. This represents an essential step towards the assessment of EV clinical translation.

Funding : PRIN 2022‐20229RMZZW (Italian Ministry of University and Research) “National Center for Gene Therapy and Drugs based on RNA Technology.”

Isolation‐ and bias‐free optofluidic platform for single extracellular vesicle characterization

Presenter: Pascal Rüedi

Institution ETH Zurich, Zurich, Switzerland

Introduction : Understanding how extracellular vesicle (EV) subpopulations relate to specific biological states and functions remains a long‐sought goal. However, EV heterogeneity poses significant challenges for their isolation, detection, and characterization. While single EV characterization techniques have advanced, they rely heavily on the quality of the input samples, making them susceptible to biases introduced during isolation and purification. Here we introduce an optofluidic platform that characterizes EV subpopulations with single particle resolution directly in complex sample matrices without the need for isolation steps, at high‐throughput and in a label‐free manner.

Methods : The optofluidic platform features a PDMS chip with parallelized single‐input, single‐output channels for multiplexing and rapid screening. As a key‐enabler for isolation‐free analysis, we developed a novel rapid immunoaffinity functionalization method (< 5 min of hands‐on time), ensuring surface passivation against non‐specific binding while selectively capturing EV subpopulations based on pan‐EV markers (CD9, CD63, CD81). The surface combines biomimetic membranes, and bio‐orthogonal antibody conjugation compatible with label‐free optical measurements. EVs from lung cancer cells (H358) were profiled, either isolated via size exclusion chromatography or directly from unpurified cell culture supernatant. Imaging was performed with a custom digital in‐line holography microscope, enabling single‐EV sensitivity and continuous monitoring of binding kinetics. The platform requires only 50 µL of sample and a 10‐min assay per channel for high‐throughput analysis.

Results : Following validation with purified EV samples and functionalized liposomes, the platform successfully characterized EVs from unpurified cell culture supernatant while maintaining near‐zero non‐specific binding, even in the presence of high concentrations of interfering molecules from other secretomes and serum, paving the way towards applications with clinical samples. Comparisons between EVs isolated via size exclusion chromatography and those directly from supernatant revealed differences in EV size and composition, demonstrating purification‐induced biases. This approach allows estimation of EV concentrations and compositions directly from large‐scale cell cultures.

Summary/Conclusion : This novel platform enables label‐free, high‐throughput single‐EV analysis without isolation or purification, advancing unbiased EV heterogeneity studies. Applications include real‐time EV secretion monitoring for quality control in therapeutics and profiling EV subpopulations from complex samples for diagnostics or treatment monitoring.

Funding : Swiss National Science Foundation, grant no. 207485.

Integrating interferometric nanoparticle tracking analysis (iNTA) with fluorescence detection for single‐molecule precision in distinguishing nanoparticle sub‐populations

Presenter: Shuhan Jiang

Max Planck Institute for the Science of Light, Erlangen, Germany

Introduction : Accurate characterization of biological particles, especially extracellular vesicles (EVs), is essential for uncovering their roles in various biological functions and diagnostic applications. The heterogeneous nature of EVs complicates the precise measurement of their biophysical and biochemical characteristics.

Methods : We recently introduced interferometric nanoparticle tracking analysis (iNTA) as a label‐free technique for analysing nanoparticles like EVs. Building on this, we present a method that integrates iNTA with single molecule sensitive fluorescence detection, enabling characterization of EVs alongside their biomolecular content, in solution. This approach enables detection of fluorescence labels on the EV surface, including lipid markers and surface proteins tagged with specific antibodies, as well as internal proteins within EVs labelled by carboxyfluorescein succinimidyl ester (CFSE). To prevent fluorescence bleaching, a flow system is used for sample introduction. Our method facilitates the simultaneous measurement of particle size, refractive index, and fluorescence intensity at the single‐particle level.

Results : To demonstrate our method, we used a mixture of 40 nm non‐fluorescent polystyrene beads and 100 nm fluorospheres, effectively distinguishing between nanoparticles with and without fluorescence labels. We further evaluated the fluorescence sensitivity of our setup with liposomes labelled with two lipid dyes, MemBright (488 nm excitation) and R18 (561 nm excitation), and confirmed that fluorescence intensity was proportional to liposome size and dye concentration. Finally, we applied our method to HEK293‐derived EVs, labelling them with anti‐CD9 and anti‐CD81 fluorescent antibodies to quantify the proportion of EVs expressing these biomarkers and labelling internal proteins with CFSE to assess internal molecular content.

Summary/Conclusion : This single‐molecule‐sensitive, multimodal approach enhances the ability to differentiate EV populations within complex samples, offering significant potential for advancing biomarker discovery and detailed analysis of heterogeneous particle systems.

Funding : Max Planck Society.

Particle detection and classification strategies in cryo‐electron microscopy images of purified cancer cell‐derived extracellular vesicles

Presenter: Roman I. I. Koning

Leiden University Medical Center, Leiden, The Netherlands

Introduction : One of the key characteristics of extracellular vesicles (EVs) is the presence of a lipid bilayer. Visualization of the lipid bilayer allows positive identification of EVs, distinguishing EVs from non‐EV particles. Cryo‐electron microscopy (cryo‐EM) is able to directly visualize lipid bilayers. Additionally, cryo‐EM can detect non‐EV particles, such as lipoproteins, lipid droplets and protein aggregates. The use of cryo‐EM is, however, limited by low throughput and complex image analysis. Therefore, we developed a workflow combining automated cryo‐EM image acquisition and supervised machine learning (sML)‐assisted detection of EVs and other particles, enabling reproducible sample imaging and comprehensive particle analysis.

Methods : EVs were purified from MDA‐MB‐231 cells using a combination of centrifugation, ultrafiltration and size exclusion chromatography. Samples were prepared for cryo‐EM by plunge‐freezing and imaged using a cryo‐electron microscope on a direct electron detector. Automated imaging was performed using EPU software, which enabled reproducible acquisition. sML and size measurements were performed using a custom web‐interface, Keras, TensorFlow, FIJI and the Hough circle transform.

Results : Automated cryo‐EM imaging combined with manual particle classification and size analysis of 200 images of purified EVs from MDA‐MB‐231 cells revealed several particle classes with distinct morphologies and sizes. Notably, ∼25% of the particles lacked a lipid bilayer, indicating they were not EVs. Cryo‐EM imaging and sML particle analysis of an additional 2000‐image dataset identified two particle classes, of which less than half were EVs. The remaining particles consisted of small amorphous structures, resembling exomeres. Additional advanced sML modelling enabled automatic detection of the lipid bilayer, as well as lipoprotein particles from blood plasma.

Summary/Conclusion : Automated cryo‐EM imaging, combined with sML particle detection, enables reproducible data collection and objective particle classification, quantification and (size) analysis of EVs. By specifically detecting EVs by their lipid bilayer, it was shown that a significant part of particles present in purified EV samples are non‐bilayer containing contaminants. The combination of cryo‐EM and sML‐assisted detection of both the lipid bilayer and particles in general, classification and analysis provides a powerful approach to explore the prevalence and detailed structures of EVs and contaminants in purified EV samples.

New microfluidic technology allows detection and high‐throughput sorting of single fluorescent extracellular vesicles

M. Couty 1 , K. Say 2 , C. Roux 2 , P. Graindorge 2 , S. Bourzeix 2 , M. Girault 2 , G. Van Niel 1

1 Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers (CRCI 2 NA), Nantes, France 2 HEKAT FLUIDICS, Angers, France

Introduction : The composition and properties of extracellular vesicles (EV) can reflect the physiopathological state of the producing cell. Once released in biological fluids, they could be used as non‐invasive biomarkers for diagnostics, prognostics or therapeutic follow‐up in several diseases. However, the diagnostic power of EVs may be hampered by the scarcity of the informative EV sub‐population. Here with Hekat, we assessed a combination of a highly sensitive optical detection system and an advanced lab on a chip device to easily detect and sort nano‐sized biological objects at high speed and meet this needle‐in‐a‐haystack challenge.

Methods : From HeLa cells expressing CD63‐mRFP or mNeonGreen lines, we have isolated EV by size exclusion chromatography (SEC) and characterized by combining transmission electron microscopy (TEM), biochemistry and size/concentration measurement approaches with various devices. EVs were then detected and sorted by the Nano‐Sorter and re‐analysed post‐sorting using similar approaches.

Results : After isolation by SEC of distinct subpopulations of CD63‐mRFP and CD63‐mNeonGreen EV, we characterized EV lots and confirmed the presence of the fluorescent reporters by biochemistry and electron microscopy. Comparison with different commercially available devices revealed that the Hekat sorting technology detects fluorescent EV in a sensitive and reproducible manner. Using single and mixed populations of CD63‐mRFP and CD63‐mNeonGreen EV, we confirmed the ability of this device to sort and collect relevant amounts of EV based on endogenous fluorescence without affecting their integrity.

Summary/Conclusion : We report here a new approach for the detection and sorting of small EVs based on their endogenous fluorescence, at a single EV level and at high speed. Using the Hekat instrument, we can detect and sort small EVs in a scalable and reproducible manner without affecting their integrity. Our ongoing study aims at refining the use of this new instrument in more complex fluids that would allow us to adapt our strategy in a clinical context in vivo to improve the use of EV as biomarkers.

Funding : This project was funded by: Hekat Fluidics and the ANRT Cifre programme (for Mickael Couty); Next Connect Talent Pays de la Loire (for Guillaume Van Niel).

Sample concentration and background signals impact single EV analysis by high‐sensitivity flow cytometry: a comparison across three different generation platforms

Estefanía Lozano‐Andrés 1 , Ye Tian 2 , Sten F. W. M. Libregts 1 , An Hendrix 3,4 , Xiaomei Yan 2 , Ger. J. A. Arkesteijn 1 , Marca H. M. Wauben 1

1 Utrecht University, Utrecht, The Netherlands; 2 Xiamen University, Xiamen, People's Republic of China; 3 Ghent University, Ghent, Belgium; 4. Cancer Research Institute Ghent, Ghent, Belgium

Introduction : High‐sensitivity flow cytometry has evolved in the last decade to accommodate the need for single EV analysis. This resulted in a variety of instruments from different generations: from analogue high‐end instruments that are tailored for EV analysis to those used for both cell and EV measurements to instruments built for small particle analysis. Hence, it is important to evaluate how results acquired with distinct instruments compare. For this purpose, we used nanoparticles (NPs) and fluorescent biological recombinant EVs (rEVs) to investigate the performance of these instruments.

Methods : We measured a range of different concentrations of non‐fluorescent silica NPs (siNP, sizes from 50 to 200 nm) by using light scatter thresholds and rEVs (average size 120 nm) by using both light scatter and fluorescence thresholds on the three different platforms, that is, a N30 nanoflow analyser (NF), an optimized BD Influx (IF) and a CytoFLEX LX (CF).

Results : Using light scatter detection, we found significant differences among instruments in relation to background signals, optimal sample concentrations and limits of detection. Only the NF was able to detect the smallest siNP (68 nm). The IF provided reliable rw‐FSC information from the 91 nm siNPs with the least background interference, while the CF detected the 91 nm siNPs based on violet‐SSC with greater background interference. Based on light‐scattering (SSC and FSC), the rEVs overlapped with the background on all instruments. Only on the NF was the rEV fluorescence sufficient to visualize a separate population, while the IF and CF showed a continuous fluorescent population on top of the background.

Summary/Conclusion : We found that with the light scatter threshold, the combination of background and low sample concentrations compromises the ability to discriminate particles of interest. The NF requires a higher sample concentration compared to the IF and CF. In addition, the CF is more affected by background interference and swarm detection compared to the IF. Furthermore, our results showed that rEVs can be used to define the optimal concentration range of a particular instrument, thereby ensuring reliable data interpretation. Therefore, the application of rEVs is useful when complex biological samples are compared by flow cytometric analysis.

Evaluation of methods for removing free fluorescent antibodies to optimize the characterization of EV surface proteins

Presenter: Tianyang Wu

Shanghai Jiao Tong University, Shanghai, People's Republic of China

Introduction : EV surface proteins can serve as biomarkers indicative of the physiological or pathological conditions of the parent cells. The characterization of EV surface proteins can be accomplished at single particle level with nanoflow cytometry (nanoFCM). Following co‐incubation with fluorescent antibodies, EVs are routinely ‘washed’ to mitigate the interference of free antibodies before nanoFCM analysis. Such ‘washing’ methods involve diluting the EV‐antibody mixture in phosphate buffered saline (PBS) and subsequent re‐extraction of EVs via ultracentrifugation (UC), size exclusion chromatography (SEC), or Exodisc, and so forth. However, there is currently a lack of comprehensive evaluation of the methods to remove free antibodies in terms of EV recovery rate and “washing” efficiency.

Methods : EVs are isolated from culture media and human urine samples with UC, SEC, or Exodisc. The isolated EVs are co‐incubated with anti‐human CD9, CD63, and CD81 fluorescent antibodies, respectively. Each mixture is subsequently diluted in PBS and re‐extracted via UC, SEC, or Exodisc. The re‐extracted EVs are subjected to nanoFCM analysis to evaluate particle counts and percentages of fluorescent particles.

Results : Regardless of EV source and isolation method, the “washing” procedure involving SEC and Exodisc yielded comparable particle counts and percentages of fluorescent particles. However, the “washing” method employing UC produced significantly lower particle counts and fluorescent rates compared to SEC and Exodisc.

Summary/Conclusion : Removing free fluorescent antibodies facilitates more accurate characterization of EV surface proteins. In terms of particle recovery rate and percentage of fluorescent particles, SEC and Exodisc demonstrate potential advantages compared to ultracentrifugation.

Funding : This work was financially supported by the National Natural Science Foundation of China (82103485, 82227801).

Advancements in MESF beads for enhanced fluorescence quantification in flow cytometry

Presenter: Boi Hoa San

Beckman Coulter Life Sciences, Indianapolis, Indiana, USA

Introduction : The quantification of fluorescence intensity in flow cytometry is important for instrument sensitivity characterization and standardization of results. One of the key tools in this regard is Molecules of Equivalent Soluble Fluorochrome (MESF) beads. These beads are essential for standardizing fluorescence measurements and ensuring consistency across different experiments and instruments.

Methods : We present here our ongoing research and latest advancements in the study of MESF beads. By leverageing new chemistry to incorporate a variety of fluorescent dyes (e.g., Pacific Blue*, SuperNova polymer dyes, FITC, APC, PE), we aim to provide a reliable method for quantifying the number of dye molecules per bead. *Pacific Blue is a registered trademark of Thermo Fisher Scientific Inc.

Results : The nano‐sized MESF beads have low fluorescence background and a low known amount of incorporated dye molecules, thus making them particularly suitable for small particle flow cytometry applications. Preliminary evaluation using the CytoFLEX nano flow cytometer has confirmed the potential utility of the new nano‐sized MESF beads.

Summary/Conclusion : Once fully developed, these beads are anticipated to become an essential tool for MESF characterization and for standardizing instruments and assays in the growing field of small particle flow cytometry.

High‐resolution characterization of HIV and extracellular vesicles by STORM‐FISHing: combining dSTORM and smFISH

Presenter: Aiden M. Jurcenko

Johns Hopkins University, Baltimore, Maryland, USA

Introduction : Super‐resolution microscopy (SRM) examines subcellular structures at high resolution and can be used to probe the surface and internal features of enveloped particles, including the human immunodeficiency virus (HIV) and extracellular vesicles (EVs). Potentially, SRM can be used to distinguish fully formed, infectious virions of viruses like HIV from non‐viral EVs that may carry a subset of viral proteins or other components, and to monitor the topology of viral markers in and on EPs.

Methods : We initially searched the literature to investigate how SRM techniques, such as dSTORM and STED, have been used to study HIV components (i.e., envelope glycoproteins or group‐specific antigen (Gag) proteins) in virion structure and assembly. We then applied dSTORM imaging to examine extracellular particles released from PM1 cells, either uninfected or infected with HIV‐1 (BaL and MN strains) and concentrated by differential ultracentrifugation. Particles were captured by antibodies against CD81 and detected with fluorescent antibodies against CD63 and gp120 in combination with anti‐HIV smFISH probes (Bal or MN).

Results : HIV‐infected cells release particles ranging from host EVs without detectable gp120 or viral RNA to fully infectious virions. Similarly, both virions and host EVs can display tetraspanins. Additionally, some particles had an unexpected topology of viral RNA: surface vs internal.

Summary/Conclusion : Our results suggest that a combination of multiple labelling techniques, particularly smFISH for RNA and fluorescent antibodies to viral proteins, may be needed to distinguish HIV virions from virus‐like particles/host EVs. Our results also emphasize that tetraspanin capture cannot easily be used to separate host EVs from viruses. Additional SRM studies are needed to examine other features that are common to or distinguish virions and host EVs.

Electrochemical biosensor for extracellular vesicle biomarker quantification with very low limits of detection

Presenter: Maria Carmen Blanco‐López

University of Oviedo, Oviedo, Spain

Introduction : Extracellular vesicles (EVs) are a source of diagnostic, prognostic, and prognostic biomarkers for several diseases. However, the methods for their isolation and subsequent characterization have not yet been standardized for clinical uses [1]. Moreover, sensitive quantification of EV and EV biomarkers is still a technological challenge. The actual techniques do not allow a full characterization due to the heterogeneity of the samples and complexity of the biological matrix. Additionally, most of these techniques require bulky and expensive instruments, and skilled operators. There is therefore an increasing need for simple methods and devices for quantification and characterization of EV biomarkers with good sensitivity and user‐friendly.

Methods : We have developed a biosensor based on the coupling of a lateral Flow immunoassay (LFIA) for EV biomarkers. By using anti‐CD9 as a capture antibody and anti‐CD63 as detection, and using only 1 mL of isolated EVs, a limit of detection of 3.19 × 10 4 EV/µL by recording the fluorescent signal of Eu NPs. On the other hand, the LFIA was coupled to screen‐printed electrodes with two methods: one of them was based on silver enhancement over gold nanoparticles, and the other one by using gold‐starch nanocomposites doped with an electrochemical probe [2].

Results : The limits of detection achieved were 26 and 39 EV/µL. These biosensors were used directly in the plasma of colorectal cancer patients for quantification of EV biomarkers.

Summary/Conclusion : Up to the authors knowledge, these are the lowest limits of detection for EV quantification and EV biomarkers characterization with these types of devices.

Funding : This work was funded by the Ministerio de Ciencia e Innovación (PID2020‐119087RB‐I00). Clara Saweres‐Argüelles acknowledges her grant FPU22/00762 from the Ministry of Science, Innovation and Universities.

Tradeoffs for rapid NTA measurements

Presenter: Julie Y. Chen

HORIBA Scientific, Piscataway, New Jersey, USA

Introduction : Nanoparticle tracking analysis (NTA) is a high‐resolution technique for analysing extracellular particle (EP) size and concentration. Unlike dynamic light scattering (DLS), which offers bulk measurements, NTA tracks individual particle movements, making it ideal for polydisperse samples like extracellular vesicles (EVs). However, longer recording times are often required to capture sufficient data before particles leave the field of view. This study examines how reduced measurement durations impact the trade‐off between data acquisition speed and result precision.

Methods : To assess the impact of shortened NTA measurements, we systematically varied the measurement duration by adjusting the number of videos and frames using NIST traceable standards (100, 150, and 400 nm polystyrene latex standards and with an EV sample). Each standard was diluted 400,000×, 100,000×, and 10,000× with 1 mM sodium dodecyl sulphate (SDS) solution. EV was diluted with phosphate‐buffered saline (PBS). Five replicates of each sample were measured per setting listed in Table 1. Video duration and number of frames were reduced incrementally to examine the threshold at which measurement precision and repeatability begin to degrade. Multiple lasers with individually adjusted laser power settings and camera gains remain constant. The particle size distributions (Dn10, Dn50, and Dn90) were compared with the supplied Certificate of Analysis, and cross‐verified with TEM; number particle concentrations were compared to fraction (1%) solids.

Results : Our results reveal that shorter measurement durations can substantially decrease the accuracy of particle size distribution data. As video length decreases, under‐sampling becomes more likely, causing increased variability and decreased precision in the measured size distributions. We observed that particle count and size distribution stabilizations improve with longer measurement durations, although diminishing returns occur beyond certain thresholds. Additionally, shorter videos also led to discrepancies in data reliability, with size distributions deviating from those obtained using longer measurement durations.

Summary/Conclusion : In summary, while reducing measurement duration in NTA can expedite analysis time, it compromises accuracy. Our findings indicate that shorter measurement times increase uncertainties in results, highlighting the necessity for carefully optimized NTA protocols that balance experimental speed with accuracy. This trade‐off analysis provides valuable guidelines for researchers looking to implement rapid NTA without compromising the integrity of particle size measurements.

Disease and therapy

Subtopic Biomarker 1

Three‐dimensional (3D) cultured umbilical cord MSC‐derived extracellular vesicles (EV) mitigate kidney damage in chronic kidney disease (CKD) models

Presenter: Naufalia Faza

Stem Cell and Cancer Institute, Kalbe Farma, Jakarta, Indonesia

Introduction : Chronic Kidney Disease (CKD) is a progressive degenerative disorder characterized by inflammation, fibrosis, and irreversible nephron loss, ultimately leading to renal failure. Current treatments are primarily centred on symptoms but ultimately fail to halt the disease progression. Recent advances in regenerative medicines have highlighted the potential of extracellular vesicles (EVs) derived from umbilical cord‐derived mesenchymal stem cells (UCMSCs) as a cell‐free therapeutic option due to their immunomodulatory and regenerative properties as an alternative to current limited treatments like dialysis and kidney transplantation. This study evaluates the potential of three‐dimensional (3D) cultured UCMSC‐derived EVs in two CKD models with distinct aetiologies: diabetic nephropathy (DN) and 5/6 subtotal nephrectomy (SN), a model of severe kidney injury.

Methods : EVs were isolated using an ultrafiltration system from UCMSCs cultured in a 3D bioreactor system under hypoxic conditions. Size and distribution profile were analysed using dynamic light scattering (DLS) and nano tracking analysis (NTA). Western Blot was used for analysing EV protein markers (CD9, CD63, CD81, TSG101). We evaluate in vitro potency of EV by quantitative measurement of pro‐inflammatory cytokines, qPCR analysis of fibrosis marker expression in TGF‐beta‐induced HK‐2 cells, and BrdU assay in oxidative stress induced HK‐2 cells. We also evaluate the in vivo potency of EV on the DN and SN rat models by albumin excretion, creatinine level, and histopathology analysis of fibrosis, glomerulosclerosis, and tubular injury.

Results : The isolated EV, with peak sizes between 138 and 183 nm, expressed cytosolic and transmembrane exosome markers (CD9, CD63, CD81, and TSG101). Functional in vitro assays demonstrated EVs were capable of reducing pro‐inflammatory cytokines (TNF‐α, IL‐6), suppressed fibrosis marker (fibronectin) expression, and enhanced HK‐2 cell proliferation post‐injury. In vivo, EV administration significantly lowered urine albumin excretion in both DN and SN models, suggesting its capability in preserving kidney function. Histopathological analysis post‐sacrifice also revealed reduced glomerulosclerosis, tubular injury, and fibrotic areas, particularly in the DN animal model.

Summary/Conclusion : These findings suggest that UCMSC‐derived EVs provide broad renal protection across different CKD pathologies, supporting their potential as a cell‐free therapeutic strategy for progressive kidney disease.

Funding : This research is funded by PT Kalbe Farma and the Indonesia Endowment Fund for Education Agency.

Subtopic Biologic activity and therapeutic potency of EVs 1

Deletion of the tissue factor‐coding F3 gene ameliorates thrombogenic adverse effects of extracellular vesicles in human and rat blood

Johannes Oesterreicher 1,2 , Magdalena Metzger 1 , Sergiu Dumitrescu 1 , Sivun Dmitry 3 , Peter Dungel 1 , Regina Grillari 4 , David Hercher 1 , Johannes Grillari 1,4 , Jaroslaw Jacak 1,3 , Marcin Osuchowski 1 , Wolfgang Holnthoner 1 , Johannes Zipperle 1

1 Ludwig Boltzmann Institute for Traumatology, Vienna, Austria; 2 BOKU University, Vienna, Austria; 3 University of Applied Sciences Upper Austria, Wels, Austria; 4 Evercyte GmbH, Vienna, Austria

Introduction : The potential role of EVs as therapeutics has attracted major attention in the scientific community. Yet, application of EVs, particularly from a mesenchymal origin, may produce deleterious side effects such as activation of coagulation via tissue factor (TF). Possible lethal thrombotic events are currently disregarded in pre‐clinical therapeutic studies using EVs. This project aimed to generate a cell line that does not pose the risk of thrombogenicity when used to generate therapeutic EVs.

Methods : We generated a TF knockout (TF KO) immortalized adipose‐derived stromal cell line using CRISPR/Cas9. Single‐cell clones were screened for loss of gene expression using RT‐PCR, western blot, and flow cytometry. EVs were enriched using tangential flow filtration and analysed via nanoparticle tracking analysis, atomic force microscopy, bead‐based flow cytometry, and dot blot. An inflammation assay using RAW 264.7 cells assessed potential changes in TF KO EV potency. Procoagulant properties of EV preparations were tested via whole blood thromboelastometry.

Results : Expression analysis confirmed the successful TF knockout. Flow cytometry revealed no difference in lineage‐specific markers. The inflammation assay resulted in no altered immune modulation capacity of generated EVs. Thromboelastometric analysis revealed a significant reduction of clotting time (human: by 39%, rat: by 59%) and clot formation time (human: by 42%, rat: by 62%) for the native EV preparations; this thrombogenic effect was abrogated by the TF‐blocking antibody (HTF‐1). The analysis of TF KO‐derived EVs showed no significant modulation of the respective coagulation parameters.

Summary/Conclusion : Our study shows the successful generation of a TF KO cell line for EV generation that diminishes previously reported possible lethal side effects. This work contributes to the development of safer therapeutic applications of EVs in pre‐ and clinical practice.

Small extracellular vesicle‐driven ferroptosis: a novel strategy to target therapy‐resistant ovarian cancer

Carmen Alarcón‐Veleiro 1 , Rocío Mato‐Basalo 1 , Sergio Lucio‐Gallego 1 , Lama Berjawi 1 , María Quindos Varela 2 , Rocío Lesta‐Mellid 2 , Isabel Santamarina‐Caínzos 2 , Sabela Varela‐Rodríguez 2 , María Fraga 3 , Marcos Quintela‐Vázquez 4 , Ángel Vizoso‐Vázquez 5 , Irene Martínez‐Martín 2 , María C. Arufe 1 , Juan A. Fafián‐Laboura 1

1 Therapy Cellular & Medicine Regenerative Group, University of A Coruña (UDC), Interdisciplinary Center for Chemistry and Biology (CICA), INIBIC—University Hospital Complex A Coruña (CHUAC), A Coruña, A Coruña, Spain; 2 Translational Cancer Research Group, A Coruña Biomedical Research Institute (INIBIC), A Coruña, Spain; Complexo Hospitalario Universitario de A Coruña (CHUAC), A Coruña, Spain; 3 Department of Anatomical Pathology, University Hospital Complex A Coruña, A Coruña, Spain; 4 European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, UK; 5 Grupo EXPRELA, Departamento de Bioloxía, Facultade de Ciencias INIBIC‐Complejo Hospitalario Universitario A Coruña (CHUAC), Centro Interdisciplinar de Química y Biología (CICA), A Coruña, Spain

Introduction : Epithelial ovarian cancer (EOC) is the deadliest gynaecological cancer, largely due to the difficulty of early diagnosis and the eventual development of resistance to treatment. Ovarian cancer cells (OVCA) exhibit altered iron metabolism, making them susceptible to ferroptosis—a form of non‐apoptotic cell death triggered by high levels of free iron. Small extracellular vesicles (sEV) play a key role in tumour communication, potentially influencing ferroptotic signalling in the tumour microenvironment (TME). However, the ability of ferroptotic OVCA‐derived EV to propagate ferroptosis in a paracrine manner remains unexplored.

Methods : To investigate ferroptosis propagation via sEV, OVCA were treated with ferroptotic modulators, and the ferroptosis phenotype was confirmed by assessing key ferroptosis markers. sEV were isolated from ferroptotic OVCA, characterized, and used to treat naïve OVCA to evaluate their ability to induce ferroptosis. Furthermore, sEV from these newly ferroptotic OVCA were collected and applied to another round of naïve OVCA to determine whether the process could be propagated further.

Results : sEV derived from ferroptotic OVCA successfully triggered ferroptosis in non‐ferroptotic OVCA. Additionally, the secondary ferroptotic OVCA released sEV that continued to propagate ferroptosis. Notably, these sEV exhibited increased Fe 2 ⁺ levels, suggesting a key role in the transmission of ferroptotic signalling.

Summary/Conclusion : This study provides evidence that ferroptotic OVCA‐derived sEV can mediate the paracrine propagation of ferroptosis, potentially offering a novel therapeutic strategy for overcoming treatment‐resistant ovarian cancer. Future research will focus on elucidating the molecular mechanisms underlying this process to explore its potential in clinical applications.

Funding : This work was supported by the Xunta de Galicia (grant number ED481D‐2021‐020), the Ministerio de Ciencia e Innovación (RYC2021‐032567‐I), funded by MCIN/AEI/10.13039/501100011033 and by the European Union «NextGenerationEU»/PRTR; the InTalent program from UDC‐Inditex for the research grant and Project PI23/01347, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union; Project PI20/00497, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. Xunta de Galicia (ED431F 2023/30). Transference Project financed FINIBIC (RESISFERRO and ENDOPROT).

Impact of genetic modification of microRNA cargo in human induced pluripotent stem cell‐derived extracellular vesicles on their pro‐regenerative activity in ischaemic tissues

Presenter: Elżbieta Karnas

Jagiellonian University, Kraków, Poland

Introduction : Growing evidence indicates stem cell (SC)‐derived extracellular vesicles (SC‐EVs) carrying bioactive molecules participate in the repair of damaged ischaemic tissue including infarcted myocardium. Cardiac fibroblasts (CFs) and endothelial cells (ECs) represent the main cellular populations responsible for scar formation and angiogenesis in the damaged tissues, and their functions may be modulated by miRNAs carried by injected EVs. However, the effects of specific miRNAs on the pro‐regenerative activity of SC‐EVs remain poorly understood. The objective of this study was to examine the regenerative abilities of induced pluripotent stem cell (iPSC)‐derived EVs (iPSC‐EVs) enriched in selected miRNAs, including miR‐1, miR‐199a or miR‐126, and their impact on ischaemic tissue repair.

Methods : EVs were harvested from genetically modified human iPSCs overexpressing miR‐1, miR‐199a or miR‐126 via sequential centrifugation, were next characterized following ISEV guidelines confirming their identity and examined for global miRNA and protein cargo. Multiple functional assays on target human cardiac cells were performed in vitro to examine the effects of iPSC‐EVs on their biological properties, while a mouse model of limb ischaemia was used to examine their pro‐regenerative potential in ischaemic tissues in vivo.

Results : We found that iPSC‐EVs enriched in miR‐199a or miR‐126 triggered cardiomyogenic and angiogenic gene expression in CFs and ECs, respectively, while miR‐1‐enriched iPSC‐EVs reduced apoptosis and profibrotic gene expression in CFs. Importantly, we found a new role of miR‐199a delivered by iPSC‐EVs into CFs in triggering the expression of several cardiomyogenic genes and ion channels involved in heart contractility. Notably, by transferring miR‐126 and triggering proangiogenic gene expression in ECs, miR‐126‐enriched EVs increased angiogenesis and tissue perfusion both in vitro and in vivo.

Summary/Conclusion : Genetically modified iPSC‐EVs carrying selected miRNAs such as miR‐1 and miR‐199a reduce apoptosis and pro‐fibrotic properties of CFs, while transferring miR‐126 enhances angiogenic capacity of ECs. These results indicate that engineered iPSC‐EVs may be used as new tools for effective and safe repair of ischaemic tissues.

Funding : This work was funded by the MAESTRO 11 (NCN; 2019/34/A/NZ3/00134) to EZS.

The impact of bovine milk extracellular vesicles on the gut: insights from human intestinal epithelial organoid models

Presenter: Yunyue Zhang

King's College London, London, UK

Introduction : Bovine milk‐derived extracellular vesicles (EVs) have demonstrated the capability to transport across the intestinal epithelium and show anti‐inflammatory properties. This makes them promising therapeutic agents for gut diseases, including inflammatory bowel disease (IBD). However, conventional in vitro intestinal models, such as Caco‐2 monolayers, fail to capture the complexity of the human intestinal environment. In this study, human intestinal epithelial organoids, which recapitulate the physiology, genetic signature, and multicellular nature of the native intestinal epithelium, as well as macrophages, with or without induction of inflammation, were used to study the effects of milk EVs. The interaction of bovine milk EVs with these models was investigated, together with how pasteurization affects the biological activity of milk EVs.

Methods : EVs were isolated from raw and pasteurized milk using ultracentrifugation combined with size‐exclusion chromatography, incorporating a chymosin‐incubation step to remove casein micelles and enhance vesicle purity. The intestinal permeability of raw and pasteurized milk EVs was compared using monolayer‐based human intestinal epithelial organoid models, which were, unconventionally, cultured with an exposed apical surface on transwells. In addition, the anti‐inflammatory effect of milk EVs was assessed in stimulated macrophages and inflamed 3D intestinal epithelial organoid models.

Results : Milk EVs isolated from raw and pasteurized milk exhibited high purity (2.30 × 10 10 particles/µg) with a size range of 100–130 nm. The human intestinal epithelial organoid monolayers were developed within 8 days of culturing, with intact tight junctions and differentiation into various cell types. Raw milk EVs demonstrated higher transport efficiency and cellular uptake in monolayer‐based intestinal epithelial models compared to pasteurized milk EVs. In stimulated macrophages, both raw and pasteurized milk EVs significantly reduced TNF‐α secretion after 72 h of inflammation induction. Similarly, both types of milk EVs alleviate inflammation in inflamed 3D intestinal epithelial organoid models by downregulating TNF‐α expression.

Summary/Conclusion : Both raw and pasteurized milk EVs demonstrate intestinal permeability and anti‐inflammatory activity, highlighting their potential in intestinal diseases. Human intestinal organoids serve as a highly biologically relevant model for studying the impact of milk EVs on the intestine.

Exploring the role of plant‐derived nanovesicles in regulating mitochondrial activity during skin inflammation

Presenter: Gabriele Kulkoviene

Lithuanian University of Health Sciences, Kaunas, Lithuania

Introduction : Mitochondrial function is increasingly recognised as a key player in the development of inflammatory skin diseases. Plant‐derived nanovesicles (PDNVs), known for their anti‐inflammatory potential, represent an emerging approach for modulating cellular responses. While PDNVs have shown promise in therapeutic applications, their effects on mitochondrial bioenergetics in inflamed skin cells remain underexplored.

Methods : Human dermal fibroblasts (BJ‐5ta) were treated with IL‐17, IL‐22, and TNF‐α to model chronic inflammation. PDNVs were isolated via polymer‐based precipitation and characterised through nanoparticle tracking analysis. Cellular viability and metabolic activity were assessed using Presto Blue, while mitochondrial respiration was analysed via Seahorse Agilent technology. Fluorescence microscopy and ImageJ software were employed to evaluate mitochondrial morphology, and inflammatory mediators were quantified using Luminex xMAP.

Results : PDNVs demonstrated a restorative effect on mitochondrial function, reversing inflammation‐induced impairments in oxidative respiration while reducing excessive glycolytic activity. Furthermore, PDNVs mitigated inflammation‐driven mitochondrial fragmentation, fostering a more interconnected mitochondrial network. Notably, they also significantly lowered the levels of key inflammatory mediators, including CXCL10, IL‐18, and IL‐4.

Summary/Conclusion : These findings highlight PDNVs as potential modulators of mitochondrial dynamics in inflammatory skin conditions such as psoriasis and atopic dermatitis. PDNVs may contribute to novel therapeutic strategies for chronic skin inflammation by improving mitochondrial structure and function. Future research should further elucidate the role of mitochondrial regulation in inflammatory disease progression.

Funding : This project has received funding from the Research Council of Lithuania (LMTLT), agreement No: S‐A‐UEI‐23‐7.

EVs secreted by pro‐oncogenic and anti‐tumour neutrophils modulate the angiogenesis process

Mateusz Smolarz 1,2 , Weronika Jurczyk 1 , Monika Pietrowska 2 , Marta Gawin 2 , Daria Kania 2 , Jadwiga Jablonska 3

1 Laboratory of Molecular Biology, Institute of Physiotherapy and Health Sciences, Academy of Physical Education, Poznań, Poland; 2 Maria Skłodowska‐Curie National Research Institute of Oncology, Gliwice Branch, Gliwice, Poland; 3 Department of Otorhinolaryngology, University Hospital Essen, University Duisburg‐Essen, Essen, Germany

Introduction : Neutrophils constitute 50%–70% of the white blood cells in the human body. It is now known that, as in the case of macrophages, neutrophils with N1 and N2 phenotypes can be distinguished. An important role in modulating the neutrophil phenotype is played by the interferon receptor, which is activated by interferon type 1. IFN‐based therapy has been shown to promote polarization of tumour‐associated neutrophils (TANs) towards the N1 phenotype. While interferon receptor silencing promotes neutrophil development towards an N2 phenotype. N2 TANs promote, directly or indirectly, tumour growth, as well as cancer cell spread by secreting ECM remodelling enzymes and proangiogenic factors that promote metastasis and angiogenesis. However, it is currently unclear how neutrophils communicate with the tumour and whether EVs are involved in this communication. Therefore, our objective was to characterise EVs secreted by pro‐oncogenic and anti‐tumour neutrophils and to compare their ability to modulate angiogenesis.

Methods : We isolated EVs using SEC. We assessed the size, concentration, morphology, and presence of typical EV markers using ZetaView, TEM, and Western Blot. Furthermore, we determined the proteome composition of neutrophils and EVs secreted by them using the SILAC technique. We verified the influence of EVs on the angiogenesis process using the aortic ring assay (permission #51/2021, Katowice, Poland) and the assessment of endothelial cell migration and proliferation.

Results : Morphology analysis using TEM and ZetaView showed the size of EVs in the range of 80‐150 nm. Furthermore, we confirmed the presence of proteins such as CD81, CD63, TSG101, CD9, and Ly‐6G in the obtained EVs samples. Proteomic analysis revealed more than 5‐fold higher levels of PTK2B, HSPH1, SLC39A8, and VPS13C proteins in N2 TAN than in N1 TAN. A similar pattern of change was observed in isolated EVs. N2 TAN‐secreted EVs also caused increased cell angiogenesis in the aortic ring assay, increased endothelial cell migration, and proliferation.

Summary/Conclusion : Knockdown of the interferon receptor in neutrophils causes changes in the levels of proteins present in EVs. The result is an increase in angiogenesis, migration and proliferation of endothelial cells stimulated by EVs secreted by N2 TAN.

Funding : The research was financed by the National Science Centre, grant no. 2023/48/C/NZ5/00001.

Comparative proteomic analyses of 2D and spheroid U87 glioblastoma‐derived extracellular vesicles in response to temozolomide: potential protein biomarker insights

Presenter: Getulio P. Oliveira

Northeastern University/Catholic University of Brasilia, Brasília, Brazil

Introduction : Glioblastoma (GBM) is the most aggressive and common primary brain tumour, with a poor prognosis and limited treatment options. The standard of care includes surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, though resistance to TMZ remains a challenge. Blood‐based biomarkers could improve diagnosis and monitoring, and extracellular vesicles (EVs) have emerged as potential candidates due to their role in tumour communication. U87 glioblastoma spheroids provide a relevant model for studying tumour behaviour and EV‐mediated biomarker discovery.

Methods : U87 cells were cultured in 2D monolayers and 3D spheroids, treated with 100 or 200 µM TMZ, and EVs were isolated from conditioned media. Proteomic analysis of cells and EVs was performed using mass spectrometry (nLC‐ESI‐MS/MS). EV size and concentration were assessed using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Apoptosis was evaluated via Annexin V/PI staining, and protein markers were analysed by flow cytometry and Western blotting.

Results : Annexin V/PI staining confirmed a dose‐dependent increase in cell death in 2D cultures, while necrosis was the dominant effect in 3D spheroids. EVs isolated from 3D cultures were significantly smaller and more abundant than those from 2D cultures, as observed in NTA and TEM analysis. Flow cytometry showed enrichment of CD9, CD63, and CD81 tetraspanins in EVs. Proteomic analysis revealed distinct profiles between 2D and 3D cells and their EVs, with TMZ treatment altering protein expression patterns. The 3D model exhibited higher expression of proteins related to stress responses and tumour progression, whereas the 2D model showed greater sensitivity to TMZ‐induced proteomic changes. Notably, NCAM1, CYBRD1, and RPL7A were identified in EVs and correlated with glioblastoma prognosis based on TCGA data.

Summary/Conclusion : Our study highlights the potential of EVs as biomarkers for glioblastoma. Differences in EV release and proteomic profiles between 2D and 3D cultures suggest that 3D models better reflect tumour behaviour. The identification of NCAM1, CYBRD1, and RPL7A as prognostic markers supports the role of EV‐based liquid biopsies in glioblastoma monitoring and personalized therapy. Future research should focus on refining EV isolation and detection methods to enhance their clinical utility.

Funding : This study was supported by UG3/UH3‐01063730 (I.G.), RO1‐01063166 (I.G.), R01CA218500 (A.R.I. and I.G.), and R35GM136421 (A.R.I.).

Combinational therapy for the treatment of ischaemic stroke

Haroon Khan 1 , Jiannan Xiao 1 , Ningqiang Gong 2 , Zhaoting Li 1 , Chuancheng Ren 1

1 The Chinese University of Hong Kong Shenzhen, Shenzhen, People's Republic of China; 2 University of Science and Technology, Hefei, People's Republic of China

Introduction : Ischaemic stroke is the leading cause of death worldwide, caused by blockage of cerebral blood flow, leading to neuronal death and disability. Fibrinolytic S100A10‐EVs, along with plasmin resistant D‐tat‐EE3 could be delivered locally by encapsulating them in thermosensitive Pluronic F127 hydrogel to mitigate both primary and secondary damage mechanisms of stroke.

Methods : S100A10 lentivirus transfected MSC‐derived EVs are isolated and characterized. Plasmin generation, clot lysis and thrombolytic assays are performed to evaluate the fibrinolytic efficiency of S100A10‐EV. Cellular uptake and in vitro ischaemic stroke mitigation efficiency are evaluated for the EVs and peptide. For animal studies, the transient middle cerebral artery occlusion model is used, and the results were confirmed through Nissl staining to examine the infarct size, and neurobehavioural tests are performed to evaluate recovery in rodents. Laser speckle imaging and immunostainings are used to examine cerebral blood flow and neuroprotection.

Results : Lentiviral transfection significantly increased S100A10 expression both in MSCs and isolated EVs, as confirmed by western blotting, qPCR, and fluorescent microscopy. In vitro studies demonstrate the fibrinolytic, neuroprotective, and anti‐reactive oxygen species capabilities of EVs and D‐tat‐EE3. Localized injection of Pluronic F127 hydrogel encapsulating S100A10‐EVs and D‐tat‐EE3 exerted distinct therapeutic effects in vivo, including enhanced cerebral blood flow and reduced neuronal death. Mice receiving the loaded hydrogel exhibit reduced infarct volumes and improved functional and behavioural outcomes.

Summary/Conclusion : This combinational therapy offers a novel and promising multi‐target approach to address the primary and secondary damage mechanisms of ischaemic stroke.

Funding : The work is supported by Guangdong Natural Science Foundation (JCYJ20240813113550064); the University Development Research Start‐up Fund (UDF01003417); the Pengcheng Peacock Project Special Fund (2023TC0190); National Natural Science Foundation of China Youth Science Foundation Project (82404527).

Subtopic Cancer progression and metastasis 1

The impact of sympathetic nerve‐derived EVs on metastatic potential in breast cancer: the role of EMT‐related microRNAs

Takaaki Tamura 1,2 , Marta Prieto‐Vila 1 , Nao Nishida‐Aoki 1,3 , Takahiro Ochiya 1

1 Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; 2 Graduate School of Medicine and School of Medicine, Chiba University, Chiba, Japan 3 Waseda Institute for Advanced Study, Waseda University, Tokyo, Japan

Introduction : Breast cancer (BCa) is the leading cause of cancer‐related mortality in women. Emerging evidence suggests that the autonomic nervous system plays a pivotal role in cancer progression. In particular, the sympathetic nervous system has been implicated in initiating the metastatic switch in BCa. Given that their paracrine signals promote tumour progression, this study aimed to determine whether extracellular vesicles (EVs) from sympathetic neuronees contribute to BCa metastasis.

Methods : We analysed 30 BCa patient specimens to assess sympathetic neuronee enrichment in patients with metastasis by immunostaining with the sympathetic marker TH by comparing its ratio to the total neuronee marker NF‐L. For in vitro functional studies, EVs were collected from PC12 rat neuronee cells differentiated into a sympathetic neuronal phenotype using nerve growth factor (dPC12), with untreated PC12 cells (uPC12) serving as a control. EVs were isolated from their culture supernatant via ultracentrifugation and characterized according to MISEV2023 guidelines. The EVs were supplemented to RMT1 rat BCa cells and characterized for their motility and metastatic phenotypes by scratch assay and next‐generation sequencing (NGS) analysis, respectively. NGS was performed to identify transcriptomic changes of RMT1 treated with dPC12‐EVs and to profile microRNAs enriched in dPC12‐EVs. Pathway enrichment analyses were performed to investigate changes in tumour cells.

Results : Clinical observations revealed that sympathetic nerve fibres are more abundant in primary tumours of BCa patients with lymph node metastases than patients with local disease. In vitro functional assays demonstrated that dPC12‐EVs significantly enhanced the migratory capacity of RMT1, as well as epithelial‐mesenchymal transition (EMT) induction. NGS analysis identified 1309 genes downregulated in RMT1 cells treated with dPC12‐EVs. Analysis of the top 12 enriched microRNAs in dPC12‐EVs identified several target genes that overlapped with these downregulated genes. Pathway analysis suggested a link to EMT and specifically highlighted the activation of the PPAR signalling pathway.

Summary/Conclusion : EVs derived from sympathetic nerves carry microRNAs that enhance EMT‐related signalling in BCa cells, contributing to the acquisition of metastatic potential in primary tumours. These findings highlight the sympathetic nervous system as a potential therapeutic target in preventing BCa metastasis.

Funding : This study was supported by the Japan AMED under Grant Number JP21fk0108575.

Long non‐coding H19 transported by colorectal cancer‐derived small extracellular vesicles promotes alternative splicing in healthy hepatocytes: new insights on the liver pre‐metastatic niche formation

Presenter: Marco Loria

University of Palermo, Palermo, Italy

Introduction : Cancer progression relies heavily on intercellular communication within the tumour microenvironment, and tumour‐derived small extracellular vesicles (TD_SEVs) play a crucial role in this interaction. It has been demonstrated that TD_SEVs enhance tumour growth, allow pre‐metastatic niche formation and promote epithelial‐to‐mesenchymal transition (EMT). The EMT is a developmental process through which cells lose their epithelial identity and acquire a mesenchymal phenotype. In cancer, this process is prodromal to tissue fibrosis, thus favouring organ colonization by metastatic cells. Colorectal cancer (CRC) is one of the most common tumours, and almost 50% of the undiagnosed patients will develop liver metastases during the disease. Recent evidence highlighted the role of alternative splicing (AS) in the gene expression rearrangement required for EMT. In CRC, however, there is currently insufficient data relating TD_SEVs to AS. Our studies aim to dissect how the cargo of colorectal cancer‐derived small extracellular vesicles (CRC_SEVs) promotes AS related to EMT in hepatocytes, to shed light on the molecular mechanisms driving liver metastases of CRC.

Methods : CRC_SEVs were isolated from the conditioned media of SW480, SW620 and HCT‐116 cells through ultracentrifugation. The SEVs were resuspended in PBS, RNA lysis buffer or RIPA buffer. RNA and protein content were assessed through qRT‐PCR and western Blot, respectively. A human healthy hepatocyte cell line, the THLE‐2, was treated with the CRC_SEVs, and the biological effects were investigated through qRT‐PCR, immunofluorescence and RNA in situ.

Results : our data demonstrated that, in CRC, the long non‐coding RNA H19 binds RBFOX2, an RNA‐binding protein involved in pro‐EMT AS, leading this to its mRNA targets. Here we found that CRC_SEVs transport both lncH19 and RBFOX2 and the treatment of the THLE‐2 with the CRC_SEVs promotes AS of RBFOX2, target mRNAs involved in cell shape remodelling. Interestingly, vesicles deprived of lncH19 also show lower levels of RBFOX2 and cannot induce the same AS mechanisms in the hepatocytes.

Summary/Conclusion : our data indicated, for the first time to our knowledge, that lncH19 carries splicing factors inside EVs, affecting AS processes in recipient cells and enforcing a pre‐metastatic microenvironment.

Proteomic profiling of tumour‐draining vein‐derived extracellular vesicles reveals novel prognostic biomarkers in lung adenocarcinoma

Jérémy Tricard 1,2 , Stéphanie Durand 2 , Amandine Rovini 2 , Amy Gateau 2 , Luc Negroni 3 , Alain Chaunavel 4 , Anaëlle Chermat 1 , Massimo Conti 5 , Hussein Akil 2 , Fabrice Lalloué 2

1 Thoracic and Cardiovascular Surgery Department, Limoges University Hospital, Limoges, France; 2 UMR INSERM 1308 CAPTuR, Limoges, France; 3 IGBMC CERBM, Illkirch, France; 4 Department of Pathology, Limoges University Hospital, Limoges, France; 5 Division of Thoracic Surgery, Laval University, Québec, Canada

Introduction : Identifying non‐invasive biomarkers for predicting relapse in non‐small cell lung cancer (NSCLC) remains a critical challenge. Tumour‐draining vein (TDV) plasma is enriched in cancer biomarkers compared to peripheral vein (PV) plasma. This study aims to characterize and compare the proteomic cargo of extracellular vesicles (EVs) derived from TDV and PV samples of lung adenocarcinoma patients to identify novel prognostic biomarkers.

Methods : This study included 20 patients with stage I‐III lung adenocarcinoma undergoing curative‐intent surgery at Dupuytren University Hospital (France) as part of the ExOnSite‐Pro trial ( NCT04939324 ). Ethical approval was granted by the independent protection committee SUD MÉDITERRANÉE III, and written informed consent was obtained from all participants. TDV and PV blood samples were collected during surgery. EVs were isolated using size‐exclusion chromatography and characterized via nanoparticle tracking analysis and flow cytometry. Proteomic analysis of TDV‐ and PV‐derived EVs and, in parallel, of paired tumour/non‐tumoural tissues was performed using nanoLC‐MS/MS on the Exploris 480 quadrupole‐orbitrap mass spectrometer at the IGBMC platform, incorporating FAIMS technology for enhanced selectivity. Bioinformatic analysis included variance normalization, k‐nearest neighbour imputation, and differential expression analysis using linear modelling with false discovery rate correction. Protein interaction networks were constructed using STRING and Cytoscape.

Results : EVs from TDV plasma were significantly smaller and more concentrated than those from PV. Proteomic profiling identified 369 differentially expressed proteins, with 176 overexpressed in TDV‐derived EVs as compared to PV‐derived EVs. Notably, SRPRB (Signal Recognition Particle Receptor Subunit Beta) was also upregulated in tumour tissues and associated with poor NSCLC prognosis. Functional enrichment analysis from deregulated proteins revealed key pathways involving protein transport, immune regulation, and cytoskeletal remodelling. Interestingly, 12 proteins were upregulated in TDV‐derived EVs but downregulated in tumour tissues, suggesting tumour cells may package tumour‐suppressive proteins into EVs.

Summary/Conclusion : This study demonstrates that TDV‐derived EVs carry a unique proteomic signature enriched with biomarkers relevant to lung adenocarcinoma prognosis and recurrence. The identification of SRPRB as a potential early‐stage biomarker underscores the utility of TDV‐derived EVs as a liquid biopsy tool for NSCLC.

Human healthy liver spheroid as a model to study the role of colorectal cancer‐derived small extracellular vesicles in priming the hepatic pre‐metastatic niche

Presenter: Gabriele Antonuccio

Università degli Studi di Palermo, Palermo, Italy

Introduction : The majority of colorectal cancer‐related deaths are caused by liver metastases (LM). It is well established that the primary CRC can prepare a supporting micro‐environment for tumour cell invasion in the liver, called the hepatic pre‐metastatic niche (h‐PMN). This occurs through the release of several factors, including small extracellular vesicles (SEVs). Our research group focuses on investigating the role of CRC‐derived‐SEVs (CRC‐SEVs) in affecting hepatocyte behaviour, thereby impacting h‐PMN. Our previous study highlighted the ability of CRC‐SEVs to induce TGFβ1‐mediated epithelial to mesenchymal transition (EMT) in hepatocytes. As the EMT is preliminary on point fibrosis, our aim is to evaluate the role of CRC‐SEVs in liver fibrosis establishment. However, fibrosis is the result of complex and highly dynamic processes that cannot be investigated using a canonical bidimensional model. Therefore, we established a tridimensional in vitro model of hepatic spheroid (HEP‐3D) where we analysed the CRC‐SEVs’ pro‐fibrotic role in h‐PMN.

Methods : The HEP‐3D model has been developed by seeding in ultra‐low attachment 96 wells the human healthy hepatocytes THLE‐2 cell line, which were subsequently treated with SEVs isolated from the CRC cell line SW480 and from non‐metastatic CRC patients’ plasma.

Results : The first evaluation of CRC‐SEVs effect on HEP‐3D confirmed the results obtained in our previous study, by revealing a decreased level of the hepatocytes functional markers Albumin and HNF4, and of the epithelial markers, cytokeratin 8/18 and E‐cadherin, as well as an increased expression of the mesenchymal markers Vimentin and N‐Cadherin. Moreover, we found that CRC‐SEVs addition to HEP‐3D caused an amplified release of the fibrosis mediators interleukin‐6, transforming growth factor β, and growth arrest specific‐6 together with an increased deposition of the extracellular matrix proteins collagen‐IV, tenascin C, and fibronectin.

Summary/Conclusion : Taken together these results show that HEP‐3D model is responsive to CRC‐SEVs addition and represents a suitable system to study liver fibrosis, providing insight into the important role of the hepatocytes in the h‐PMN shaping.

An investigation of the effect of hypoxia on extracellular vesicles released from triple negative breast cancer cell lines

Róisín Daly 1,2,3 , Niamh McNamme 1,2,3 , Ledezma‐Gallegos Fabricio 1,2,3 , O'Driscoll Lorraine 1,2,3

1 School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin, Ireland; 2 Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; 3 Trinity St. James's Cancer Institute, Trinity College Dublin, Dublin, Ireland

Introduction : Our group was the first to demonstrate that anti‐cancer drug resistance can be transmitted via extracellular vesicles (EVs) using pre‐clinical models and patient samples. This finding highlights the significant role of EVs in cancer biology, particularly in the context of hypoxia, a key feature of the tumour microenvironment known to enhance malignant traits in cancer and alter the cargo of EVs. Additionally, triple negative breast cancer (TNBC) is an aggressive sub‐type of breast cancer, whose EVs have been associated with the promotion of metastasis and invasiveness. Given the critical role of EVs and hypoxia in cancer progression, this study aims to investigate and compare the effect of EVs collected under hypoxic and normoxic conditions on TNBC cells through functional assays.

Methods : The TNBC cell lines MDA‐MB‐231, MDA‐MD‐468, and Hs578T were cultured in EVs‐depleted FBS‐containing medium under normoxia (21% O 2 ) or hypoxia (1% O 2 ). EVs were collected from conditioned media by differential ultracentrifugation using a 70Ti fixed‐angle rotor at 120,000 × g at 4°C. EVs were characterised following the MISEV guidelines using TEM, NTA, image flow cytometry, and immunoblots. Wound healing and transwell migration assays were performed by treating cells with autologous normoxic or hypoxic EVs.

Results : In this study, hypoxic conditions did not show an increase in the release or size of EVs in any of the cell lines. The exposure to EVs obtained from hypoxic conditions significantly enhanced the migration of cells as depicted by a higher percentage of wound healing ( p = 0.01) compared to cells exposed to normoxic EVs. Additionally, treatments with hypoxic EVs significantly enhanced the invasion of MDA‐MB‐231 and Hs578T cells ( p = 0.01) compared to the untreated and normoxic EVs treated counterparts.

Summary/Conclusion : Our findings suggest that hypoxic EVs enhance migratory and invasiveness potential, supporting the role of hypoxia‐induced EVs in promoting an aggressive phenotype. The dynamics of EVs release in hypoxic conditions is crucial for understanding tumour progression and metastasis.

Funding : This research was supported by the Research Council Advanced Laureate Award EVIC to LOD [IRCLA/2019/49].

Subtopic Disease/therapy relevant mechanism/s of action 1

Chemotherapy induced painful peripheral neuropathy alters Schwann cell derived extracellular vesicles isolated from mouse peripheral nerves

M. Vecchitto 1 , M. Norimoto 1 , K. Funk 1 , Z. Wang 1 , T. Arai 1 , S. Martellucci 1 , S. L. Gonias 2 , W. M. Campana 1,3

1 Department of Anesthesiology, University of California, San Diego, La Jolla, California, USA; 2 Department of Pathology, University of California, San Diego, La Jolla, California, USA; 3 San Diego VA HealthCare System 3 , San Diego, California, USA

Introduction : Painful peripheral neuropathy (PPN) is a severe side effect of chemotherapy, manifested as nerve toxicity and neuropathic pain. In cancer patients with PPN, treatment adherence is often compromised, resulting in suboptimal cancer therapy. Treatment options for PPN are limited. We hypothesized that extracellular vesicles (EVs), which participate in cell‐to‐cell communication, may regulate PPN and thus, represent a therapeutic target.

Methods : We describe a novel method for capturing peripheral nerve‐derived EVs (pn‐EV) from extracellular spaces of the sciatic nerve. We applied this method to inducible turboGFP‐EV reporter (TIGER)‐P0‐Cre mice, which express tGFP only in Schwann cells (SCs). Pn‐EVs were isolated by centrifugation and size exclusion chromatography. nanoparticle tracking analysis (NTA) and electron microscopy revealed a concentration of ∼8.0 × 10 10 particles/mL, a modal diameter of 147 nm and crescent morphology. Pn‐EVs contain EV biomarkers, flotillin and TSG101, and glial biomarkers, CD9 and P0. Cellular proteins GM130, β‐actin and βIII‐tubulin were not detected. SC EVs were identified by tGFP using a ZetaView QUATT and super resolution microscopy with direct stochastic optical reconstruction (dSTORM).

Results : Fluorescence NTA revealed that > 30% of pn‐EVs were derived from SC EVs with a size of 109 nm. When pn‐EVs were injected into mouse hind‐paws, no pain‐related behaviours were identified. However, when pn‐EVs were injected prior to inducing acute pain with capsaicin, pain‐related behaviours increased ( p < 0.05), suggesting that pn‐EVs exacerbate pain states. Next, we treated mice with the chemotherapeutic, paclitaxel (PTX), which induces mechanical and cold hypersensitivity. Pn‐EVs isolated after PTX treatment demonstrated no change in size or concentration. However, dSTORM and fluorescence NTA demonstrated a decrease ( p < 0.05) in pn‐EV fluorescence following PTX, indicating a decrease in the fraction of pn‐EVs derived from SC EVs. The modal size of PTX‐treated SC EVs was larger ( p < 0.05). Naïve and PTX‐treated pn‐EVs showed differential Akt/ERK1/2 signalling in primary sensory neuronees, suggesting distinct biological properties.

Summary/Conclusion : Collectively, these studies establish a novel method for isolating peripheral nerve EVs in vivo, show that SCs are major contributors to the pn‐EV population in the peripheral nerve, and demonstrate that the cellular source and activities of pn‐EVs may change in PPN.

Funding : This study was supported by Veterans Administration I101RX003363 to WM Campana.

Exosomes and hair disorder

Presenter: Abdullah M. Aleisa

DERMA, Riyadh, Saudi Arabia

Introduction : Exosomes are small membrane sacs or vesicles secreted by eukaryotic cells. Initially discovered over 30 years ago, they were once thought to be merely cell waste products. However, it is now understood that naturally produced exosomes contain nucleic acids, such as genomic DNA, various forms of RNA, proteins, and lipids, which can carry information to other cells, sometimes even to those far from the originating cell. In recent years, the medical community has increasingly recognized the therapeutic potential of exosomes, establishing them as a powerful tool in various medical specialities. In an upcoming presentation, we will focus on the impact of human‐derived exosomes on hair abnormalities.

Methods : To treat different hair conditions, we utilize 100% pure umbilical cord blood stem cell conditioned media, such as Curestem or ASCE adipose tissue‐derived mesenchymal stem cells.

Results : This includes reversing the ageing of hair, which presents as grey hair, back to its original black colour. Additionally, we can enhance new hair growth in androgenetic alopecia by prolonging the anagen phase and decreasing the duration of the telogen phase. Even in the case of early‐stage scarring alopecia, initiating treatment can stop the progression of hair loss and prevent permanent scarring. We have treated over 100 patients and will present our experiences in choosing and using exosomes, along with our clinical results. Keywords : exosomes, hair loss, eukaryotic cells, medical innovation, hair treatment.

Summary/Conclusion : Exosome are one of the extravesicular vesicles that have a promising effect on hair disorders.

Plasma extracellular vesicles regulated the function of ILC2s via miRNA‐150‐5p in patients with allergic rhinitis

Presenter: Zhi‐Rou Zhou

The First Affiliated Hospital of Sun Yat‐Sen University, Guangzhou, People's Republic of China

Introduction : Group 2 innate lymphoid cells (ILC2s) are novel key players in the induction and maintenance of allergic inflammatory responses in airways. Various extracellular vesicles (EVs) are released into plasma in allergic airway inflammation, correlating with impaired airway function and severe inflammation. EV‐derived miRNAs are extensively involved in intracellular communication. However, the interaction between plasma EVs and immunocytes in this complex network remains to be further elucidated. Our study aimed to characterize the miRNA profiles in plasma EVs from healthy controls and AR patients, to evaluate the effect of plasma EVs on ILC2s and to identify miRNAs potentially contributing to ILC2 dysfunction in AR.

Methods : Plasma EVs were isolated from healthy controls and AR patients by using size exclusion chromatography (SEC), and differential miRNA expression profiles were obtained through miRNA sequencing. Peripheral blood mononuclear cells (PBMCs) were exposed to plasma EVs and miRNA mimics and miRNA inhibitors to assess the effect of plasma EVs and the underlying mechanisms.

Results : We found that EVs from HC and AR patients exhibited comparable characteristics in terms of concentration, structure, and EV marker expression. Both HC‐EVs and AR‐EVs were efficiently internalized by ILCs. AR‐EVs significantly promoted the differentiation and activation of ILC2s compared to HC‐EVs. Unique miRNA signatures of HC‐EVs and AR‐EVs were revealed by miRNA sequencing in diverse biological processes, among which miR‐150‐5p, miR‐144‐3p, miR‐10a‐5p, and miR‐10b‐5p were identified as potential contributors to AR‐EVs’ effects on ILC2s. The level of IL‐13+ ILC2s was only increased by mimics of the miR‐150‐5p and was significantly decreased after the treatment of miR‐150‐5p inhibitor.

Summary/Conclusion : Our study demonstrated that plasma EVs from patients with AR exhibited a pronounced capacity to significantly enhance the differentiation and activation of ILC2s, which was correlated with an elevated expression of miR‐150‐5p in AR‐EVs. These findings contribute to the advancement of our comprehension of EVs in the pathogenesis of AR and hold the potential to unveil novel therapeutic targets for the treatment of AR.

Funding : This study was supported by National Natural Science Foundation of China.

Calcium ionophore enhances the release of neutrophil‐derived extracellular vesicles with therapeutic potential for sepsis

Yumi Kumagai, Etsuo A. Susaki, Isao Nagaoka

Juntendo University, Tokyo, Japan

Introduction : Extracellular vesicles (EVs), secreted in response to microbial infections, play a pivotal role in modulating immune and inflammatory responses. Sepsis, a life‐threatening condition characterized by multiple organ dysfunction due to a dysregulated systemic inflammatory response to infection, presents significant challenges in therapeutic management. Despite extensive research, effective treatment strategies remain elusive. Our previous study demonstrated that EVs derived from mouse neutrophils stimulated with LL‐37, a human cathelicidin host‐defence peptide, improved survival rates in a septic mouse model induced by caecal ligation and puncture (CLP). However, the low yield of EVs posed a limitation of EV for therapeutic use. In this study, we explored strategies to enhance EV release from neutrophils with the aim of advancing the potential for clinical application of EV‐based therapies for sepsis.

Methods : EVs were isolated from the supernatant of neutrophils incubated with LL‐37 and/or the calcium ionophore A23187 using ultracentrifugation. The EVs were characterized by flow cytometry, interferometric microscopy, and western blotting. Their antibacterial efficacy was assessed by incubating them with Escherichia coli isolated from the mouse cecum. To evaluate therapeutic potential, CLP mice were intraperitoneally administered either EVs or phosphate‐buffered saline (PBS) as a control, and survival rates and body weight changes were monitored over 1 week.

Results : The calcium ionophore A23187 enhanced EV secretion from LL‐37‐stimulated mouse neutrophils. These EVs (PMN‐LL‐37Ca‐EV) exhibited greater antibacterial activity compared to EVs derived from neutrophils incubated with LL‐37 alone (PMN‐LL‐37‐EV). In the CLP mouse model, administration of PMN‐LL‐37Ca‐EV resulted in a higher survival rate or a faster recovery compared to PMN‐LL‐37‐EV when an equivalent number of EVs was intraperitoneally injected. Additionally, neutrophils isolated from human donors were stimulated with LL‐37 to release EVs with antimicrobial activity. These human‐derived EVs also demonstrated sepsis‐ameliorating effects when administered to CLP mice.

Summary/Conclusion : Calcium ionophore enhances the secretion of neutrophil‐derived EVs, as well as their antimicrobial activity and sepsis‐ameliorating effects. These findings provide valuable insights into the development of EV‐based therapeutic strategies for sepsis.

The microRNAs contained in extracellular vesicles derived from foam cells promote atherosclerosis in cells around plaques

Presenter: Okamura Akihiko

Tokyo Medical University, Tokyo, Japan

Introduction : To prevent ischaemic heart disease (IHD), it is essential to elucidate the molecular mechanisms involved in the progression of atherosclerotic plaques. This study aimed to investigate the effects of extracellular vesicles (EVs) secreted by foam cells (FCs), the main components of plaques, on cells surrounding the plaques.

Methods : Smooth muscle cells (SMCs) and macrophages were treated with enzymatically modified non‐oxidized LDL to induce the formation of FCs. FC‐derived EVs (FC‐EVs) were then purified from the culture supernatant using ultracentrifugation in a serum‐free medium. FC‐EVs were applied to macrophages, SMCs, and endothelial cells (ECs) to analyse atherosclerosis‐related gene expression by qPCR, and to assess cell migration and adhesion using a live‐cell analysis system. Additionally, ECs co‐cultured with macrophages and FC‐EVs were evaluated for the macrophage adhesion capacity to ECs. We performed microRNA (miRNA) analysis contained in FC‐EVs using next‐generation sequencing. Furthermore, we transfected mimics of those miRNAs into plaque surrounding cells and analysed their target genes using next‐generation sequencing. Finally, FC‐EVs were administered to atherosclerosis model mice, and the aortic plaque area was measured.

Results : FC‐EVs enhanced cell‐cell adhesion between ECs and macrophages and increased the migration and proliferation of SMCs and macrophages. Specific miRNAs enriched in FC‐EVs were identified, and transfection of mimics of these miRNAs into cells surrounding the plaque led to an in vitro enhancement of atherosclerosis‐related phenotypes, such as increased migration of SMCs and macrophages and enhanced cell‐cell adhesion between macrophages and ECs. FC‐EV administration increased the aortic plaque area in atherosclerosis model mice.

Summary/Conclusion : Our findings suggest that FC‐EVs contribute to atherosclerosis by enhancing EC‐monocyte adhesion and promoting the accumulation of macrophages and SMCs around plaques. The miRNA cargo of FC‐EVs plays a crucial role in these effects, highlighting both EVs and their miRNAs as potential therapeutic targets for atherosclerotic diseases. Furthermore, we are currently analysing the target genes of these miRNAs to further elucidate the detailed mechanisms underlying their pro‐atherogenic effects.

Exosomes derived from bone marrow mesenchymal stem cells alleviate neurovascular dysfunction following ischaemic stroke injury

Presenter: Zyad Mushtaq

University of Louisville, Louisville, Kentucky, USA

Introduction : Ischaemic stroke (IS) is a major global cause of morbidity and mortality, primarily affecting the central nervous system (CNS), but also causing multi‐organ dysfunction. Bone marrow mesenchymal stem cells (BMMSCs) are key in regulating inflammation and tissue repair after ischaemic injury. This study investigates whether exosomes derived from BMMSCs (BMMSC‐Exo) can reduce inflammation and neurovascular damage following IS injury in a mouse model.

Methods : Male and female mice, aged 10–12 weeks, underwent middle cerebral artery occlusion (MCAO) followed by reperfusion. BMMSC‐Exo was administered intravenously 2 h after MCAO and continued for 7 days. The experimental groups included: (1) Sham; (2) Sham + BMMSC‐Exo; (3) IS; (4) IS + BMMSC‐Exo. Neuroprotective effects were evaluated using a modified neurological severity score (mNSS), triphenyltetrazolium chloride (TTC) staining, brain water content, the Novel Object Recognition Test (NORT), and the Rotarod test. Additionally, Western blotting and quantitative PCR (qPCR) were performed.

Results : The IS + BMMSC‐Exo treatment group showed significant improvements, including reduced neurological deficits, infarct volume, and oedema compared to the untreated IS group. BMMSC‐exosome treatment in the IS group resulted in decreased inflammation and reduced expression of N‐methyl‐D‐aspartate (NMDA) receptors. Furthermore, BMMSC‐Exo alleviated the expression of glial fibrillary acidic protein (GFAP) in astrocytes and enhanced the presence of NeuN‐positive neuronees in IS‐injured mice. Additionally, BMMSC‐Exo treatment restored tight (ZO‐1, claudin‐5, and occludin) and adherens junction proteins (VE‐cadherin) in the vascular endothelium of IS‐injured mice when compared to untreated IS‐injured mice.

Summary/Conclusion : These findings suggest that BMMSC‐derived exosomes facilitate neurovascular restoration by modulating inflammation following ischaemia‐reperfusion injury in mice. This therapeutic approach may offer a promising strategy for treating ischaemic stroke and its associated neurovascular dysfunction.

Funding : The study was supported by (1) TPA‐971566/American Heart Association and (2) 24TPA1304527/American Heart Association.

Topically applied engineered ASC‐derived exosomes decrease inflammation and stimulate tissue repair mechanisms that provide treatment for intractable eczema

Presenter: Robin L. Smith

Exotropin, LLC, New York, New York, USA

Introduction : More than 31 million Americans have some form of eczema that ranges from mild to severe. The standard of care includes emollients and topical steroid treatments combined with bandages and oral therapeutics. Many cases are recurrent, complicated with chronic inflammation and infection, and remain persistent, raising the need for well‐tolerated and efficacious treatments.

Methods : Our data suggest that bioactive extracellular vesicles from human adipose stromal cells can be tuned, purified, and delivered to the skin via topical emulsions to effectively reprogram skin cells to reduce the symptoms associated with eczema. We have developed a patented scalable bioprocess to engineer pro‐healing EVs specifically tuned to target inflammatory skin diseases and enhance healing processes. By controlling the cellular environment in a closed‐system bioreactor, we can stimulate production and release of EVs that are biologically active and configured with a distinct miRNA cargo.

Results : We demonstrate that our proprietary engineered EVs, EXO‐3, contain a unique activity profile that reduces inflammatory cytokines and stimulates collagen production, as well as activates keratinocyte and fibroblast proliferation. Research has shown that in the context of eczema, decreased levels of specific miRNAs, like miR‐29b and miR‐143, are often observed in lesional skin, which contributes to the disrupted skin barrier function and increased inflammation characteristic of the disease. Low expression of these miRNAs can exacerbate symptoms. We have identified a proprietary EXO‐3 miRNA cargo signature which includes miR‐29b and miR‐143. Replenishing decreased levels of protective miRNAs via exosomal cargo potentially offers a new treatment strategy for eczema. Under informed consent, we evaluated our EXO‐3 exosomes formulated in emulsion compositions and topically applied to afflicted skin. Our tuned EXO‐3 exosomes show a dramatic reduction of eczema symptoms, including discomfort, redness, scaling, and itchiness.

Summary/Conclusion : EXO‐3 emulsion compositions are personal care formulations that are registered with the FDA under the Modernization of Cosmetics Regulation Act of 2022. EXO‐3 products were initially used solely to improve the condition and appearance of the skin. These findings were unexpected; they are supportive of engineered exosomes having targeted activity and usefulness as miRNA replacement therapies that alleviate symptoms and provide overall skin health improvement. Further exploration is warranted.

Funding : This study was supported by Exotropin, LLC.

Engineered pro‐healing EV cargo targeting chronic wounds by manipulating the cellular environment in a closed‐system bioreactor

Presenter: Jodi P. Gurney

Exotropin, LLC, New York, New York, USA

Introduction : Stem cell therapies have recently evolved to exploit stem cell‐derived exosomes as a biologic‐derived therapy, rather than delivering transient stem cells to treat chronic wounds. Secreted extracellular vesicles (EVs), such as exosomes, are packed with potent pro‐repair proteins and RNA cargos that are both inducible and cell type‐specific, providing a compelling means of tissue repair and wound healing.

Methods : We have developed a bioreactor‐based production system for manufacturing Human Adipose Stromal Cell (ASC) exosomes using a highly controlled and tunable environment that can modify EV production and cargo. The patented innovation in this abstract focuses on engineering novel pro‐healing EVs with therapeutic activity and using a topical formulation to treat diabetic ulcers. Chronic wound tissue is associated with inflammation, collagen fibre degeneration, and degradation of the extracellular matrix.

Results : Herein we demonstrate that our proprietary engineered ASC EVs, EXO‐3, contain a unique activity profile that reduces inflammatory cytokines and stimulates collagen production, as well as activates keratinocyte and fibroblast proliferation and migration. When formulated with an emollient and topically applied to an in vivo excisional wound model, tuned EVs demonstrated enhanced wound closure, increased keratinization, collagen deposition, and overall improved recovery rate. In a clinical case study addressing non‐healing diabetic foot ulcers, conducted under informed consent, topical treatment with engineered EXO‐3 EVs formulated in a proprietary gel emulsion showed complete wound closure and dermal regeneration.

Summary/Conclusion : EXO‐3 personal care formulations are registered in compliance with the FDA under the Modernization of Cosmetics Regulation Act of 2022. This product described is not a drug product and was initially used solely to improve the condition and appearance of the skin. These reported findings were unexpected; however, they support the ability to engineer pro‐healing EVs that provide therapeutic benefit beyond traditional pharmacological treatments. Further investigation is warranted.

Funding : This study was supported by Exotropin, LLC.

Engineering Yulangsan‐derived extracellular vesicles can target the treatment of ischaemic stroke through anti‐inflammatory and anti‐apoptotic pathways

Presenter: Chunxia Chen

The People's Hospital of Guangxi Zhuang Autonomous Region & Guangxi Academy of Medical Sciences, Nanning, People's Republic of China

Abstract Unavailable

Subtopic Cancer biomarkers 1

Extracellular vesicle‐associated heparan sulphate proteoglycans drive prostate cancer growth and have potential application as biomarkers of clinically significant disease

Demi Pritchard 1* , Alex Shephard 2,3* , Sara Veiga 4,5,6 , Haiyan An 1 , Gokul V. KandaSwamy 7 , Aled Clayton 2 , Jason P. Webber 1

1 Swansea University, Swansea, UK; 2 Cardiff University, Cardiff, UK; 3 Tozaro, Cardiff, UK; 4 Massachusetts General Hospital, Boston, Massachusetts, USA; 5 Harvard Medical School, Boston, Massachusetts, USA; 6 The broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; 7 Swansea Bay University Health Board, Swansea, UK *Demi Pritchard and Alex Shephard contributed equally to this study .

Introduction : Prostate cancer (PCa) is the most common cancer to affect men. Many patients have slow growing, clinically insignificant tumours, but some have aggressive, clinically significant disease. We previously demonstrated that heparan sulphate proteoglycans (HSPGs) are required for extracellular vesicle (EV)‐mediated activation of a disease‐supporting stromal phenotype. Here we explore the impact of HSPG attenuation on EV‐mediated stromal cell activation and subsequent impact on tumour growth. Furthermore, we assessed whether EV‐associated HSPGs have potential as biomarkers for early detection of clinically significant PCa.

Methods : EVs were isolated from PCa cells (LNCaP, PC3 and Du145) by sucrose cushion ultracentrifugation and characterised in accordance with guidelines published within the Journal of Extracellular Vesicles. EV‐HSPG expression was assessed by immunoaffinity assay. Expression of detectable HSPGs was attenuated by lentiviral delivery of shRNAs. Stromal cell activation was assessed by immunofluorescent detection of alpha smooth muscle actin (αSMA) within fibroblasts incubated with EVs for 72 h. Tumour growth was assessed in vivo following subcutaneous implantation of EV‐deficient (Rab27aKD) Du145 cells with fibroblasts into the hind flank of athymic nude mice, and subsequent administration of HSPG‐deficient, or control, EVs every 7 days. EVs were isolated from PCa patient serum (supplied by the Wales Cancer Bank; n = 100) and assessed for HSPG expression. Validation of EV‐HSPG expression was performed on an independent cohort of serum samples from patients ( n = 150) undergoing diagnostic testing for PCa within the Swansea Bay University Health Board (NHS REC 23/WA/0087).

Results : Syndecan 3, Syndecan 4, Glypican 1, Glypican 6 and Betaglycan were detectable on the surface of PCa EVs. HSPG attenuation resulted in a loss in the ability of EVs to trigger stromal cell activation and a reduction in tumour growth in vivo. All five HSPGs were detectable on EVs within patient serum; however, only the detection of Syndecan 4, Glypican 6 and Betaglycan was elevated on EVs from patients with clinically significant PCa compared to those with clinically insignificant disease.

Summary/Conclusion : Syndecan 4, Glypican 6 and Betaglycan are required for EV‐mediated activation of stromal cells to a disease‐promoting phenotype and have potential as biomarkers for early detection of clinically significant PCa.

Funding : This study was supported by Prostate Cancer UK.

Identifying extracellular vesicle‐based biomarkers distinguishing responders from non‐responders in EndoBARR clinical trial patients

Presenter: Sunila Pradeep

Medical College of Wisconsin, Milwaukee, Wisconsin, USA

Introduction : Endometrial cancer (EC) incidence and mortality are increasing, necessitating the development of more effective therapies. The EndoBARR trial ( NCT03694262 ) investigated a novel three‐drug regimen of bevacizumab, atezolizumab, and rucaparib in patients with recurrent or persistent EC after 1–2 prior lines of therapy. Rucaparib, a PARP inhibitor, induces double‐strand DNA breaks and inflammation, enhancing immunogenic responses through PD‐L1. Atezolizumab, a PD‐L1 inhibitor, boosts immune activity by blocking inhibitory signals to T cells, while bevacizumab inhibits VEGF‐A‐mediated angiogenesis. The trial demonstrated a 42% overall response rate, with a median response duration of 5.8 months. This study aims to identify factors distinguishing responders from non‐responders by analysing extracellular vesicles (EVs) isolated from patient serum and characterizing their molecular profiles.

Methods : EVs were isolated from plasma using the SmartSECTM Mini EV Isolation Kit or differential centrifugation. Characterization was performed using nanoparticle tracking analysis (NTA) and immunoblotting for EV markers, including TSG101, CD63, ALIX, and HRS. The quality and enrichment of EVs were further validated via sucrose gradient centrifugation. After EV isolation, the protein profiles of EVs were analysed using mass spectrometry (MS). MS facilitated the detection of novel biomarkers and assessment of proteomic changes before and after the therapeutic trial. KLE, HEC1B, and AN3CA endometrial cancer cell lines were chosen based on their mutational profiles. Pathway analyses were conducted using EndoBARR treatments alone and in combination.

Results : The EndoBARR trial involving 30 subjects demonstrated that two participants achieved complete response with progression‐free survival, while nine showed partial response with temporary progression‐free survival. The remaining participants either exhibited no response to treatment, had stable disease, or experienced progressive recurrence. Notably, some partial responders later developed disease progression after multiple treatment cycles. Preliminary mass spectrometry analysis of clinical trial samples revealed distinct protein expression patterns between responders and non‐responders, particularly in proteins involved in adhesion, migration, and the complement pathway.

Summary/Conclusion : EC cell line KLE demonstrated greater sensitivity to rucaparib in vitro compared to HEC1B and AN3CA. Mass spectrometry results show differential response to the triplet. EndoBARR therapy may be related to differential expression of complement factor and proteins associated with metastasis.

Higher percentages of CD221 (IGF‐1 receptor)‐positive EVs detected by bead‐based and single‐EV flow cytometry in blood from breast cancer survivors: preliminary results during lifestyle intervention

Mariele Montanari 1 , Daniele Lopez 1 , Giovanna Panza 1 , Sara Biagiotti 1 , Faiza Abbas 1 , Rita Emili 2 , Michele Guescini 1 , Fabiana Fanelli 1 , Mauro De Santi 1 , Paola Lanuti 3 , Elena Barbieri 1 , Ludovica Di Fabrizio 1 , Giosuè Annibalini 1 , Barbara Canonico 1

1 Department of Biomolecular Sciences, University of Urbino Carlo Bo,Urbino, Italy 2 , U.O.C. Oncologia Medica, Ospedale Santa Maria della Misericordia, AST Pesaro Urbino, Urbino, Italy, 3 University “G.d'Annunzio,” Chieti‐Pescara, Italy

Introduction : Among techniques for single EV analysis, flow cytometry (FC) has a high potential for clinical application due to its high throughput and multiplex fluorescence capability. Indeed, bead‐based FC has also been used to interrogate EV surface proteins. Particularly, IGF‐1R (CD221), a key member of the IGF axis, known for its oncogenic role in multiple cancer lineages, was investigated on both cells and EVs in blood from breast cancer survivors since the IGF‐1/IGF‐1R pathway is highly breast cancer subtype context‐dependent.

Methods : An FC method for analysing the expression and activation of the IGF‐1 receptor is currently set up in our Laboratory, giving good results on myeloid cells. The plasma of donors and BC survivors was investigated at different time points for EV detection and CD221 positivity. BC patients are enrolled in the MoviS Trial (ClinicalTrials.gov: NCT04818359 ) and follow a 3‐month aerobic exercise and nutritional education programme. All reagents were spun at 21,000 × g , following general guidelines. We stained blood plasma (centrifuged at 2500 × g ) with a lipophilic cationic dye (LCD, BD Biosciences) recently identified as a generic EV tracer, highly useful when samples containing heterogeneous EV population need to be analysed or when EVs may not express the classical tetraspanin markers (e.g., CD9, CD81, CD63). Rosetta calibration beads and the application of either an SSC or fluorescent trigger threshold were employed. The capture bead technique was applied using Exostep (Immunostep) for bead‐based evaluation.

Results : Preliminary results show higher percentages of CD221+ EVS in blood from BC survivors concerning age‐matched donors. Furthermore, these CD221+ EVS mainly belong to the pool of CD81 negative EVs. Although further tests are needed (also to increase adherence to the MISEV guidelines), this finding is confirmed by Exostep data. Other experiments are ongoing to confirm data and to add more MISEV technical approaches.

Summary/Conclusion : The IGF‐1 receptor is an important immunomodulator, emerging as an attractive target for cancer therapy. These preliminary findings will be integrated with the characterization of the isolated EVs and nanoparticle tracking analysis, aiming at consolidating current results and coupling them with data on the expression and activation of the IGF‐1 receptor on immune cells.

TP53 isoform proteins and mRNAs in extracellular vesicles: potential biomarkers for colorectal cancer detection

Martina Presicci 1 , A. E. Martinez 2 , C. Di Battista 3 , M. Gironella 3 , J. C. Bourdon 4 , T. van Wezel 1

1 Dept of Pathology, Leiden University Medical Center (LUMC), Leiden, The Netherlands; 2 Dept of Cell and Chemical Biology, Leiden University Medical Center (LUMC) Leiden, The Netherlands, 3 Gastrointestinal & Pancreatic Oncology group, FRCB‐IDIBAPS; Experimental Pathology Dept. IIBB‐CSIC; Facultat de Medicina i Ciencies de la salut, Universitat de Barcelona, Spain, 4 School of Medicine, Cancer Division, University of Dundee, Dundee, Scotland, UK

Introduction : Colorectal cancer (CRC) is one of the leading causes of cancer‐related deaths worldwide, emphasizing the need for non‐invasive biomarkers to enable early detection and improve survival outcomes. Extracellular vesicles (EVs) carry proteins, RNA, and other biomolecules that reflect the functional state and molecular profile of their cells of origin. These unique properties make EVs promising candidates for biomarker discovery. Mutations in TP53, a critical tumour suppressor gene, are frequently implicated in CRC and are linked to cancer progression and resistance to therapy. This study investigates the presence of TP53 isoform proteins and mRNAs in EVs isolated from the conditioned media of lymphocyte B cell lines derived from individuals with hereditary cancer predisposition disorder. Our goal is to evaluate their potential as biomarkers for familial CRC detection.

Methods : EVs were isolated and purified from serum‐free conditioned media of lymphocyte B cells derived from individuals with hereditary cancer predisposition disorder. EVs were characterized via nanoparticle tracking analysis (NTA) and Western blotting for positive and negative EVs markers. TP53 isoform proteins were detected using validated TP53 isoform‐specific antibodies, while mRNA was analysed using reverse transcription (RT) and droplet digital PCR (ddPCR) with probes specific for different TP53 splice variant transcripts.

Results : Western blot analysis confirmed the presence of EV markers in size exclusion chromatography (SEC)‐enriched fractions, validating the isolation protocol. Preliminary findings revealed TP53 isoform protein enriched in EVs. RT‐ddPCR analysis identified specific TP53 splice variant mRNA in EV‐enriched fractions. Notably, the abundance of TP53 splice variant mRNA and p53 protein isoforms was higher in EVs derived from CRC cell lines associated with Li‐Fraumeni syndrome compared to those from sporadic CRC cell lines.

Summary/Conclusion : This study indicates the presence of specific TP53 isoform proteins and mRNAs in EVs, underscoring their potential as biomarkers for CRC. Our findings suggest that EVs act as carriers of some TP53 splice variants and isoform‐specific signals. This highlights the intricate nature of this tumour suppressor gene, whose complexity has been observed in various cancer types but is not yet fully understood.

Funding : Funded from the European Union's Horizon Europe research and innovation programme under the Marie Skłodowska‐Curie Doctoral Network Grant Agreement No. 101072448.

Subtopic EV biomarker clinical translation 1

Extracellular vesicle‐encapsulated microRNA_9985 inhibits lung adenocarcinoma growth by targeting GCC2

Ga Yoon Kim 1,2 , Byeong Hyeon Choi 1,2 , Chang Geun Kim 1,2 , Jun Hee Lee 1,2 , Hyunku Shin 3 , Yeonho Choi 3,4 , Hyun Koo Kim 1,2*

1 Department of Thoracic and Cardiovascular Surgery, Korea University Guro Hospital, College of Medicine, Korea University, Seoul, Republic of Korea; 2 Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea; 3 EXoPERT Corporation, Seoul, Republic of Korea; 4 Department of Biomedical Engineering, Korea University, Seoul, Republic of Korea

Introduction : Lung adenocarcinoma (ADC) accounts for 40% of non‐small cell lung cancer cases. Previously identified GRIP and coiled‐coil domain‐containing 2 (GCC2) as an extracellular vesicle (EV) biomarker that can be used to diagnose ADC and demonstrated its therapeutic potential. miRNAs have emerged as promising therapeutic targets in ADC due to their ability to regulate the expression of specific proteins by binding to their mRNA. miRNA_9985 is expected to target mRNA of GCC2, but its mechanisms of action in lung ADC remain unclear. Here, we investigated the anti‐cancer mechanisms of miRNA_9985 mediated GCC2 inhibition.

Methods : The miRNA targeting GCC2, miRNA_9985, was selected using the miRDB database. EVs from bone marrow mesenchymal stem cells (BMMSC_EVs) were isolated from the culture medium using polymer‐based precipitation and size exclusion chromatography. miRNA_9985 was encapsulated in BMMSC‐EVs using a heat shock method, and encapsulation efficacy was quantified using quantitative real‐time PCR (qRT‐PCR). The delivery of miRNA_9985‐loaded BMMSC‐EVs into lung ADC cells was evaluated using confocal microscopy. Protein expression changes following miRNA_9985 mediated GCC2 knockdown were assessed using cell‐signalling arrays. A total of 16 nude mice were individually exposed to the following treatments: (1) PBS, (2) control‐EVs, (3) miRNA_9985, or (4) miRNA_9985‐inhibitor ( N = 4 per group). Tumour formation was induced by subcutaneously injecting mice with a number of 1 million H1650 cells, and 2 weeks after tumour formation, individual treatments were administered weekly through the tail vein. Tumour growth and toxicity were monitored across groups.

Results : qRT‐PCR revealed high encapsulation efficiency of miRNA_9985 in BMMSC‐EVs. Confocal microscopy confirmed the efficient intracellular delivery of miRNA_9985. Cell‐signalling arrays and western blotting revealed that miRNA_9985‐mediated GCC2 knockdown downregulated HDAC1 and BCL‐2, ultimately leading to increased c‐PARP levels, a marker of apoptosis. Co‐immunoprecipitation assays revealed an interaction between GCC2 and HDAC1, suggesting that GCC2 knockdown activates the HDAC1‐mediated intrinsic apoptosis pathway. In vitro assays confirmed that GCC2 knockdown induced anti‐cancer effects. In vivo, miRNA_9985‐loaded BMMSC‐EVs effectively suppressed tumour growth without any side effects or toxicity.

Summary/Conclusion : miRNA_9985‐loaded EVs suppress tumour growth by targeting GCC2 and inducing apoptosis, offering potential for therapeutic development.

Identification of miRNAs associated with immunotherapy response in mouse pancreatic cancer

Juntaro Matsuzaki 1,2 , Taisuke Koreeda 2 , Yukiko Asakawa 2 , Rina Shibagaki 2 , Marina Nakamura 2 , Chihiro Oikawa 2 , Masaki Kimura 2 , Yoshimasa Saito 2

1 Division of Interdisciplinary Genetics and Nanomedicine, Research Center for Drug Discovery, Keio University Faculty of Pharmacy, Tokyo, Japan; 2 Faculty of Pharmacy, Division of Pharmacotherapeutics, Keio University, Tokyo, Japan

Introduction : Although cancer immunotherapy is widely used today, its efficacy against pancreatic cancer is still limited. Agonistic CD40 antibodies are one of the immunotherapies expected to have therapeutic effects against pancreatic cancer (Lancet Oncol 25:853, 2024). We explored miRNAs in extracellular vesicles (EV‐miRNAs) as potential biomarkers associated with the response to agonistic CD40 antibodies in mouse pancreatic cancer.

Methods : Two distinct mouse pancreatic cancer cell clones, 2838c3 and 6419c5, established from KPCY (LSL‐KrasG12D; Trp53LSL‐R172H/+; Pdx1‐cre; LSL‐Rosa26YFP/YFP) mice were used. Higher levels of T cell infiltration into the tumour and sensitivity to immunotherapy were reported in 2838c3 compared to 6419c5 when transplanted into C57BL/6 mice (immunity 49:178, 2018). Intracellular and extracellular miRNA profiles with these two KPCY cell clones were compared. Small EVs released from each cell line were collected by ultracentrifugation of culture supernatants after the 0.22 µm‐filtration. The particle number and size of EVs were checked by nanoparticle tracking analysis. Supernatant EV‐RNAs and intracellular RNAs were extracted using miRNeasy Kits (QIAGEN). The efficacy of agonistic CD40 antibodies for xenograft tumours developed by subcutaneously transplanted KPCY cells with modified miRNA expression was investigated.

Results : Regarding the intracellular miRNA profiles, 93 miRNAs had more than twice higher expression levels in 6419c5 than in 2838c3, whereas 70 miRNAs had more than twice higher expression levels in 2838c3 than in 6419c5. Similar differences were also observed for some miRNAs in the culture supernatant EVs. Specifically, 17 miRNAs showed more than twice the expression level in 6419c5 compared to 2838c3, and 10 miRNAs showed more than twice the expression level in 2838c3 compared to 6419c5 in the culture supernatant. We further focused on 10 highly expressed miRNAs (> 16 times) in 6419c5 compared to 2838c3. When we overexpressed the 10 miRNAs in 2838c3, the resistance to agonistic CD40 antibodies was acquired.

Summary/Conclusion : We identified the candidates of extracellular miRNA biomarkers associated with the response to agonistic CD40 antibodies in mouse pancreatic cancer.

Funding : This work was supported by JSPS KAKENHI Grant‐in‐Aid for Scientific Research (B).

From Glioblastoma to the blood: tenascin‐C positive EVs as clinical biomarkers and vehicles of circulating tumour DNA

Amanda Salviano‐Silva 1* , Kathrin Wollmann 1 , Santra Brenna 2 , Rudolph Reimer 3 , Julia E. Neumann 4,5 , Matthias Dottermusch 4,5 , Laura Woythe 6 , Cecile L. Maire 1 , Berta Puig 2 , Ulrich Schüller 4,7,8 , Manfred Westphal 1 , Richard Drexler 1 , Lasse Dührsen 1 , Jens Gempt 1 , Dieter H. Heiland 9 , Katrin Lamszus 1 , Franz L. Ricklefs 1*

1 Department of Neurosurgery, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 2 Neurology Department, Experimental Research in Stroke and Inflammation, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 3 Leibniz Institute for Experimental Virology, Hamburg, Germany; 4 Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 5 Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 6 Oxford Nanoimaging Limited (ONI), Oxford, UK, 7 Department of Pediatric Hematology and Oncology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany, 8 Children's Cancer Research Center Hamburg, Hamburg, Germany; 9 Department of Neurosurgery, Medical Center University of Freiburg, Freiburg, Germany

Introduction : Extracellular vesicles (EVs) are mediators of intercellular communication, transferring tumour‐derived biomolecules into the bloodstream and offering a minimally invasive approach for disease monitoring. This is particularly relevant for glioblastoma, an aggressive and highly heterogeneous brain tumour with limited real‐time monitoring options. Here, we aimed to evaluate glioblastoma‐associated surface antigens on blood circulating EVs and characterize their biomarker utilities.

Methods : EVs were isolated from plasma samples of 37 newly diagnosed glioblastoma patients (pre‐ and matched post‐surgery), 11 matched patients with recurrent glioblastoma, and 22 healthy donors (HD) by differential ultracentrifugation. EVs were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Imaging flow cytometry (IFCM) was employed to immunophenotype tetraspanins (CD9, CD81, CD63) in combination with glioma‐associated markers (Tenascin‐C [TNC], Integrin‐β1 [ITGB1], CD44, CD131, Profilin‐1 [PFN1], SPARC, GPNMB, and HLA class‐II), selected based on previous proteomic screening. Double‐positive EV subpopulations were corrected per millilitre input plasma and compared among the groups by Mann‐Whitney tests.

Results : TNC+ EVs exhibited the most significant differences between glioblastoma patients and non‐tumour subjects. Specifically, TNC+/CD9+ EVs were markedly elevated in newly diagnosed (fold change [FC] = 7.6, p < 0.05).

Summary/Conclusion : TNC+ EVs are promising biomarkers for glioblastoma, and their selective isolation improves the detection of tumour‐specific mutations, highlighting their potential utility in liquid biopsy applications.

Funding : This study was supported by Deutsche Forschungsgemeinschaft and Rudolf Bartling Stiftung.

EVs in liquid biopsies: do different size exclusion chromatography approaches yield different results?

Szilárd‐Krisztián Belényesi 1,2,3 , Sean Patmore 1,2,3 , Faye Lewis 3,4,5 , Brian Henderson 3,4,5 , Volga M. Saini 3,6,7 , Ezgi Oner 3,6,7 , Sinead Hurley 3,5,6,7 , Marika Kanjuga 3,4,5 , Kathy Gately 3,6,7 , Sharon O'Toole 3 4 5 , John J. O'Leary 3,4 5 , Lorraine O'Driscoll 1,2 3

1 School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin, Ireland; 2 Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; 3 Trinity St. James Cancer Institute, Trinity College Dublin, Dublin, Ireland; 4 Department of Obstetrics and Gynaecology, School of Medicine, Trinity College Dublin, Ireland; 5 Department of Histopathology, School of Medicine, Trinity College Dublin, Dublin, Ireland; 6 Thoracic Oncology Research Group, Trinity Translational Medicine Institute, St James's Hospital, Dublin, Ireland; 7 Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Ireland

Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed particles released by a plethora of cell types. Importantly, EVs play a role in cancer progression by facilitating cell‐to‐cell communication and delivering cargo molecules within the tumour microenvironment. Emerging evidence suggests that EVs can be collected from minimally invasive samples, such as plasma, where they hold promise as potential disease biomarkers. However, conventional isolation methods such as ultracentrifugation are often impractical in clinical settings. To address this, we employ size exclusion chromatography (SEC), a more clinically feasible technique, to collect and characterise EVs from two cancer patients’ cohorts (lung and ovarian) to subsequentially investigate their diagnostic, prognostic, and predictive potential.

Methods : After ethical approval was obtained, plasma samples were acquired from patients with either lung or ovarian cancer. EVs were collected by SEC, using columns with either a 35 nm cut‐off (qEV35) or a 70 nm cut‐off (qEV70). The collected EVs were characterised by five parameters: transmission electron microscopy, nanoparticle tracking analysis, immunoblotting, flow cytometry, and ELISA.

Results : SEC reproducibly collected EVs from the plasma of patients with both cancer types. The resulting EVs consistently expressed similar physical and biochemical traits, as evidenced by morphology, size, and biomarker expression (tetraspanins, syntenin). Flow cytometry analysis of EVs isolates showed an increased amount of CD9+, CD63+ and CD81+ EVs in qEV35 isolates, when compared to qEV70, in both cancer types. The amount of particles and protein was also significantly higher in qEV35 isolates, while the amount of ApoB contamination did not differ between the two columns. Interestingly, the purity ratio (particles/protein) was higher in EVs collected with qEV70 from lung cancer patient samples ( p < 0.01), and a similar trend could be observed in ovarian cancer patient EVs ( p = 0.06).

Summary/Conclusion : SEC may be a suitable method for collecting EVs from liquid biopsies, with potential for the study of biomarkers in cancers. However, it must be considered that different columns may collect different EVs sub‐populations. Furthermore, certain columns might increase EVs yield, while others may collect purer populations.

Funding : This study was supported by HEA's North‐South funding of the All‐Ireland Cancer Liquid Biopsies (CLuB).

Glycosylation profiling of small extracellular vesicles in head and neck squamous cell carcinoma reveals potential biomarkers

Presenter: Alicja Głuszko

Medical University of Warsaw, Warsaw, Poland

Introduction : Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer‐related morbidity and mortality, with limited early detection methods and prognostic biomarkers. Small extracellular vesicles (sEVs), including exosomes, are key mediators of intercellular communication and have emerged as potential sources of biomarkers. Glycosylation, a post‐translational modification, plays a critical role in cancer progression and metastasis. In this study, we explore the glycosylation profiles of sEVs derived from HNSCC to identify potential biomarkers for diagnosis and prognosis.

Methods : Three HNSCC cell lines (FaDu, PCI‐30, and SCC‐25) and normal keratinocytes (HaCaT) were cultured under normoxic (21% O 2 ) through 72 h. sEVs were isolated from supernatants of those cells as well as from HNSCC patient plasma ( n = 10) and healthy donors ( n = 10), using size exclusion chromatography. sEVs were characterized by western blot, electron microscopy and nanoparticle tracking analysis. Flow cytometry was used to assess the sEV‐associated glycan pattern. Mass spectrometry and TCGA‐based database analysis were used to assess modifications in glycosylation‐associated proteome and transcriptome. For correlation analysis, vesiculation‐associated gene sets were used.

Results : sEVs range in size from 85 to135 nm, carry CD63, CD9, TSG101, and Alix, but not Calnexin nor Grp94. Bulk RNAseq analysis revealed significant correlation with patient survival for LG3BP ( p = 0.042), LEG1 ( p = 0.014), OST48 ( p = 0.042), DAD1 ( p = 0.017), B4GALNT1 ( p = 0.039), PYGL ( p = 0.023), ALG3 ( p = 0.017), EXT2 ( p = 0.0069), and KDELC1 ( p = 0.0061). Compared to HPV‐positive tumours significantly higher expression of LG3BP, LEG1, B4GALNT1, PYGL, EXT2, and KDELC1 was observed in HPV(−) tumours. These genes correlated significantly with tumour stage and with the vesiculation‐associated gene set. Single‐cell RNA‐seq revealed upregulation of LEG1 in immune cells subsets. MS‐based comparative analysis between tumour and normal cells revealed upregulation of mannose receptor (MPRD) and downregulation of oligosaccharyl transferase (OST48) and dolichyl‐diphosphooligosaccharide‐protein glycosyltransferase (RPN1) in tumour cells, with galectin‐3 binding protein (LG3BP) enriched in sEVs. HNSCC patient sEV glycan profiling revealed significant downregulation of ConA lectin binding compared to normal controls.

Summary/Conclusion : In conclusion, the glycosylation profiles of small extracellular vesicles in head and neck squamous cell carcinoma hold promise as potential biomarkers for early detection and prognosis. Further clinical validation is needed to establish their role in improving patient outcomes.

Funding : This study was supported by Medical University of Warsaw subvention for science.

Subtopic Biomarker technologies 1

Extracellular vesicles as activity‐based biomarkers: a novel, versatile approach to analyse EV‐associated protease activity

Mengke Zhao, Jan Van Deun, Andreas Baur

Department of Dermatology, Universitätsklinikum Erlangen, FAU Erlangen‐Nürnberg, Deutsches Zentrum Immuntherapie(DZI), Erlangen, Germany

Introduction : Extracellular vesicles (EVs) are released by cells as crucial mediators of intercellular communication. Proteases are integral to various biological processes, including protein maturation, cellular communication and immune modulation. Their activity is tightly controlled and often dysregulated in disease. Physiological, plasma EV‐associated protease activity is therefore emerging as a promising functional biomarker. We are developing a novel method for measuring endogenous EV‐associated protease activity with increased sensitivity and specificity.

Methods : We have constructed a bacterial expression vector that places a substrate sequence of choice between a SpyTag‐GFP and SNAP‐tag domain. After functionalizing magnetic beads with SpyCatcher, we capture our construct from bacterial lysates. We then add benzylguanine‐capped DNA barcodes, which will bind to the SNAP domain. Upon incubation with proteases or protease‐containing EVs, DNA barcodes are released into the supernatant and can be quantified by qPCR.

Results : As proof‐of‐concept, we have selected ADAM17 and the corresponding cleavage domain of TNFα. We could demonstrate successful capture of the SpyTagged construct by flow cytometry for GFP and fluorescently‐labelled SNAP. We evaluated its resistance to non‐specific cleavage by using a construct without TNFαdomain and incubating with recombinant protease and protease‐containing EVs. Conversely, we could show that the presence of the TNFα domain results in cleavage and release of barcoded fragments into the supernatant.

Summary/Conclusion : We hypothesize that our probes could improve the sensitivity and specificity of protease activity detection compared to traditional FRET‐based approaches. Future work will include multiplexing for several protease/substrate combinations and testing on cohorts of plasma‐derived EV samples to determine biomarker potential.

Enhancement of extracellular vesicles production for personalised medicine

Presenter: Ilaria Menon

CNR NANOTEC, Lecce, Italy

Introduction : Over the past decade, extracellular vesicles (EVs) have emerged as key mediators of intercellular communication, influencing physiological and pathological processes. According to MISEV2023 nomenclature, EVs are lipid bilayer‐enclosed particles incapable of autonomous replication, classified as small or large. EVs encapsulate bioactive molecules, including proteins, nucleic acids, and lipids, playing roles in cancer progression, immune modulation, and cellular homeostasis. Their presence in biofluids makes them promising candidates for liquid biopsy biomarkers. EVs originate from distinct biogenesis pathways, often regulated by the ESCRT machinery. Increasing interest surrounds their potential as diagnostic and therapeutic tools in personalized medicine.

Methods : With the support and development of LOC, this project aims to: characterize EV biogenesis pathways by analysing ESCRT machinery, develop an on‐chip protocol to enhance EV release, evaluate the effects of selected drugs (for example tipifarnib and doxorubicin) on EV production, characterize EVs generated under different ESCRT pathway modulations and establish a precision medicine tool for autologous EV production.

Results : At CNR NANOTEC, a microfluidic device enables on‐chip cell culturing under dynamic conditions. Preliminary results based on comparative studies of static vs. dynamic cultures showed that dynamic conditions significantly increased EV production—6‐fold for small EVs and 3‐fold for large EVs—while maintaining comparable cell viability and apoptosis rates. This effect will be tested in combination with drugs for modulating the ESCRT pathway to enhance EVs production. Comparison with standard flow cytometry and specific membrane markers analysis will confirm the stimulation of EVs production.

Summary/Conclusion : A deeper understanding of EV biogenesis and its molecular regulation is critical for advancing precision and personalized medicine. EVs hold promise as therapeutic and prognostic biomarkers, especially in oncology, with potential applications in drug development and biogenesis modulation. The in vitro production of autologous EVs could enable patient‐specific drug delivery with minimal side effects. This approach could be scaled to hospital settings, where patient‐derived cells cultured in microfluidic devices produce EVs for drug loading and reinfusion, aligning with the principles of personalized medicine.

Funding : This study was supported by PRIN 2022 Project—financed by the European Union—Next Generation EU‐PNRR M4.C2.1.1. RESOLVE—an innovative platform based on field‐flow fractionation and sample on‐chip detection to unravel extracellular vesicles heterogeneity (grant number: 202233FTW8).

Subtopic Biologic activity and therapeutic potency of EVs 1

Extracellular vesicles derived from clonally expanded immortalized mesenchymal stromal cells reduce neuroinflammation in pre‐clinical sepsis

Presenter: Maria Carolina Barbosa da Silva

University of Toronto, Toronto, Canada

Introduction : Sepsis is an exacerbated host response against an infection. Sepsis‐associated encephalopathies (SAE) are neurological complications which occur during or after sepsis events. Currently, there is no treatment available for SAE‐induced neurological damage. Therefore, it is necessary to investigate new therapeutic approaches. Mesenchymal stromal cells (MSCs) have a well‐established immunomodulatory capacity. Our group demonstrated beneficial effects of MSC therapy in reducing neuroinflammation and cognitive damage caused by SAE; further, MSCs seem to act in a paracrine way. Extracellular vesicles (EVs) are lipid bilayer‐delimited particles carrying proteins, nucleic acids, and lipids released by cells. EVs from MSCs (MSC‐EVs) are promising candidates for different types of therapy due to their capacity to carry and deliver mediators from MSCs.

Methods : Our aim was to evaluate the effects of MSC‐EV therapy in an experimental model of sepsis. Our readouts were survival, peripheral inflammation and neuroinflammation. For this, adult male C57Bl/06 mice underwent surgery for sepsis induction. Six h after surgery, mice were treated with MSC‐EVs intravenously.

Results : MSC‐EV therapy improved survival rates 72 h after surgery. At the same timepoint, MSC‐EV therapy reduced peripheral inflammation. In the cortex and hippocampus, also at 72 h, MSC‐EV therapy led to a reduction in astrogliosis and microglial activation. Despite the reduction in neuroinflammation, we did not observe any changes in the local synthesis of cytokines or blood‐brain barrier integrity. In cultured microglial‐BV2 cells, stimulated with LPS, MSC‐EVs reduced the expression of IL‐6 and IL‐1b, suggesting an anti‐inflammatory effect. Finally, this effect could be blocked by Dynasore—an inhibitor of clathrin/dynamin‐mediated endocytosis.

Summary/Conclusion : Thus, we can conclude that, in our model, a single dose‐therapy with MSC‐EVs reduced peripheral inflammation and neuroinflammation, and improved survival rates. Further, using an in vitro model, we show that these EV‐mediated effects are likely dependent on their uptake by the cells, highlighting the potential of EVs as tissue‐specific targeted therapies.

Red blood cell‐derived extracellular vesicles as alternative vehicles of anti‐fibrotic miRNA mimics and their effect in in vitro models of liver fibrosis

Presenter: Elena Cecotti

University of Torino, Turin, Italy

Introduction : Liver fibrosis is a wound‐healing response that results from an imbalance in extracellular matrix (ECM) synthesis and degradation following liver injury. Several studies evaluated the anti‐fibrotic capacity of extracellular vesicles (EVs) derived from different sources and highlighted the correlation with the EV‐miRNA content. Red blood cell‐derived EVs (RBC‐EVs) represent a natural delivery system with several advantages over EVs from other sources. They are biocompatible, obtained in a cost‐effective way and with low horizontal gene transfer. In this study, we evaluate the effects of RBC‐EVs loaded with specific anti‐fibrotic miRNA mimics in bi‐dimensional (2D) and three‐dimensional (3D) models of hepatic fibrosis.

Methods : RBCs were obtained from healthy donors, and EVs were purified by ultracentrifugation and characterized in accordance with ISEV guidelines through NTA, transmission electron microscopy, flow cytometry and western blot. RBC‐EVs were loaded by electroporation with anti‐fibrotic miRNA mimics and their therapeutic effect was tested on TGF‐β1 (10 ng/mL)‐activated human hepatic stellate cells (LX‐2) and in a 3D model of liver spheroids. Different RBC‐EV preparations were obtained by loading a single anti‐fibrotic miRNA mimic, and the EV treatment was performed with a single EV dose (5000 EV/cell, 10,000 EV/cell, 20,000 EV/cell) or with a combination of two RBC‐EV preparations loaded with different miRNA mimics. By real‐time PCR and western blotting, we evaluated the expression of the pro‐fibrotic markers alpha‐SMA and COL1A1.

Results : By real‐time PCR, we assessed the presence of anti‐fibrotic miRNA mimics inside electroporated RBC‐EVs, confirming the efficacy of the loading protocol. In activated LX‐2 cells and in 3D liver spheroids, engineered RBC‐EVs significantly downregulated the expression of the pro‐fibrotic markers alpha‐SMA and COL1A1 both at RNA and protein levels. In particular, the treatment with specific combinations of RBC‐EVs seems to augment their anti‐fibrotic potential with respect to the treatment with the single EV preparation.

Summary/Conclusion : Engineered RBC‐EVs represent a natural RNA‐delivery vehicle that can be directly transferred to the target cells, modulating their activated pro‐fibrotic phenotype and opening new perspectives for future clinical application.

Funding : This study was funded by the European Union—Next Generation EU, Mission 4, Component 2, Investment line 1.4, CUP B93D21010860004.

Extracellular vesicles to fight dementia: a journey across the blood‐brain barrier

Diogo Fortunato 1 , Henrique Nogueira Pinto 1 , Stephanie Beekhuis‐Hoekstra 1 , Leontien Bosch 2 , Susanne van der Pol 1 , Manon Karsten 1 , Negisa Seyed Toutounchi 1 , Remco Klaassen 3 , Joke den Haan 1 , August B. Smit 3 , Ruud Toonen 4 , Vivi Heine 5,6 , Michiel Pegtel 2 , Elga de Vries 1

1 Department of Molecular Cell Biology and Immunology, Amsterdam UMC location Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 2 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC location Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 3 Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 4 Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 5 Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 6 Department of Child and Adolescent Psychiatry, Emma Center for Personalized Medicine, Emma Children's Hospital, Amsterdam UMC location Vrije Universiteit, Amsterdam Neuroscience, The Netherlands

Introduction : Neurodegenerative diseases (NDs) are particularly devastating, with no available cure and only a few, largely palliative therapeutic options. ND patients display variable degrees of progressive neuroinflammation, neurodegeneration and blood‐brain barrier (BBB) dysfunction. Most promising recently approved therapies are based on monoclonal antibodies. Such therapies manage symptoms but fail to halt disease progression. Extracellular vesicles (EVs) hold significant potential to revolutionize ND therapeutics. Depending on donor cells and composition, EVs can cross the BBB via transcytosis, dampen inflammation and promote regeneration. Here, we quantified the transport of brain cell‐derived EVs across the BBB and characterized their cargo.

Methods : Human‐induced pluripotent stem cells (hiPSC) were differentiated into brain cells and used for small EV (sEV < 200 nm) production. Pre‐cleared conditioned medium was concentrated with ultrafiltration, and sEVs were purified by size‐exclusion chromatography. sEVs were characterized using tunable resistive pulse sensing (size, concentration) and cryogenic electron microscopy. EV‐associated markers were detected by capillary western blot, single‐particle flow analysis (MIFlowCyt‐EV compliant) and microscopy. The BBB was modelled in vitro using brain microvascular endothelial cells (BMEC), hCMEC/D3, or hiPSC‐derived BMEC barrier monolayers on the apical side of cell culture inserts, with or without brain pericytes on the underside. Uptake, transport and permeability of fluorescently labelled EVs and liposomes in in vitro BBB models were quantified by single‐particle flow analysis.

Results : BMEC barrier remained intact upon sEV treatment during the timeframe of transport assays. sEVs quickly bound to the apical BMEC membrane; uptake gradually increased and peaked at 4 h. Transcytosis mirrored uptake dynamics, peaking at 6 h after 30 min CFSE‐labelled EV pulses. Blockage of endo/exocytosis and intracellular transport mechanisms using different pharmacological inhibitors evidenced important pathways for sEV transcytosis across BMEC monolayers. EVs derived from BMECs, astrocytes and neuroepithelial stem cells showed higher BBB transcytosis capacity. Proteomics of transcytosed EVs revealed shared pro‐transcytotic mediators, but also distinct immunomodulatory cargo across different EV types tested.

Summary/Conclusion : We devised a robust screening assay to measure paracellular and transcellular EV transport across in vitro BBB with single‐vesicle resolution. EV types showing high BBB transcytosis capacity will be tested in vivo to assess brain penetrance and anti‐inflammatory/regenerative cargo delivery.

Targeted Migration of Milk‐Derived Exosomes to Brain Inflammation Sites: Implications for Neurological Therapeutics

Presenter: Arwa Qasem

Tel Aviv University, Tel Aviv, Israel

Introduction : Milk‐derived exosomes have emerged as potential therapeutic vehicles for brain injuries due to their ability to cross biological barriers. This study investigates the migration of milk exosomes to inflammation sites in the brain.

Methods : Using a mouse model, we induced localized inflammation in the striatum by injecting either lipopolysaccharide (LPS) or endothelin‐1. Milk exosomes were administered intranasally or orally (per os) to assess their biodistribution.

Results : Our findings demonstrate that milk‐derived exosomes successfully migrate to the lesion site in the brain, regardless of the administration route.

Summary/Conclusion : This targeted accumulation of exosomes at the site of inflammation suggests their potential as natural nanocarriers for delivering therapeutic agents to damaged brain areas. The ability of milk exosomes to reach inflamed brain regions after non‐invasive administration highlights their promise as a novel approach for treating various neurological disorders and brain injuries.

Subtopic Immunotherapy 1

Harnessing collagen binding separates two functionally distinct subpopulation of milk extracellular vesicles regarding bone regeneration

Presenter: Peng Wang

Radboud UMC, Nijmegen, The Netherlands

Introduction : Difference in the composition of the protein corona formed outside at the extracellular vesicle (EVs) membrane have gained interest, as this potentially determines the functional differences of EVs subpopulations. Emerging evidence indicates that EVs inherently interact with the meshwork of the extracellular matrix (ECM). In this study we utilized collagen binding to separate two functional subpopulations of EVs from cow milk (mEVs) regarding osteogenesis ex vivo and bone regeneration using a bone defect model in osteoporotic rats. As Annexin V (AnxV) is present in cow milk and known as an EV marker that can bind to collagen, we next studied its role in mEVs‐induced osteogenesis.

Methods : Two fluorochromes (calcein green and alizarin red) were utilized for bone formation labelling in the ex vivo human bone defect model. The uptake of collagen‐bound mEVs (cb+ mEVs) by human mesenchymal stem cells (hBMSCs) was assessed using confocal microscopy. The expression of AnxV on mEVs was validated by western blotting, and depletion of the AnxV‐expressing mEV subpopulation, AnxV‐depleted mEVs (AnxV− mEVs) was obtained by immunomagnetic separation. Collagen hydrogels loaded with mEVs, collagen‐unbound mEVs (cb− mEVs) and AnxV‐mEVs were implanted in femoral condyle bone defects to evaluate their functional performances on bone regeneration.

Results : mEVs stimulated mineral apposition as more incorporation of fluorochromes was observed. cb+ mEVs, but not cb‐ mEVs, strongly promoted hBMSCs mineralization cultured on collagen‐coated plates or in 3D collagen hydrogel. Removal of the cb+ mEVs led to a reduction of AnxV protein level in mEVs. Addition of AnxV neutralizing antibodies or depleting AnxV‐positive EV markedly diminished mEV‐induced mineralization by hBMSCs. In vivo, micro‐CT analyses at 4 weeks post‐implantation revealed that only the mEVs‐col group exhibited a 2.37‐fold increase in bone tissue volume/total tissue volume (BV/TV%) and a slight increase in bone mineral density (BMD) compared to the control‐col group.

Summary/Conclusion : Utilizing the collagen‐binding property of EVs realizes selection of a subpopulation of mEVs that is positive for AnxV and has robust stimulatory effects on bone regeneration. This approach of selecting functional EV subpopulations is an important step forward in their therapeutic application to enhance bone regeneration.

Leverageing extracellular vesicles for enhanced imaging: insights from preclinical research

Sara Mathlouthi 1 , Beate Rinner 2 , Stefano Salmaso 1 , Paolo Caliceti 1 , Lukasz Kuryk 1,3 , Mariangela Garofalo 1

1 University of Padua, Padua, Italy; 2 Medical University of Graz, Graz, Austria; 3 National Institute of Public Health ‐ NIH, Warsaw, Poland

Introduction : Nano‐ and micro‐sized extracellular vesicles (EVs) are naturally occurring carriers of regulatory macromolecules that play a crucial role in physiological intercellular communication. EVs derived from cancer cells prove marked tropism for tumours, as shown by numerous in vivo studies.

Methods : Encapsulating cancer cell‐derived EVs with fluorescent molecules or anticancer agents offers a promising approach for cancer treatment. By labelling plasma‐derived EVs with indocyanine green (ICG) and tracking their biodistribution through in vivo and ex vivo imaging technologies, as a detection system for the characterization of the whole‐body biodistribution of the EV‐formulations, we provided evidence that a selective homing of cancer‐derived EVs to the neoplastic tissue originating the vesicles exists.

Results : Our results demonstrate the presence of nanoparticles in the blood of advanced melanoma patients that exhibit a selective homing to the neoplastic tissue, a trait not observed in healthy individuals. This remarkable ability facilitates the precise labelling of cancer cells with fluorescent markers, delivering a diagnostic fluorescent agent directly into the neoplastic tissue when administered systemically, thus enabling the accurate localization of metastases. Our findings obtained in in vivo preclinical studies show that EVs generated from a tumour type have a tropism for cancers originating from different tissues, and even from different species. These heterologous and cross‐species homing capabilities suggest for EVs a wider role in cell‐to cell communication during tumour progression, which was not expected before.

Summary/Conclusion : Collectively, this research could potentially be combined with the administration of drugs or radiotherapeutic isotopes to address tumour micrometastasis, thus opening new avenues for the selective delivery of diagnostic/therapeutic agents to solid tumours.

Mycobacterial EVs from different strains as potential tuberculosis vaccine candidates

Han‐Gyu Choi 1 , Dahee Shim 2 , Sang Chul Park 2 , Hwa‐Jung Kim 1

1 Chungnam National University, Daejeon, South Korea; 2 Hallym University, Chuncheon, South Korea

Introduction : Tuberculosis (TB) remains a global health concern, and there is an urgent need for effective vaccines. Extracellular vesicles (EVs) derived from mycobacteria have emerged as promising vaccine candidates. This study investigated the immunomodulatory properties and protective efficacy of EVs derived from different mycobacterial strains (BCG, Ra, and Rv).

Methods : Mycobacterial strains were initially cultured in 7H9 medium (seed culture, OD600 = 1.0), followed by main culture in Sauton's medium (OD600 = 0.1) under static conditions for 3 weeks. EVs were isolated from culture supernatants using sequential filtration (TFF/UF/DF 300 kDa) and concentrated to 15 mL with PBS dialysis. EV characterization included NTA for size distribution and concentration analysis, and microBCA for protein quantification. Immunological properties were assessed by analysing DC surface markers and cytokine production upon EV treatment. T cell proliferation was evaluated using OT2 mouse splenocytes. Additionally, in vitro Mtb growth inhibition assays and in vivo prophylactic vaccination studies were performed.

Results : Isolated EVs showed characteristic size distributions and protein content. EV treatment induced significant changes in DC surface markers and cytokine profiles, demonstrating their immunomodulatory potential. OT2 mouse splenocytes showed enhanced proliferation in response to EV treatment. Notably, EVs demonstrated significant inhibition of Mtb growth in vitro. In prophylactic vaccination studies, EV‐immunized mice showed reduced bacterial burden upon Mtb challenge.

Summary/Conclusion : Our findings demonstrate that mycobacterial EVs possess immunomodulatory properties and provide protective immunity against Mtb infection, suggesting their potential as novel TB vaccine candidates. These results provide a foundation for further development of EV‐based TB vaccines.

Funding : This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS‐2024‐00463203).

Extracellular vesicles from plasma of patients with gastric cancer modulate T cell immune phenotype

Presenter: Yashna Chabria

Trinity College Dublin, Dublin, Ireland

Introduction : Gastric cancer (GC) remains a significant global health burden with a poor prognosis. Advances in immune checkpoint inhibitors benefit only a subset of patients, highlighting persistent immune evasion that limits efficacy. Our previous findings demonstrated that drug‐resistant breast cancer cells and their EVs exhibit increased PD‐L1 and TGFβ1 levels, suppressing T‐cell activity. Additionally, plasma EVs‐associated TGFβ1 levels inversely correlated with HER2‐targeted therapy response (1). Therefore, this study aims to examine the impact of EVs from GC patients’ plasma, collected pre‐ and post‐FLOT chemotherapy, on activated T cells.

Methods : Plasma samples from patients with GC undergoing biweekly cycles of neoadjuvant fluorouracil, leucovorin, oxaliplatin and docetaxel (FLOT) chemotherapy were procured longitudinally, including both pre‐ and post‐treatment, following ethical approval and informed consent. EVs were collected using size‐exclusion chromatography and characterised by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, immunoblotting, and ELISA. To evaluate the influences of EVs on immune cells, PBMCs from healthy donors were isolated using Ficoll density gradient centrifugation, activated, and co‐cultured with EVs at varying concentrations for 24 h. The impact of EVs was assessed by analysing cell viability and immune cell markers (CD3, CD4, CD8, CD69, CD25, CD62L) using flow cytometry.

Results : Initial results highlighted successful collection of EVs in the size range of 200–650 nm, had detectable EVs surface markers CD9 and syntenin and an absence of calnexin. No substantial variations in EVs concentrations from plasma samples were observed. Increased levels of activation markers CD69 and CD25 were observed in both CD4⁺ and CD8⁺ T‐cell populations treated with GC‐EVs isolated from patients undergoing chemotherapy. Notably, these PBMCs were activated prior to EVs treatment; therefore, further increased CD69 and CD25 expression suggests an additional modulatory effect elicited by EVs on T‐cell activation.

Summary/Conclusion : This study highlights the role of GC‐EVs in promoting T‐cell activation and immune modulation, thereby potentially shaping the tumour microenvironment. Further research is warranted to explore EVs patterns during GC progression and their impact on immune function.

References : (1) V. G. Martinez et al. Oncoimmunology. 2017;6(12): e1362530. (2) J. A. Welsh et al. Journal of Extracellular Vesicles (MISEV2023). 2024;13(2): e12404.

Funding : This study was supported by HEA's North‐South funding of AICRIStart (aicri.org/aicristart), CLuB (clubcancer.ie), and Trinity College Dublin's BOOST Award.

Subtopic EV in metabolic diseases 1

Artemisiae scopariae Herba‐derived extracellular vesicle‐like particles confers anti‐inflammatory effects in liver injury via regulating IL‐17 signalling pathways

Presenter: Weiyin Zeng

Guangzhou University of Chinese Medicine, Guangzhou, People's Republic of China

Introduction : The incidence of alcoholic liver disease (ALD) is increasing in China; however, no effective natural ingredient‐based drugs have been approved for market release to date. Traditional Chinese medicine, specifically Artemisiae scopariae Herba, is a natural plant that has demonstrated significant efficacy in alleviating hangovers, revitalizing the spleen, and protecting both the intestines and liver. Chinese herbal derived extracellular vesicle‐like particles (CHM‐EVLP) are characterized by their stability, target specificity, and bioactivity, retaining the pharmacological properties of their source plants. They are readily absorbed by macrophages across various organs and serve as natural drug carriers within traditional Chinese medicine. This study investigates the anti‐hangover and hepatoprotective effects of Artemisiae scopariae Herba, leverageing the unique properties of CHM‐EVLP. Our objective was to explore the potential therapeutic effects and underlying mechanisms of action of Artemisiae scopariae Herba derived extracellular vesicle‐like particles (AH‐EVLP) in mitigating alcoholism and safeguarding the liver against alcohol‐induced damage in vivo.

Methods : AH‐EVLP were isolated from dried Artemisiae scoparia e Herba juice using ultracentrifugation and characterized by TEM and nano‐flow analysis. Their proteins, RNAs, and lipids were identified using silver‐staining, agarose gel, and thin‐layer analysis. The alcohol detoxification effect was evaluated through recovery reflex and serum alcohol and acetaldehyde levels. To study hepatoprotective effects against acute alcoholic liver injury (AALI) in mice, liver injury markers, inflammation, oxidative stress, damage, and apoptosis were monitored. Potential active targets and mechanisms for AALI treatment were investigated using WB, qRT‐PCR, IHC, RNA sequencing, LC‐MS, and network pharmacology.

Results : AH‐EVLP resemble mammalian extracellular nanovesicles in size and shape. Their administration alleviated toxic symptoms and liver injury in a dose‐dependent manner. They are also anticipated to reduce alcohol‐induced liver inflammation and oxidative stress‐related hepatocyte death by inhibiting the IL‐17 signalling pathway and lowering Ripk4 expression in mice with acute alcohol intoxication.

Summary/Conclusion : AH‐EVLP has the potential to mitigate alcohol‐induced liver injury by reducing the inflammatory response, suggesting its development as an innovative nutraceutical or pharmacological intervention for the treatment of alcoholic liver disease (ALD).

Plant‐derived extracellular vesicles ameliorate aGVHD after allo‐HSCT by inhibiting NLRP3 inflammatory vesicle activation

Presenter: Liping Liu

Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China

Introduction : Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is currently regarded as the most effective therapeutic approach for haematological malignancies. However, acute graft‐versus‐host disease (aGVHD) remains a major obstacle to the advancement of allo‐HSCT. Recent studies have reported that plant‐derived extracellular vesicles significantly inhibits the proliferation of T and B lymphocytes. However, whether Plant‐Derived Extracellular Vesicles can exert therapeutic effects on aGVHD following allo‐HSCT remains unclear. The aim of this study was to investigate the efficacy and potential mechanisms of plant‐derived extracellular vesicles (PDEVs) in the prevention and treatment of aGVHD. We demonstrated that PDEVs effectively prevent the onset and progression of aGVHD in mice. The underlying mechanism may involve immunomodulation and inhibition of NLRP3/IL‐1β pathway activation.

Methods : Plant‐derived extracellular vesicles were extracted and purified using traditional ultracentrifugation and density gradient centrifugation. To establish a humanized aGVHD mouse model, we utilized immunodeficient NOD‐PrkdcscidIL2rPnull mice. PDEVs were administered by oral gavage every other day, starting one week prior to irradiation, while mice in the aGVHD control group received the same volume of PBS. The therapeutic efficacy of PDEVs on aGVHD in mice was assessed through clinical scoring, survival rate analysis, and histopathological examination of target organs. The effects of PDEVs on T lymphocyte subsets in target organs and peripheral blood of GVHD mice were analysed by flow cytometry.

Results : (1) We demonstrate that PDEVs exhibit immunomodulatory properties by inhibiting the proliferation of effector T cells and promoting the differentiation of T cell subsets into regulatory T cells (Tregs). (2) PDEVs significantly alleviated aGVHD clinical symptoms and prolonged the survival time of aGVHD mice, reduced the pathological tissue damage. (3) PDEVs modulated the proportion of T‐cell subsets in aGVHD mice. Eventually it induced T cell differentiation towards immune tolerance. (4) PDEVs down‐regulated the expression of NLRP3 and its downstream ASC, Caspase‐1. It suggests that PDEVs may ameliorate intestinal barrier damage and attenuate aGVHD immune response by inhibiting NLRP3/IL‐1β activation.

Summary/Conclusion : PDEVs prevented the development of aGVHD in mice, which may improve the intestinal barrier damage by inhibiting the activation of NLRP3/IL‐1β, thereby attenuating the aGVHD.

Day 2, 25th April Disease and therapy

Subtopic Biologic activity and therapeutic potency of EVs 2

The interaction between host and gut next‐generation probiotics via extracellular vesicles in Alzheimer's disease

Hui‐Chun Lin 1 , Bao‐Hong Lee 2 , Tang‐Long Shen 3 , Xiao‐Yang Chu 1 , Yi‐Wen Wang 1 , Ya‐Chun Yang 1 , Wei‐Hsuan Hsu 1 *

1 Department of Food Safety/Hygiene and Risk Management, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2 Department of Horticulture, National Chiayi University, Chiayi, Taiwan; 3 Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan

Introduction : This study explores the interaction between the host and gut next‐generation probiotics (NGP), focusing on the role of extracellular vesicles (EVs) in Alzheimer's disease (AD). In Alzheimer's patients, it has been observed that gut barrier function is impaired, and there is a significant imbalance in gut microbiota, characterized by a decrease in anti‐inflammatory bacteria and an increase in pro‐inflammatory bacteria. Literature suggests that supplementation with NGP can alleviate the progression of AD, but there are few studies examining the impact of NGP‐EVs on AD. Therefore, we investigated the protective mechanisms of NGP‐derived EVs against AD.

Methods : NGP‐EVs were isolated using gradient centrifugation and size exclusion chromatography (SEC). Aβ was induced in Caco‐2 cell models to simulate cellular damage in AD. APP knock‐in transgenic mice (TG mice) were also used to model AD, with faecal transplantation to reconstruct their gut microbiota.

Results : The results showed that NGP‐EVs significantly increased cell survival rates and improved gut barrier function, indicating protective effects. In animal experiments, administering NGP‐EVs to TG mice via oral gavage improved their gut microbiota composition by increasing the proportion of anti‐inflammatory bacteria and reducing pro‐inflammatory bacteria. This beneficial microbiota composition was sustained for a period of time and concurrently reduced the cognitive impairment caused by AD in the animals.

Summary/Conclusion : These findings suggest that NGP‐EVs enhance the interaction between the host and gut microbiota along the gut‐brain axis, alleviating cellular damage and cognitive decline associated with Alzheimer's disease. This research supports the development of NGP‐EVs as a novel microbial agent for AD treatment and underscores the potential of gut microbiota in neurodegenerative diseases.

Influence of a static magnetic field on mesenchymal stromal cell‐derived extracellular vesicles

Joseph Alexander 1 , Emma Jackson 1 , Justine Clarke 1 , Evangelia Bochti 1 , Margaret Mullin 2 , Savvas Ioannou 1 , Catherine Berry 1

1 Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, UK; 2 Glasgow Imaging Facility, University of Glasgow, Glasgow, UK

Introduction : Mesenchymal stromal cells (MSCs) are widely used in disease models and clinics for bone tissue engineering (BTE). MSC phenotype can be controlled through cell culture conditions, such as exposure to a static magnetic field, which has been proposed for use in stem cell therapy due to the promotion of osteogenic differentiation. It has been established that MSC therapeutic benefits are largely driven by paracrine activity that modulates tissue regeneration and repair, particularly mediated through the release of MSC‐derived extracellular vesicles (EVs). EVs carry functional cargo, such as proteins, RNA and metabolites, which mediate cell‐cell communication. Recent research has focused on characterising EV cargo and functional relevance, with a view toward potential therapeutics. Here, we aim to understand how exposing MSCs to a constant static magnetic field may influence MSC‐EV cargo, in terms of osteogenic mediators for BTE.

Methods : MSCs were cultured in EV‐depleted FBS/DMEM media, either exposed to a constant static magnetic field (340 mT) or not, with media collections every 48 h. MSC‐derived EVs were isolated using differential centrifugation and ultrafiltration, further undergoing size exclusion chromatography. The resultant MSC‐EVs were characterised in terms of morphology, size and concentration by transmission electron microscopy (TEM), microBCA (total protein), dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). MSC‐EV cargo was assessed at a cytokine level, with further evaluations via RNA‐seq to identify any key differences between our EV populations.

Results : MSC‐EVs were successfully isolated from parent MSCs which were cultured either with or without an SMF. Differences were noted in the isolation technique used, with ultracentrifugation generating EV preparations with less protein contamination than ultrafiltration. MSC‐EVs were in the size range of exosomes and microvesicles, with similar concentrations derived from both parent cell populations. Cytokine analysis indicated possible differences when parent cells are exposed to a SMF, whilst RNA‐seq analysis is ongoing.

Summary/Conclusion : MSC‐derived EVs are considered to play a crucial role in tissue repair and regeneration. MSC preconditioning can lead to improved yields, stability and functional cargo. Here, an SMF is shown to generate MSC‐EVs with differing cargo, which may have potential in BTE.

Funding : This study is supported by EPSRC (UKRI).

Intestinal Lactobacillus johnsonii‐ derived extracellular vesicles suppress Clostridium scindens abundance and reduce toxic bile acid production

Presenter: Chih Yao Hou

National Kaohsiung University of Science and Technology, Taiwan, People's Republic of China

Introduction : Gut microbiota dysbiosis and altered bile acid metabolism are linked to neurodegenerative diseases like Alzheimer's disease (AD). While most studies focus on colonic bacteria, we propose that the small intestine microbiota, key in bile acid recycling, plays a more significant role in AD pathology. This study examines the impact of small intestine microbiota changes on bile acid metabolism in AD, using amyloid precursor protein knock‐in (APPNL‐G‐F/NL‐G‐F) transgenic mice.

Methods : APPNL‐G‐F/NL‐G‐F transgenic mice were used to explore the relationship between small intestine microbiota changes and bile acid metabolism under beta‐amyloid (Aβ) accumulation. The effects of Lactobacillus johnsonii ‐derived extracellular vesicles (EVs) on inhibiting Clostridium scindens and toxic bile acid production were tested at concentrations of 10 1 ⁰ and 10 1 2 /mL. Small RNA removal from EVs was conducted to investigate the mechanism of their inhibitory action.

Results : Beta‐amyloid accumulation correlated with a decrease in Lactobacillus johnsonii and an increase in Clostridium species, which produce toxic bile acids. L. johnsonii ‐derived EVs effectively inhibited C. scindens growth and lithocholic acid (TLA) production at non‐cytotoxic concentrations. The removal of small RNA from EVs eliminated their inhibitory effect, suggesting a mechanistic role of small RNA.

Summary/Conclusion : The small intestine microbiota appears more impactful than colonic microbiota in AD pathology, where disruptions can alter bile acid metabolism, exacerbating AD. L. johnsonii ‐derived EVs show promise as probiotics for inhibiting toxic bile acid production, presenting a potential therapeutic pathway for AD.

Funding : This research was primarily funded by E‐DA Hospital (Grant No. NCKUEDA11008) and the National Science and Technology Council (NSTC), Taiwan (Grant Nos. NSTC 111‐2636‐B‐006‐014, 112‐2221‐E‐992‐002‐MY3, 110‐2320‐B‐992‐001‐MY3, and 111‐2221‐E‐328‐001‐MY3). Additional support was provided by the Higher Education Sprout Project, Ministry of Education, to National Cheng Kung University (NCKU). The authors also acknowledge technical support from the Bioimaging Core Facility of the National Core Facility for Biopharmaceuticals, NSTC, Taiwan.

TIPS microcarriers induce increased pro‐angiogenic properties of MSC‐derived small extracellular vesicles in vitro

Sinziana Popescu 1 , Haley Stang 2 , Richard Day 2 , Costanza Emanueli 1

1 National Heart and Lung Institute, Imperial College London, London, UK; 2 Department of Ageing, Rheumatology & Regenerative Medicine, UCL Division of Medicine, University College London, London, UK

Introduction : Small extracellular vesicles (sEVs) produced by cultured mesenchymal stem cells (MSCs) are currently investigated as advanced therapy medicinal products in cardiology. Among several cardioprotective activities, MSC‐sEVs pro‐angiogenic properties are of interest for treating ischaemic vascular disease. Environmental cues, such as the attachment substrate, modulate the MSC secretome. Here, we developed polymeric microspheres with highly tunable physical properties to act as cell substrates for 3D cultures and explored how the microcarrier size range impacts the MSC‐sEV yield, formulation and in vitro pro‐angiogenic activity.

Methods : Porous microcarriers were produced from poly(lactic‐co‐glycollic) acid using thermally induced phase separation (TIPS) within three size ranges: small = 250–355 µm, medium = 425–500 µm, and large = 600–710 µm. TIPS ultrastructural features, porosity, and morphology pre‐ and post‐cell attachment were examined (scanning electron microscopy and Morphologi‐G3, Malvern). Suspension cultures of MSCs attached to the microcarriers were maintained in vertical‐wheel bioreactors (PBS Biotech) for up to 26 days. For sequential MSC‐secretome harvesting (at days 6, 12, 21 and 26), the culture media was temporarily switched (24 h) to serum‐free. MSC‐sEVs were isolated by size exclusion chromatography (qEV columns; Izon). MSC‐sEV size‐distribution and concentration were determined by Nanosight (Malvern). The tetraspanins expression profile of the MSC‐sEVs was assessed by Exoview‐R100 (NanoView). The MSC‐sEV proangiogenic potential was assessed by studying their capacity to induce proliferation (WST‐1 assay, Roche) of human primary cardiac microvascular endothelial cells (ECs) prepared from both male and female donors.

Results : Ultrastructural analysis of the microcarrier surfaces revealed similar hierarchical structures for all microcarrier groups. Particle diameters were not significantly affected by the culture. Similar MSC‐sEV yields were recovered from all microcarriers, with diameter peaks at 100–117 nm. Tetraspanin expression changed with the TIPS size: at Day‐6, CD63 expression was 74.5% in small, 58.8% in medium and 65.6% in large TIPS; CD81: 13.2% in small, 8.9% in medium and 5.2% in large TIPS; CD9: 12.5% in small, 29.8% in medium and 20.9% in large TIPS. All MSC‐sEVs preparations induced EC proliferation, with maximal potency observed for MSCs‐EVs collected at Day‐6 (80.6% for Large TIPS) and Day‐12 (88.8% for small TIPS).

Summary/Conclusion : The growth of MSCs on TIPS microcarriers increases the pro‐angiogenic properties of MSC‐derived sEVs.

Development of a scalable isolation process for therapeutic applications of extracellular vesicles from human milk

Presenter: Patricia Hrasnova

Paracelsus Medical University, Salzburg, Austria

Introduction : The benefits of human milk in preventing the outcome of inflammatory gastrointestinal diseases sparked interest in its composition. Current research results suggest human milk extracellular vesicles (HMEVs) as a potential therapeutic candidate for reducing inflammation and inducing tissue regeneration. Multiple protocols for HMEV isolation were reported. However, for clinical use, scalable and robust manufacturing processes meeting the criteria for compliance with good manufacturing practices are required. Our lab recently developed an industrial isolation process for large‐scale production of cow milk derived EVs. Here we adapt it to the isolation of HMEVs.

Methods : Human milk obtained from a built‐up biobank was defatted, casein was depleted by coagulation, and the EVs were enriched by automated tangential flow filtration, followed by high‐resolution FPLC‐SECs. EV‐enriched fractions obtained during the purification were characterised by their physico‐chemical properties (NTA, TEM, zeta potential), molecular composition (protein, lipid, RNA, endotoxin content) and tested for biological activity (uptake into relevant cells, epithelial barrier penetration and anti‐inflammatory properties). Furthermore, we investigated how different storage conditions of human milk affect final EV products to optimize them for clinical use.

Results : By adapting a process previously developed for large‐scale isolation of cow milk EVs from industrial whey, we established a protocol for isolating HMEVs with high yields, reproducibility, and scalability to litres of starting material. Adding an HR‐FPLC‐SEC step eliminated co‐enriched non‐vesicular components, including lactoferrin and immunoglobulins, that could interfere with the biological function of EVs. HMEVs obtained this way exhibited biological activity, assessed by anti‐inflammatory functions in human primary intestinal epithelial cells, uptake into relevant cell lines, and transcytosis across gastrointestinal epithelial monolayers in vitro, including extravasation and tissue cell uptake after iv injection in mice. Comparisons of EVs isolated from fresh human milk and frozen at different stages allow for long‐term storage implementable in a clinical setting.

Summary/Conclusion : We established a large‐scale process for HMEV isolation, suitable for upscaling with higher yields compared to existing protocols. The HMEVs showed higher anti‐inflammatory activity compared to non‐EV co‐isolated fractions, corroborating bona fide therapeutically relevant activity of the vesicles. This protocol provides a starting point for the development of GMP‐compliant HMEV production for clinical applications.

Funding : This study was supported by CONSONANT (Salzburg, WISS2025; F2200397‐KZP).

Extracellular vesicles originated from mesenchymal cells of peritoneal dialysate reduce fibrosis

Presenter: Beáta Szebeni

Semmelweis University, Budapest, Hungary

Introduction : There are several data about the beneficial effect of cell therapy in different experimental fibrosis models. Using extracellular vesicles (EVs) as an alternative to cell therapy may have benefits, including lower immunogenicity, the ability to cross the blood‐brain barrier, and not inducing acute immune rejection.

Methods : Mesenchymal cells (MSCs) were isolated from peritoneal dialysate (PDE) of patients treated with continuous ambulatory peritoneal dialysis ( n = 3). Isolated MSCs were cultured in serum‐free DMEM‐F12 media for 24 h, and EVs were purified from their supernatant (300 mL) by tangential flow filtration (polysulphone hollow fibres, 20 nm pores), followed by size exclusion chromatography using 70 nm qEV columns. Characterization steps, such as nanoparticle tracking analysis, transmission electron microscopy, flow cytometry and western blot provided information about EV size/particle numbers, morphology, surface markers and protein content, respectively. After testing the internalization of DiI pre‐labelled EVs by primary peritoneal fibroblasts (pFB) isolated from peritoneal biopsy collected during removal of the Tenckhoff catheter, their importance in the regulation of fibroblast proliferation, collagen production and migration was tested.

Results : The isolated mesenchymal cells showed CK‐18, α‐SMA, CD73, CD105 and CD90 positivity and lack of CD34, HLA‐DR, CD45 and CD19 markers demonstrated by immunofluorescent staining and RT‐PCR. The separated EVs also showed stem cell marker and CK18 positivity, suggesting that their donor cells are the (isolated) MSCs, which had undergone mesothelial mesenchymal transition. EVs successfully internalized into pFBs and reduced their PDGF‐B‐induced proliferation, as demonstrated by the MTT assay. Moreover, these EVs decreased the TGF‐β‐induced collagen accumulation and EGF‐induced migration of these cells, as shown by Sirius Red and TAS assays, respectively.

Summary/Conclusion : Based on the potential antifibrotic properties of these EVs they may have therapeutic significance; however, in vivo testing is also necessary to justify this assumption.

Funding : This research was supported by the National Research, Development and Innovation Office (NKFIH) K‐142728, 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021, Semmelweis University, TKP2021‐EGA‐24, TKP2021‐EGA‐31, RRF‐2.3.1‐21‐2022‐00003, EKÖP‐2024‐53, EKÖP‐2024‐160, EKÖP‐2024‐162, Hungarian Research Network, ELKH‐POC‐2022‐024, Hungarian Academy of Sciences, and János Bolyai Research Scholarships.

Response of activated microglia to MSC‐derived EVs obtained from various isolation protocols

Lara Bieler 1,2 , Rodolphe Poupardin 3 , Fabian Blessing 1,2 , Eva Grasmann 4 , Daniela Auer 4 , Stefan Rund 4 , Carina Kals 4 , Christina Folie 4 , Elisabeth Bayer 4 , Eva Rohde 6,7 , Mario Gimona 4,6,7 , Sebastien Couillard‐Despres 1,2

1 Institute of Experimental Neuroregeneration, Paracelsus Medical University, Salzburg, Austria; 2 Austrian Cluster for Tissue Regeneration, Salzburg, Austria; 3 Institute of Experimental and Clinical Cell Therapy, Paracelsus Medical University, Salzburg, Austria; 4 GMP Laboratory Paracelsus Medical University, Salzburg, Austria; 5 Department of Transfusion Medicine, University Hospital Salzburg, Salzburg, Austria; 6 Research Program for Nanovesicular Therapies, Paracelsus Medical University Salzburg, Salzburg, Austria; 7 Ludwig Boltzman Institute for Nanovesicular Precision Medicine, Salzburg, Austria

Introduction : At present, no effective therapies are available for traumatic spinal cord injury (SCI). Following the initial trauma, persistent inflammation exacerbates neuronal and functional losses. Our recent findings highlight the potential of acute treatment with MSC‐derived extracellular vesicles (MSC‐EVs) in mitigating inflammation and its consequential damages after SCI. However, with numerous protocols available for EV preparation from MSC secretomes, we sought to identify the most efficacious method for generating EV‐preparations capable of reducing the pro‐inflammatory polarization of microglia.

Methods : We assessed the potential and biological activity of EV fractions produced with several protocols involving various pore sizes for tangential flow filtration (TFF), in optional combination with size exclusion chromatography, or ultrafiltration, and supplementation of the culture medium with human platelet lysate. EV fractions were applied in vitro on LPS‐treated BV‐2 microglia. Thereafter, cells and medium were harvested for analyses.

Results : We observed that all EV fractions reduced the production of nitric oxide by LPS‐treated BV‐2 microglia comparably, regardless of the preparation protocol used. Nevertheless, EVs enriched via small pore size (100 kDa) TFF led to higher anti‐inflammatory activity based on PCR analyses of pro‐inflammatory cytokine expression. We conducted a transcriptomic analysis comparing samples obtained from BV2 microglia which were activated with LPS and treated with the most and least active EV preparation or left untreated, which are currently being analysed. Elucidating the specific signalling pathways activated by potent anti‐inflammatory EV‐fractions, will provide valuable insights to further refine EV preparation protocols and identify molecular targets for combating inflammatory conditions.

Summary/Conclusion : Elucidating the specific signalling pathways activated by potent anti‐inflammatory EV‐fractions, will provide valuable insights to further refine EV preparation protocols and identify molecular targets for combating inflammatory conditions.

Matrix‐bound vesicles offer a potent alternative for regenerative applications in bone

Presenter: Genevieve Anghileri

Loughborough University, Loughborough, UK

Introduction : Matrix‐bound vesicles (MBVs) function as early sites of mineral formation in developing tissues such as cartilage and bone. However, therapeutic studies continue to prioritise media vesicles (MVs) isolated from suspension. Consequently, the therapeutic potential of MBVs remains largely undefined. This study sought to characterise MBVs and evaluate their therapeutic potential using in vitro and in vivo models.

Methods : MBVs and MVs isolated from primary human bone marrow MSCs cultured under osteogenic conditions were profiled using ZetaView, western blotting, super resolution microscopy (ONI), and mass spectrometry. Pro‐mineralisation capacity and mineral composition were assessed using alkaline phosphatase (ALP) assay, phosphate hydrolysis assays, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). Association with collagen and decellularized/demineralised bovine bone hydrogels (dECM) was determined to identify an appropriate material delivery system. In vitro models assessed the pro‐mineralisation potential of MBV delivery, and in vivo subcutaneous rodent models were applied to quantify EV clearance (DiR labelled) and de novo mineralisation potential when administered with collagen sponges.

Results : Significantly higher numbers of MBVs (2.56 × 10 12 ± 8.50 × 10 11 particles/mL) were liberated from the ECM compared to MVs recovered from conditioned medium (1.54 × 10 11 ± 2.73 × 10 10 particles/mL) ( p < 0.001). Phosphate hydrolysing enzymes and calcium channelling proteins required for mineralisation were elevated in MBVs, with confirmation of their localisation with tetraspanins confirmed at the single vesicle level by super‐resolution microscopy. MBVs rapidly hydrolysed polyphosphates (ATP) and significantly enhanced mineralisation in MSC cultures when compared with MVs as confirmed by turbidity assays and alizarin red staining ( p < 0.05). MBVs were comparatively enriched in electron dense material (TEM imaging), which was identified as hydroxyapatite by FTIR. In vitro and in vivo analysis confirmed MBVs exhibited a high binding affinity for collagen and dECM scaffolds (60%–70% binding in vitro), while MVs exhibited minimal binding (< 10%). DiR‐labelled MBVs were detectable on collagen sponges when implanted subcutaneously over a period of 4 weeks.

Summary/Conclusion : MBVs represent a comparatively abundant and potent source of vesicles for hard tissue regeneration and are compatible with collagen‐based delivery systems for sustained delivery in vivo.

Funding : This work was supported by the Academy of Medical Science, the EPSRC and the British Council.

Exosomes from periodontal ligament stem cells under mechanical stimuli promote lymphangiogenesis via lncRNA NEAT1 to alleviate alveolar bone disuse osteoporosis

Presenter: Yue Wang

China‐Japan Friendship Hospital, Beijing, People's Republic of China

Introduction : External mechanical stimuli are crucial for bone remodelling. After tooth loss, the alveolar bone loses physiological occlusal loading, leading to a degenerative condition known as alveolar bone disuse osteoporosis (ABDO), which affects bone mineral density and integrity, impacting oral function and posing challenges for dental restoration. Periodontal ligament stem cells (PDLSCs) are vital for alveolar bone remodelling under mechanical stress. While previous studies indicate that external stimuli enhance lymphangiogenesis in bone tissue, the role of lymphatic vessel formation in alveolar bone remodelling and the regulatory function of PDLSCs remain unclear.

Methods : A unilateral tooth loss model was established in Sprague–Dawley rats; microCT and immunofluorescence analyses were conducted to evaluate alveolar bone remodelling of opposing teeth and lymphatic markers after 8 weeks. In vitro, compression force was added to PDLSCs, and exosomes secreted under forces were extracted and applied to lymphatic endothelial cells (LEC) to assess changes in their lymphangiogenic capacity. A bilateral tooth loss rat model was developed, with exosomes injected into the periodontal ligament of the opposing teeth on one side and the other side as a control. Samples were collected after 4 and 8 weeks to evaluate bone remodelling and lymphatic markers. The differential expression of NEAT1 in exosomes was validated, and NEAT1 was knocked down in LEC to confirm its role in lymphatic vessel formation. RNA high‐throughput sequencing analysed gene expression changes post‐NEAT1 knockdown.

Results : After 8 weeks in the tooth loss model, the alveolar bone of the opposing teeth showed reduced BMD and BV/TV, along with the decreased bone markers OCN and RUNX2 and the lymphatic markers LYVE‐1 and PROX1 compared to controls. Exosomes derived from pressure‐stressed PDLSCs enhanced migration and tube formation in LEC. Following exosome injection, the BMD and BV/TV of the injection side increased, alongside the increased expression of OCN, RUNX2, LYVE‐1, and PROX1. LEC with NEAT1 knockdown exhibited decreased expression of lymphatic vessel markers. High‐throughput sequencing revealed that NEAT1 is highly associated with the VEGF‐VEGFR pathway, which is crucial in lymphangiogenesis.

Summary/Conclusion : Exosomes from PDLSCs under mechanical stress promote local lymphangiogenesis via NEAT1, facilitating bone formation and alleviating ABDO. Further investigation is needed to explore the underlying mechanisms.

Selective packageing of tumour suppressor miRNAs in extracellular vesicles of a parasitic whipworm

Presenter: Bradley Whitehead

Aarhus University, Aarhus, Denmark

Introduction : Trichuris trichiura , infects over 500 million people. T. suis , a whipworm of pigs provides a large mammal model of human infection with Trichuris spp. T. suis ova have been assessed in clinical trials for the treatment of inflammatory diseases, including ulcerative colitis, Crohn's disease, multiple sclerosis and allergic rhinitis. T. suis excretory/secretory (ES) products potently inhibit inflammatory responses in vitro; however, the role of extracellular vesicles (EVs) remains unknown.

Methods : EVs were isolated from adult and L1 T. suis ESP and characterised using NTA, AFM and LC‐MS/MS. EVs and supernatants (SN) were subjected to miRNA sequencing to identify and quantify miRNAs enriched in EVs. miRNAs were aligned to human miRNAs to identify orthologues of known miRNAs. Immune effects of T. suis were assessed in LPS stimulated THP‐1 cells. Functional effects of T. suis miRNAs were determined by transfection of SW480 colon cancer cells followed by proteomic analysis at 48 and 72 h.

Results : T. suis adults and L1 produce EVs as assessed by nanoparticle tracking analysis and AFM to confirm the presence of vesicular particles enriched from ESP. EV isolation was further confirmed by proteomic analysis. EVs did not inhibit TNF production by LPS stimulated THP‐1 cells, whereas ES and EV depleted fractions did. Analysis of T. suis secretory miRNA profiles revealed selective packageing of miRNAs in EV and SN and also by life stage. Tsu‐miR‐22‐3p and tsu‐miR‐29‐3p were enriched in EVs and their homologous miRNAs are well‐described tumour suppressor miRNAs. Therefore, the functionality of these miRNAs was tested in SW480 colon cancer cells. Proteomics at 48 and 72 h identified specific downregulation of proteins that are experimentally validated targets of hsa‐miR‐22‐3p and hsa‐miR‐29b‐3p and include well‐known tumour suppressors and are associated with poor outcomes in colon cancer.

Summary/Conclusion : This work shows that T. suis produces EVs but unlike that observed for A. suum , they do not contribute to the immune effects of T. suis ES. However, T. suis EVs are enriched in miRNAs that are functional mimetics of human tumour suppressor miRNAs, suggesting a role for EVs beyond host‐immune modulation.

Myofibroblast‐derived small EVs mediate cardiac cell crosstalk as regulators of cardiomyocyte function and are potential therapeutic targets in hypertrophic cardiomyopathy

Presenter: Rahul Sanwlani

University of Surrey, Guildford, UK

Introduction : Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic cardiomyopathy. Previously, we reported paracrine signalling‐mediated regulation of hiPSC‐derived cardiomyocyte electrophysiology by myofibroblasts. The role of extracellular vesicles (EVs) has been increasingly highlighted as mediators of pathophysiology.

Methods : HCM patient biopsies were processed for cardiac fibroblast (CF) isolation from tissue explants. To understand the role of CFs and their EVs in regulating cardiomyocyte function, in this study, we used the ALK5 inhibitor SD‐208 to characterise its effects on HCM CFs. Utilising this model of revering CF activation, we studied the effect of HCM CF small EVs in regulating cardiomyocyte function and electrophysiology.

Results : SD‐208 treatment did not significantly alter the viability and metabolic potential of HCM CFs. However, molecular characterisation suggested a significant reduction in mRNA levels of activation markers ACTA2, POSTN, FAP, COL1A1, IL‐6 and IL‐11 ( p < 0.0001) following treatment. A significant reduction in α‐SMA positive cells ( p = 0.0043) in flow cytometry and reduction in total collagen secretion ( p = 0.00062) occurred following treatment. CF EVs collected using size exclusion chromatography were observed to be in the small EV range and were enriched with EV marker proteins. Interestingly, SD‐208 treatment led to a 6.5‐fold reduction in EV particles secreted by HCM CFs ( p < 0.0001). Similarly, SD‐208 treatment had no significant impact on viability and metabolic potential of HEK‐293 cells but led to a dose‐dependent reduction in EV yield (< 0.01). HCM CF EVs were observed to regulate contractility and calcium signalling in hIPSC derived cardiomyocytes. Following treatment, the cardiomyocytes demonstrated the HCM phenotype.

Summary/Conclusion : Overall, these findings demonstrate antifibrotic activity of SD‐208. As SD‐208 treatment reverted myofibroblasts to fibroblasts, this allowed for investigations to understand the role of CF EVs in regulating cardiomyocyte function. These findings have also highlighted the potential role of SD‐208 as a regulator of EV biogenesis and secretion.

The therapeutic potential of differentiating myoblast‐derived extracellular vesicles to combatting inflammation in sarcopenia

Soyeon Won 1 , Kuo‐Fen Lee 2 , Ji Eun Lee 1

1 Sungkyunkwan University, Suwon, South Korea; 2 Salk Institute for Biological Studies, La Jolla, California, USA

Introduction : Treatment of sarcopenia remains a significant challenge in modern medicine. Stem cell therapy shows promise, but the hostile microenvironment of aged muscle tissue often compromises the survival and function of transplanted cells. This limitation underscores the need for alternative therapeutic strategies. Muscle‐derived extracellular vesicles (EVs) transfer regulatory factors that support muscle development and maintenance to neighbouring cells and their surrounding microenvironment for signal propagation. During myogenesis, muscle cells undergo significant changes in their membrane composition and protein expression patterns. Additionally, these cells show distinct patterns of EV secretion at different stages of differentiation. The ability to deliver multiple bioactive molecules simultaneously can ameliorate the hostile conditions commonly found in aged muscle tissue by normalizing inflammatory responses, enhancing protein synthesis, and improving myogenic cell function. This approach could potentially overcome limitations associated with current therapeutic strategies while providing a more effective treatment option for sarcopenia.

Methods : For isolation of differentiated myoblast‐derived EVs, C2C12 myoblasts were cultured in DMEM supplemented with exosome‐depleted 2% horse serum. Culture media was cleared of debris by centrifugation and concentrated using a 100 kDa MWCO filter. EVs were isolated from the concentrated media using total exosome isolation reagent. EVs samples were characterized using Nanosight and tandem mass spectrometry. EVs were added to LPS‐treated C2C12, followed by the induction of differentiation. Western blot was performed to examine the changes in inflammation and myogenic differentiation markers. Additionally, morphological observations were conducted to assess the degree of myogenic differentiation.

Results : Our analysis revealed that EVs secreted by differentiated myoblasts were enriched with distinct protein cargos, including anti‐inflammatory proteins, muscle fusion‐related proteins, and growth‐associated proteins, compared to those from non‐differentiating conditions. Treatment with differentiated myoblast‐derived EVs reduced LPS‐induced inflammatory responses in vitro, subsequently enhancing myogenic fusion and promoting the formation of mature myotubes.

Summary/Conclusion : In this study, we demonstrate that differentiated myoblast‐derived EV proteins have anti‐inflammatory properties, suggesting the potential of EVs as a therapeutic strategy for age‐related muscle loss in sarcopenia.

Funding : This study was supported by the CCAD lab of Ji Eun Lee at Sungkyunkwan University and supported by the National Research Foundation of Korea MSIP (RS‐2024‐00335208 and 2024‐00509145 to J.E.L., RS‐2024‐00450260 to S.W.).

Extracellular vesicles of probiotic Escherichia coli O83:K24:H31 decorated with allergens using the SpyCatcher‐SpyTag system for testing in allergy

Viktor Černý 1,2 , Stefan Heinl 3 , Eliška Krčmářová 2 , Michael Thaler 1 , Agnieszka Razim 1 , Jiří Hrdý 2 , Aleksandra Inic‐Kanada 1 , Ursula Wiedermann 1 , Irma Schabussova 1

1 Institute of Specific Prohylaxy and Tropical Medicine, Medical University of Vienna, Vienna, Austria; 2 First Faculty of Medicine, Charles University and General University Hospital in Prague,, Prague, Czech Republic; 3 BOKU University of Natural Resources and Life Sciences, Vienna, Austria

Introduction : Probiotic bacteria such as Escherichia coli O83:K24:H31 (EcO83) can help prevent allergy. Extracellular vesicles (EVs) retain the strain‐specific effect since they carry proteins, lipids, nucleic acids and MAMPs from the parent bacteria. Engineered vesicles expressing SpyCatcher can be decorated with SpyTag‐marked proteins, enabling the construction of probiotic EVs with bound allergen.

Methods : EcO83 strains with pMS470 plasmid encoding outer membrane protein A—SpyCatcher, mCherry‐SpyTag, and ovalbumin‐SpyTag were generated. OmpA‐SpC marked EcO83 extracellular vesicles (EcO83‐EVs) were isolated by ultracentrifugation (3 h, 150,000 × g , 4°C; Beckman Coulter 45Ti rotor) and purified using SEC (IZON automated fraction collector). Vesicles were characterised using Zetasizer. SpyTag‐marked proteins were expressed and gravity‐purified using NEBExpress Ni resin. Expression of ompA‐SpC, mCherry‐SpT and ovalbumin‐SpT in the respective strains was confirmed using western blotting (WB). OmpA‐SpC EcO83‐Evs were decorated with SpT‐marked proteins, purified using SEC, and the binding was tested with WB using antibodies against OmpA, mCherry and ovalbumin.

Results : We successfully isolated wild‐type EcO83‐EVs (mean size 115.6 nm) and ompA‐SpC EcO83‐EVs (mean size 107.8 nm) and confirmed the presence of protein, LPS and RNA. SpyTag‐marked protein expression as well as the successful binding of these proteins to OmpA‐SpC EcO83‐EVs were confirmed by WB.

Summary/Conclusion : SpC‐SpT decoration of EcO83‐EVs with ovalbumin will enable the study of effects probiotic strain‐derived EVs have after intranasal administration in the murine model of ovalbumin‐induced allergic airway inflammation. mCherry‐tagged EcO83‐EVs will be used for ex vivo uptake studies.

Funding : The work was supported by the OPJAC project, MSCA fellowships CZ‐UK2 (Reg. No. CZ.02.01.01/00/22_010/0008115), and OEAD (CZ 0772023).

Correlation of particle counts with ovarian function and IVF outcomes in couples with poor ovarian response in different groups of intraovarian platelet rich plasma, exosome, and the combination

Presenter: Yigit Cakiroglu

Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey

Introduction : Decrease in the follicular cohort pool labelled as ‘poor ovarian response’ (POR) is one of the common factors related to infertility and the management of these couples still poses a challenge. The aim of the study was to investigate the correlation of particle counts with ovarian function and IVF outcomes in couples with poor ovarian response in different groups of intraovarian injection of platelet‐rich plasma, exosome, and the combination.

Methods : This retrospective cohort study was conducted on POR patients who underwent intraovarian PRP (Group 1), exosome (Group 2) and the combination (PRP + exosome; Group 3). The PRP was prepared by centrifugation after separation of the plasma. Exosomes were isolated from human plasma and urine. Particle size distribution of the plasma and urine samples was analysed with the nanoparticle tracking analysis (NTA) technique. PRP, exosomes or both were administered to the ovarian tissue under transvaginal ultrasound guidance.

Results : Data of a total of 139 patients with the diagnosis of POR were analysed, retrospectively ( n = 50 for Group 1; n = 44 for Group 2; and n = 45 for Group 3). Mean particle concentration of the plasma was higher in exosome group compared to exosome + PRP group (25.4 × 10 9 ± 12.3 × 10 9 vs. 14.4 × 10 9 ± 9.5 × 10 9 ; p < 0.01). Mean particle concentrations of plasma and urine among patients with positive pregnancy tests revealed higher levels in the exosome group compared to the exosome + PRP group (23.4 × 10 9 ± 8.9 × 10 9 vs. 12.6 × 10 9 ± 8.0 × 10 9 , p < 0.01; 27.6 × 10 9 ± 15.0 × 10 9 vs. 11.3 × 10 9 ± 8.5 × 10 9 , p < 0.01, respectively). Analysis of ovarian reserve markers has revealed a statistically significant increase in AFC and AMH and a decrease in FSH levels in all the groups compared to pre‐procedure values. Total pregnancies were highest in Group 3 compared to Groups 1 and 2 (37.8% vs. 26.0% and 25.0%, respectively) without statistical significance.

Summary/Conclusion : In this study, mean particle concentrations of plasma and urine among patients with positive pregnancy test revealed higher levels in the exosome group compared to the exosome + PRP group. The total pregnancy rate was found to be highest among the patients who were performed the combination of exosome and PRP. Intraovarian injection of autologous PRP, exosome and the combination might be considered as an alternative strategy in women with POR.

B7‐H3 CAR‐T‐derived extracellular vesicles display antitumour activity in pancreatic cancer cells

Presenter: Rosalba Florio

University “G. d'Annunzio”, Chieti‐Pescara, Italy

Introduction : Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A new frontier of cell‐based immunotherapy in haematological malignancies is represented by chimeric antigen receptor (CAR)‐T cells. Unfortunately, their efficacy in solid tumours is still limited. Extracellular vesicles (EVs) emerged as regulators of the immune system. In this context, CAR‐T‐derived EVs represent a promising development of CAR‐T immunotherapy approaches. In this study, we explored the antitumour activity of CAR‐T‐derived EVs targeting B7‐H3, which is overexpressed in PC cells and resulted in safety in clinical trials.

Methods : B7‐H3 CAR‐T and control T cells (CTRL) were co‐cultured with a positive B7‐H3 PC cell line (L3.6pl) to evaluate the cytotoxicity of engineered T cells. EVs from B7‐H3 CAR‐T and CTRL‐T cells were isolated by cell sorting followed by ultracentrifugation and phenotypically characterized by a flow cytometry method patented by our Laboratory. EVs were characterized by atomic force microscopy (AFM), western blot (WB), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The antitumour activity of B7‐H3 CAR‐T‐derived EVs was evaluated by both MTT and killing assays. Furthermore, their cargo was studied by proteomics.

Results : B7‐H3 CAR‐T‐derived EVs characterization revealed that EVs displayed a globular shape and an average size of 140 nm. Furthermore, B7‐H3 CAR T‐derived EVs expressed characteristic EV markers (CD63 and Flotillin‐1) and were negative for cytochrome C and other typical contaminants (i.e., apolipoproteins). Co‐culture experiments showed that B7‐H3 CAR‐T cells induced 36% (*** p < 0.01) of killing on L3.6pl cells after 24 h. B7‐H3 CAR‐T‐derived EVs showed a time‐dependent killing activity against L3.6pl cells. In particular, the killing percentage of L3.6pl cells was 10% ( p = 0.05) and 22% (* p < 0.05) after 24‐ and 48‐h treatments with 150 µg B7‐H3 CAR‐T‐derived EVs, respectively. In line with killing assay results, B7‐H3 CAR‐T‐derived EVs significantly affected PC cell viability by MTT, compared to control.

Summary/Conclusion : Our preliminary results showed a relevant antitumour activity of B7‐H3 CAR‐T‐derived EVs on PC cells in vitro, encourageing further studies to evaluate their potential use in PC therapy.

Funding : This study was supported by the European Union—NextGenerationEU, under the National Recovery and Resilience Plan (NRRP), Project Title: “National Center for Gene Therapy and Drugs based on RNA Technology,” CUP: D73C22000810006.

Biotherapy of B cell lymphoma by healthy donor NK cell‐derived extracellular vesicles

Serena Cecchetti 1 , Cristina Federici 1 , Rossella Canese 1 , Egidio Iorio 1 , Veronica Huber 2 , Maria Elena Pisanu 1 , Mattea Chirico 1 , Elisabetta Iessi 1 , Serena Camerini 1 , Marialuisa Casella 1 , Andrea Matteucci 1 , Daniele Macchia 1 , Massimo Spada 1 , Ann Zeuner 1 , Luana Lugini 1

1 Istituto Superiore di Sanità, Italy, 2 Istituto Nazionale dei Tumori, Rome, Italy

Introduction : Extracellular vesicles of natural killer cells (NKEV) exert an antitumour effect towards haematopoietic and solid tumours and have an immune‐modulating effect, suggesting a promising role in immunotherapy. In this study, we demonstrated a network by mass spectrometry analysis between healthy donor‐derived NKEV protein cargo and antitumour effects in vivo against human B cell lymphoma.

Methods : We isolated NKEV, either exosomes or microvesicles, from in vitro amplified healthy NK cells by differential centrifugation and then characterized them by NanoSight, immunoelectron microscopy, western blot and mass spectrometry (Proteome Xchange, PXD014894). NKEV cytotoxic effect against B cell lymphoma was studied by cytometry analyses, SCID mice xenograft, in vivo MRI/MRS measurements, ex vivo MRS analyses and immunohistochemistry.

Results : Human healthy NKEV, both exosomes and microvesicles, have a significant and direct cytotoxic effect against human B cell lymphoma in in vitro and in vivo conditions. We observed a remarkable NKEV cytotoxic effect, mainly by apoptosis, on B cell lymphoma in vitro when exosomes and microvesicles were administered simultaneously. In vivo results showed metabolic alterations in SCID mice xenografts after NKEV treatment, associated with a significant reduction of tumour growth (64%). In the in vivo 1 H MR spectra we found a significant increase in the tumour lipid/lactate and in taurine signals, both considered as apoptosis markers. Ex vivo lymphoma metabolomics revealed a significant increase in the fatty acid (FA) pool and a decrease in unsaturated and monounsaturated FA in treated groups, as compared to the control one, thus suggesting an alteration of tumour homeostasis. Immunohistochemistry analyses confirmed the reduction of B‐cell lymphoma proliferation rate, as well as the induction of apoptosis following the NKEV treatment.

Summary/Conclusion : This study underscores the importance of NKEV as a novel biological acellular tool for B‐cell lymphoma treatment, probably having a greater effect on combined treatment regimens. These nanovesicles have an extraordinary potential in innovative cancer immunotherapy, representing a safe and efficient tool naturally circulating in healthy individuals and ready to maintain the immune homeostasis and therefore a good organism healthy state.

Funding : This study was supported by AIRC IG (Project IG 29148) 2023, Italy, to AZ and Ministero della Salute, Italy, WFR GR‐2011‐02351400 to LL.

Extracellular vesicles are responsible for bystander immunity in targeted radionuclide therapy

J. Constanzo 1 , T. David 1 , J. Karam 1 , S. Braga‐Cohen 1 , I. Vila 2 , N. Laguette 2 , M. Morille 3 , J.‐P. Pouget 1

1 Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Université de Montpellier, Nuclear Medicine Department, Institut régional du Cancer de Montpellier (ICM), Montpellier, France, Équipe Labellisée Ligue Contre le Cancer. 2 Molecular Basis of Inflammation, Institute of Molecular Genetics of Montpellier (IGMM), CNRS, Université de Montpellier, Montpellier, France; 3 ICGM, Université de Montpellier, ENSCM, CNRS, Montpellier, France

Introduction : Targeted radionuclide therapy (TRT), a systemic radiotherapy using radiopharmaceuticals, is known for its tumouricidal effects. Recent research, however, reveals its potential to boost antitumour immunity by promoting immunogenic cell death. Here, we examine evidence that TRT promotes the release of extracellular vesicles (EV), playing a critical role in modulating this antitumour immune response.

Methods : B16F10 melanoma cells were subcutaneously injected into C57BL/6J and athymic mice. Mice received intraperitoneal injections of TA99 mAb targeting TYRP‐1/gp75 tumour antigen radiolabelled with lutetium‐177 (177Lu‐TA99) or intratumoural injection of EVs purified from B16F10 cells exposed to 177Lu‐TA99 (TRT‐EVs) or from non‐treated cells (NT‐EVs). EVs were purified and analysed according to MISEV guidelines. Dendritic cells (DCs) were differentiated from mice bone marrow. Maturation of DCs was assessed by flow cytometry (e.g., MHC‐II marker).

Results : In vivo, TRT significantly delayed tumour growth in immunocompetent mice (** p = 0.001), requiring T‐cells for adaptive immunity, while no effect was observed in athymic mice. Nanoparticle tracking analysis revealed that TRT‐exposed B16F10 cells released larger EVs (average size shifted from 132.8 ± 4.4 nm to 153.9 ± 8.2 nm, *** p = 0.0001), indicating potential changes in EV biogenesis and cargo. When TRT‐EVs were administered intratumourally, they significantly delayed tumour growth and improved survival in immunocompetent mice (*** p = 0.0007), suggesting that these EVs may stimulate antitumour immunity. Notably, we observed that EVs from TRT‐treated cells promote DC maturation in vitro by carrying an immunestimulatory protein cargo, a key step in orchestrating effective antitumour immunity. In B16F10 cells exposed to TRT, Anxa1 gene expression was upregulated, while Arrdc1 expression remained unchanged over time post‐irradiation. The role of large EV in the effectiveness of TRT in vivo was confirmed using two inhibitors: imipramine, which prevents the translocation of acid sphingomyelinase (aSMase), and WRW4, which inhibits the Annexin A1 receptor FPR2. By blocking these pathways, the release of large EVs through membrane burgeoning was reduced, ultimately causing TRT to fail.

Summary/Conclusion : This study highlights EV as a modulator of TRT‐induced immune responses. These insights advance our understanding of how EVs contribute to TRT‐mediated immune activation, positioning EVs as potential biomarkers and therapeutic targets to improve cancer treatment strategies.

Subtopic Cancer initiation and promotion 2

The edible fungus Ganoderma lucidum is a novel modulator of cell‐cell communication based on extracellular vesicles in a triple‐negative breast cancer cell model

Presenter: Sandra L. Romero‐Cordoba

National Autonomous University of Mexico, Mexico City, Mexico

Introduction : There is increasing evidence that diet can influence cancer, but its role in tumour behaviour remains unexplored, mainly in edible mushrooms and their biotechnological derivatives. In this study, we characterized the impact of extracellular vesicle (EV)‐based communication influenced by extracts of the edible mushroom Ganoderma lucidum (Gl) on triple‐negative breast cancer cells. Our previous research shows that Gl extracts regulate systemic and hepatic cholesterol metabolism and demonstrate antitumour activity in in vitro and in vivo models.

Methods : Conditioned mediums from MDAMB231 cells treated with Gl1 or Gl2 extracts or vehicle at half inhibitory concentration or non‐treated cells were collected, and EVs were isolated using PEG precipitation and differential ultracentrifugation. Large (10,000 × g —lower speed centrifugation) and small (100,000 × g —higher speed centrifugation) EVs were characterized both physically and biochemically. MDAMB231 cells were exposed to 3 daily doses of 15,000 small and large EVs/cell prior to functional assays, which tested proliferation, viability, apoptosis, migration, and drug response. RNA‐seq of the EVs and conditioned cells was performed, correlating findings with molecular mechanisms influenced by Gl extracts in cell‐to‐cell communication via EVs.

Results : Characterization of small EVs showed the presence of CD9 and CD63 proteins, and the absence of exclusion markers like calnexin and B‐actin. We also observed a differential size between the two populations of EVs obtained by nanosight. In vitro assays showed a conditioning‐dependent inhibitory effect with both EVs populations isolated from cell cultures treated with the extracts on cell viability, proliferation and apoptosis rate, with some more attenuated responses in the population of large EVs. Chemopharmaceutical response assays showed that EVs from cells treated with the extracts, sensitized recipient cells. In all assays, EVs isolated from the conditioned medium of GL2‐treated cells showed a greater effect on the recipient cells. The study of RNA delivery by EVs and the transcriptional portrait established by EV conditioning identified relevant mechanisms associated with tumour programmes.

Summary/Conclusion : Our results indicate that GL extracts and their modulation of cell–cell communication mechanisms could prevent signalling pathways associated with tumour programmes established by EV conditioning in triple‐negative breast cancer cells.

Funding : This study was supported by PAPIIT projects IA206422 y IA207624, UNAM.

Decoding Cellular Dust of Breast Cancer: Functional Analysis of EVs in Breast Cancer

Gizem Sunar Cetin 1,2 , Fatih Inci 1,2

1 UNAM ‐ National Nanotechnology Research Center, Bilkent University, Ankara, Turkey; 2 Institute of Materials Science and Nanotechnology, Bilkent University, Ankara, Turkey

Introduction : Extracellular vesicles (EVs), initially regarded as insignificant cellular debris or ‘cellular dust,’ have since been recognized as nano‐sized particles with a pivotal role in mediating intercellular communication, particularly within cancer. EVs serve as vehicles for the transfer of bioactive molecules, and their cargo composition reflects their parental cells, impacting recipient cells and influencing tumour microenvironment. In breast cancer progression, EVs play multifaceted roles by contributing to the establishment of pre‐metastatic niches, drug resistance, angiogenesis, and immune evasion. However, the cellular information transferred by EVs, which plays a crucial role in breast cancer regulation and progression, requires further exploration. Herein, this study investigates the effects of EVs on alterations in gene expression profiles between breast cancer cells and non‐tumourigenic breast cells, aiming to elucidate the molecular mechanisms through which EVs influence breast cancer progression.

Methods : EVs were collected from MDA‐MB‐231 breast cancer cells and MCF‐12A non‐tumourigenic cells using a hybrid microfluidic chip that combines dead‐end and cross‐flow processes for efficient EV isolation. After isolation, EVs from MDA‐MB‐231 cells were introduced to MCF‐12A cells to evaluate their impact on cellular behaviour and gene expression profiles. The isolated EVs were characterized through nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), and Western blot analysis. Subsequently, we introduced isolated tumour‐derived EVs to non‐tumourigenic cells. Following that, we compared differences in gene expression levels between untreated and EV‐treated non‐tumourigenic mammary cells with qRT‐PCR analysis through epithelial‐mesenchymal transition (EMT), apoptotic, and anti‐apoptotic markers.

Results : Since breast cancer cells have distinctive assets from non‐tumourigenic mammary cells, the mammary cells incubated with tumour‐derived EVs have been examined for cancer cell characteristics, suggesting that EV‐mediated molecular transfer may promote malignancy‐associated transformations.

Summary/Conclusion : Consequently, our findings highlight the complex roles of EVs in breast cancer, particularly their ability to alter gene expression profiles in non‐tumourigenic cells toward cancerous characteristics. This approach enables significant insights into EV‐mediated communication within EV‐regulated pathways.

Funding : The authors gratefully acknowledge support from the Scientific and Technological Research Council of Turkey (TÜBİTAK) 2232 International Fellowship for Outstanding Researchers (Project No: 118C254), the Outstanding Young Scientists Award (GEBİP) from the Turkish Academy of Sciences, and Young Scientist Awards Program (BAGEP) from the Science Academy.

SMAC mimetics stimulate anti‐cancer cytotoxic T cell responses through small extracellular vesicles

Jordan J. Yin 1,2,3,4 , Daniel Panting 1,2,3,4 , Maria Dimancheva 1,2,3,5 , Mikaela van der Merwe 1,2,3,4 , Mariam Hakoum 1,2,3,4 , Martine St‐Jean 1 , Nathalie Earl 1 , Eric LaCasse 1,2 , Robert Korneluk 1,4 , Dylan Burger 6,7 , Rostyslav Horbay, 1,2,3,4 , Shawn Beug 1,2,3,4

1 Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Canada; 2 Centre for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, Canada; 3 Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Canada; 4 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada; 5 Department of Biology, University of Ottawa, Ottawa, Canada; 6 Kidney Research Centre, The Ottawa Hospital Research Institute and University of Ottawa, Ottawa, Canada; 7 Department of Cellular and Molecular Medicine and School of Pharmaceutical Sciences, University of Ottawa, Ottawa, Canada

Introduction : The inhibitor of apoptosis proteins (IAPs) are crucial in regulating programmed cell death by directly inhibiting caspases and promoting cell survival. These proteins are key negative regulators of the alternative NF‐κB pathway and its downstream, pro‐inflammatory and immune responses. A class of drugs termed SMAC mimetic compounds (SMCs) has been developed to antagonize the IAP proteins. Consequently, these SMCs can promote cell death and stimulate immune responses. This study aims to evaluate the ability of SMCs to promote anti‐tumour immune responses driven by the intercellular transfer of antigen‐presenting small extracellular vesicles (sEVs).

Methods : sEVs were isolated from cancer cells treated with SMCs via ultracentrifugation and characterized per the latest ISEV recommendations via Zetaview NTA, immunoblotting and NTA. Subsequently, dendritic cells were treated with different combinations of sEVs and SMCs and analysed via flow cytometry for immune activation and tumour‐antigen presentation. SMC and sEV‐treated dendritic cells were also co‐cultured with SMC‐treated, antigen‐specific CD8+ T cells, which were analysed for activation with flow cytometry.

Results : The release of sEVs from SMC‐treated cancer cells was increased by ∼30‐fold compared to control groups. These sEVs were positive for syntenin, a classical ESCRT‐dependent sEV biogenesis biomarker. Furthermore, the sEVs extracted measured between 50 and 200 nm according to NTA results. Dendritic cells co‐treated with SMC and sEVs from SMC‐treated cancer cells showed increased tumour‐antigen presentation and activation compared to control groups. Upon co‐culturing tumour antigen‐specific CD8+ T cells with these activated dendritic cells and SMCs, T cell activation was increased over ∼2‐fold when compared to controls.

Summary/Conclusion : This research provides insight into the potential synergistic effects of SMCs and sEVs on a specific, host‐mediated immune response against cancer cells. Future directions include investigating the mechanism linking SMC and sEV release, as well as determining if these anti‐tumour responses can be replicated upon SMC and sEV treatments in vivo.

Funding : This study was supported by CIHR (#PJT‐169126) and CRS (#935202)

Insights into the role of endothelial extracellular vesicles in pre‐metastatic niche formation and metastasis in breast cancer

Presenter: Kübra Bekar

ULiege (GIGA Research Center), Liège, Belgium

Introduction : Breast cancer (BC) is one of the most common cancers worldwide. While it can be treated, metastatic BC remains incurable. Prior research from our group demonstrated that endothelial cell‐derived extracellular vesicles (EVs) enriched in miR‐142‐5p, miR‐183‐5p, and miR‐222‐3p (i.e., ‘miR‐TAM’) participate in the polarization of macrophages towards an M2‐like phenotype, promoting tumour growth in a BC mouse model (Njock et al. 2022, JEV). However, the impact of miR‐TAM in metastasis remains unexplored. A crucial step before metastasis is the formation of a pre‐metastatic niche (PMN), which is notably initiated by EVs. This study aims to unravel the role of miR‐TAM in PMN formation and metastasis both in vitro and in vivo.

Methods : Mouse endothelial cell‐derived EVs were isolated using a differential ultracentrifugation method and characterized through western blotting and nanoparticle tracking analysis (NTA). EV uptake was assessed in macrophages (i.e., RAW264.7) and fibroblasts (i.e., MEF; mouse embryonic fibroblasts) via confocal microscopy. Subsequently, changes in PMN‐related gene expression profiles following miR‐TAM‐enriched EV treatment of these cells were assessed by quantitative PCR. To further investigate the impact of miR‐TAM in 4T1 tumour‐bearing mice, peritumoural administration of miR‐TAM‐ or control microRNA‐enriched EVs was performed, followed by flow cytometry and immunohistochemical analysis.

Results : Notably, isolated EVs were enriched for EV markers CD63, CD81, and syntenin but lacked the negative marker cytochrome C. Furthermore, endothelial EVs exhibited a major peak in the size range of small EVs (< 200 nm) and were successfully internalized by recipient cells. Interestingly, treatment with miR‐TAM‐enriched EVs upregulated the expression of pro‐tumourigenic genes Csf3, Cxcl1, Col3a1, Il‐1β, and Ccl3 in macrophages and fibroblasts, which are two major cell types in PMN formation. Strikingly, repeated peritumoural administration of miR‐TAM‐enriched EVs resulted in a significant reduction of CD4+ and CD8+ T cells in the lungs of 4T1 tumour‐bearing mice, establishing an immunosuppressive environment that favours metastatic outgrowth.

Summary/Conclusion : Here, we elucidate a previously unrecognized role of miR‐TAM‐enriched endothelial EVs in advancing metastasis both in vitro and in vivo, identifying them as potential modulators of PMN formation. These preliminary findings highlight promising avenues for novel BC therapeutic strategies.

Dual facets of MSC‐derived EVs: regulatory insights into antitumour mechanisms in pancreatic ductal adenocarcinoma

Kacaroglu Demet 1 , Ulaslı Alper Murat 2 , Yaylacı Seher 1

1 Faculty of Medicine, Department of Medical Biology, Lokman Hekim University, Ankara, Turkey; 2 Interdisciplinary Stem Cell and Regenerative Medicine Department, Stem Cell Institute, Ankara University, Ankara, Turkey

Introduction : Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense, fibrotic, immunosuppressive, and desmoplastic extracellular matrix. Due to this dense microenvironment, conventional therapeutics often fail to effectively reach target tumour cells, making nanosized drug carriers a promising alternative. Mesenchymal stem cell (MSC)‐derived extracellular vesicles (EVs) have emerged as a novel therapeutic strategy, given their capacity to be engineered with antitumour agents such as chemotherapeutic drugs and RNAs. However, the potentially conflicting effects of MSC‐EVs on tumour cells need to be considered. This study aimed to evaluate the mechanistic effects of MSC‐EVs on PDAC.

Methods : Wharton's Jelly (WJ) MSC‐derived EVs were isolated using the differential ultracentrifugation(dUC) method and analysed through nanoparticle tracking analysis (NTA), flow cytometry, and transmission electron microscopy (TEM). EVs were added to Panc‐1 cells at concentrations of 4000, 8000, and 12,000 EVs per cell. After 24 h, apoptosis and cell cycle analyses were performed. The expression of epithelial‐mesenchymal transition (EMT)‐related genes (CD44, CDH1, VIM, ZEB1, and CLDN1) was assessed by quantitative real‐time PCR (qRT‐PCR). Additionally, the expression of immune‐related genes (IL‐6, IL‐10, TNF‐α, IFN‐γ, IL‐1α, IL‐1β) was analysed by qRT‐PCR.

Results : NTA analysis indicated a mean EV size of approximately 104.6 nm, with over 89% of particles being double‐positive for CD63 and CD9. Cell cycle analysis showed an increase in the G1 phase from 31% in the untreated group to around 35‐36% in treated groups. Apoptosis analysis revealed similar viable and necrotic cell percentages between the control (around 80% viable) and treated groups(approximately 78%–79% viable). The treated groups exhibited downregulation of CD44, VIM, and ZEB1 genes compared to the control. CLDN1 and CDH1 were upregulated at the lowest EV concentration but downregulated at higher concentrations. Immune gene analysis showed downregulation of pro‐inflammatory cytokines (IL‐6, TNF‐α, IFN‐γ, IL‐1α, IL‐1β) and upregulation of the anti‐inflammatory cytokine IL‐10 in treated groups relative to the control.

Summary/Conclusion : This study revealed the dual role of WJ‐MSC‐EVs in PDAC. While they suppressed cell proliferation and modulated EMT markers, indicating antitumour potential, their induction of an immunosuppressive profile, marked by increased IL‐10 and reduced pro‐inflammatory cytokines, could hinder immune responses. These findings highlight both the promise and challenges of using WJ‐MSC‐EVs as therapeutic agents, necessitating further studies to optimize their application and balance their effects.

Breast cancer MDA‐MB‐231 cell‐derived extracellular vesicles express the purinergic receptor P2X4 and induce immunotolerant monocyte‐derived dendritic cells by decreasing the surface expression of HLA‐DR and glycogen content

Presenter: Mohammed Ismail Youssef Elmallah

University of Tours, Tours, France

Introduction : Tumours are well known to promote the induction of immunosuppressive microenvironments by recruiting the surrounding cells to serve their own survival and growth. This could be assigned to the release of soluble factors by tumour and large numbers of heterogenic extracellular vesicles (EVs) of different origins, including microvesicles and exosomes. Tumour‐derived EVs gain the attention of several research groups in terms of their immunomodulatory effect, as they encompass immunosuppressive proteins and microRNAs targeting suppressive pathways in recipient cells.

Methods : EVs were isolated from MDA‐MB‐231 by differential ultracentrifugation then characterized by nanoparticle tracking analysis and western blot. PBMCs were isolated from the blood of healthy donors, and then CD14+ monocytes were selected. CD14+ cells were treated with EVs derived from the wild‐type MDA‐MB‐231 cells that express P2X4 (MDA‐EXO +/+P2X4). The cells were incubated for 7 days at 37°C and 5% CO 2 in the presence of IL‐4 and GM‐CSF. The change in the surface expression of both differentiation and maturation markers was monitored by flow cytometry.

Results : Flow cytometric analysis revealed that CD14+ cells treated with MDA‐EVs ± P2X4 showed a significant reduction in the expression of the cell surface receptor HLA‐DR compared to the positive control (cells only treated with IL‐4 and GM‐CSF). In addition, no significant differences regarding the surface expression of CD86, CD33, and CD14 as well as the expression of the released cytokines were observed. Interestingly, we found that CD14+ cells treated with MDA‐EVs ± P2X4 displayed a significant reduction in the glycogen content compared to the positive control using electron microscopy. We are planning to test the same experiments by treating CD14+ cells with MDA‐EVs deprived of the P2X4 receptor (MDA‐EVs ‐/‐ P2X4) in order to investigate whether P2X4 plays a central role in the induction of an immunosuppressive environment and subsequent cancer progression and metastasis.

Summary/Conclusion : MDA‐MB‐231‐derived EVs inhibit the maturation of monocyte‐derived dendritic cells by decreasing the cytoplasmic content of glycogen and decreasing the surface expression of HLA‐DR. we conclude that our results may provide new insight toward the immunosuppressive as well as toward the immunomodulatory role of tumour‐derived EVs.

Funding : This study was supported by INCa PLBIO_16110 “PURINO4EXO” to S.R., Grant Region Centre‐val de Loire “EXOGRAL” to S.R.

Hyperglycaemia‐induced changes in extracellular vesicles produced by endometrial cancer cells

Anastasiia Artuyants 1,2 , Zi Yin Enid He 1 , Bianca Nijmeijer 1 , Sophia Bebelman 1 , Martin Middleditch 3 , Deanna Shea 3 , Cherie Blenkiron 1,2

1 Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand; 2 Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand; 3 School of Biological Sciences, The University of Auckland, Auckland, New Zealand

Introduction : Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries, with rising incidence partly driven by obesity and metabolic disorders like type 2 diabetes. Hyperglycaemia, a defining feature of diabetes, contributes to a pro‐tumourigenic environment through enhanced cellular growth, inflammation, and oxidative stress, which can accelerate cancer progression. However, the impact of hyperglycaemia on extracellular vesicles (EVs)—crucial mediators of cell‐to‐cell communication in the tumour microenvironment, remains underexplored. In this study, we investigated how high glucose conditions affect EV production, protein cargo, and functionality in EC cell lines, potentially revealing mechanisms through which hyperglycaemia may influence EC progression.

Methods : Three EC cell lines—MFE280, MFE296, and Ishikawa, representing different EC grades, were cultured in 3D bioreactor systems under normal and high‐glucose conditions for 10 to 23 weeks. EVs were isolated twice weekly via differential centrifugation and size exclusion chromatography. Cell tolerance to long‐term high glucose exposure was monitored through particle counts, glucose and pH testing. Selected EV samples were analysed using proteomics and functional assays.

Results : 3D bioreactor models enabled continuous culture and large‐scale EV production. High glucose conditions did not significantly change EV particle count or size, though a pH reduction in the conditioned media suggested increased lactate production. Proteomic analysis revealed significant alterations in EV protein composition under high glucose conditions, indicating potential shifts in pathways related to extracellular matrix remodelling, immune response, energy metabolism, stress signalling, and oxidative stress. These changes suggest that hyperglycaemia may promote modifications in the tumour microenvironment by altering cell signalling, supporting metabolic adaptation, and enhancing survival mechanisms within cancer cells. The effects on EC line proliferation, activity, colony formation and adhesion are being analysed.

Summary/Conclusion : These findings indicate that hyperglycaemia induces specific changes in EV protein cargo that may alter the tumour microenvironment, potentially facilitating EC progression. This research provides insights into the role of EVs in hyperglycaemia‐related tumour biology. Further work could clarify the role of EV‐mediated signalling in hyperglycaemic conditions and its implications for EC progression.

Extracellular vesicle release from tumour cells: endoplasmic reticulum stress regulates amount, composition and function

Viviane Ponath, Judith Röttering, María Gómez‐Serrano, Bilal Alashkar Alhamwe, Christian Preußer, Witold Szymanski, Johannes Graumann, Elke Pogge von Strandmann

Philipps University Marburg, Marburg, Germany

Introduction : Initially, the release of extracellular vesicles (EVs) from cells was regarded as a major cellular strategy to dispose of obsolete proteins, and this is probably of particular relevance within the context of endoplasmaic reticulum stress (ER stress) and the unfolded protein response (UPR). However, the relevance of the ER stress and the UPR on protein secretion via vesicle release is largely unknown. We hypothesize that ER stress and EV release are functionally and mechanistically linked and that the chaperone BAG6 is a crucial molecular player within this scenario. Of note, BAG6 is not only involved in vesicle cargo loading [1] but it is also known as a critical component of the endoplasmic‐reticulum‐associated protein degradation (ERAD) [2].

Methods : BAG6‐wildtype and BAG6‐deficient human pancreatic cancer cell lines were designed and treated with thapsigargin and tunicamycin to induce ER stress. The cell supernatant was collected to measure the particle release with nanoFCM. EVs were purified using differential ultracentrifugation and subjected to label‐free proteomic analysis using high‐resolution mass spectrometry.

Results : In line with our hypothesis, we observed that ER stress induced a significant increase in vesicle release. Of note, this increase was abolished in BAG6 deficient cells, indicating that (1) ER stress triggers the release of sEVs and (2) that this process is regulated by BAG6. Mass spectrometry data revealed that ER stress resulted in changes in EV composition. Most strikingly, 257 proteins were specifically enriched in EVs from BAG6 knockout cells upon ER stress. The top biological function assigned to these proteins was the regulation of cell polarity, representing a crucial process in tumour initiation and progression.

Summary/Conclusion : Pancreatic cancer cells frequently experience ER stress, however a link to tumour cell‐mediated EV release was largely unknown. Here we show that ER stress impacts the amount and composition of EVs in response to ER stress in a BAG6‐dependent manner.

References : (1) Alashkar Alhamwe, B., Ponath V, et al. 2024. Cellular & Molecular Immunology 21, no. 8. (2) Ganji, R., Mukkavalli S, et al. 2018. Molecular & Cellular Biology 38, no. 13).

The immunomodulatory effects of colorectal tumour‐derived extracellular vesicles on primary human monocytes

Presenter: Kirsty M. Danielson

University of Otago, Dunedin, New Zealand

Introduction : Extracellular vesicles (EVs) play an important role in modulating the tumour microenvironment (TME) and affecting the initiation and progression of cancer. Circulating monocytes can encounter tumour‐derived EVs in the circulatory system or when they extravasate into the TME, where they, and their derivative macrophages, can exhibit both tumour‐suppressive and tumour‐promoting properties. One way in which their fate may be determined is through interactions with CRC‐EVs, which may predispose circulating monocytes towards phenotypes that promote a favourable TME for tumour survival. This study aimed to investigate the effects of tumour‐derived EVs on monocytes from healthy volunteers.

Methods : This study was approved by the Health and Disability Ethics Committee NZ, and all participants gave written informed consent. Plasma samples were obtained from CRC patients prior to any treatment and matched controls had normal bowel confirmed by colonoscopy. EVs were isolated from plasma or from colorectal cell lines (DLD‐1 and SW‐837) using qEV size exclusion columns, quantified by tunable resistive pulse sensing, and imaged by cryo‐SEM. Protein and RNA contents were characterized by Western blotting and RT‐qPCR. EVs were then co‐cultured with monocytes from healthy human volunteers to determine their effects on gene and protein expression.

Results : Western blotting confirmed the presence of EV‐associated markers CD9 and CD63, and cryo‐SEM showed spherical vesicles ranging from 40 to 300 nm in size. EV counts increased in CRC patients in a stage‐dependent manner compared to controls. EVs from cell lines had no significant effect on monocyte metabolic activity across the dose range. EVs from DLD‐1 and SW‐837 cells differentially affected the mRNA expression of genes related to pro/anti‐inflammatory functions, inflammasome proteins, and pattern recognition receptors.

Summary/Conclusion : EVs from different colorectal cell lines had differential effects on gene expression in primary human monocytes, indicating that their immunomodulatory effects are heterogenous across cultures.

Funding : This work was supported by the Wellington Medical Research Foundation.

Subtopic Cancer progression and metastasis 2

Investigating the role of CD133‐enriched protrusion‐derived extracellular vesicles in triple‐negative breast cancer aggressiveness

Mireia Gómez‐Duro 1 , Graça Raposo 1,2 , Ptissam Bergam 2 and Gisela D'Angelo 1

1 Institut Curie, PSL Research University, CNRS, UMR144, Paris, France; 2 Institut Curie, CNRS, UMR144, PSL Research University, Cell and Tissue Imaging Facility (PICT‐IBiSA), Paris, France

Introduction : Cancer spreading from tumours to tissues and distant organs is the primary cause of cancer morbidity and mortality. Recent data suggest that cancer cells release extracellular vesicles (EVs), membranous nanocarriers containing proteins and genetic material, which may stimulate the invasion and dissemination of tumour cells to target cells. CD133 is linked to aggressive cancer and poor outcomes. We have shown that CD133 enhances the production and secretion of a novel EV subtype protrusion‐derived EVs (PD‐EVs), with functional implications in development. But their role in cancer remains unexplored. The aim is to correlate the expression of CD133 in breast cancer cells to EV secretion and their EV‐mediated invasive and metastatic properties.

Methods : Examine the influence of CD133 on PD‐EV protein content by overexpression of CD133, EM, EVs isolation, characterization, mass spectrometry; tubulogenesis. Investigate the impact of Eng‐containing PD‐EVs on tubulogenesis by reduction of Eng levels in MDA‐MB‐468 cells, EVs isolation, tubulogenesis assay, lipid‐raft‐fractionation, IP‐assay, and subcellular‐distribution of Eng. Uncover the organ tropism and metastatic potential of PD‐EVs in vivo.

Results : We observed that BL‐TNBC cells (MDA‐MB‐468, HCC70, and HCC38) display membrane protrusions that bear the CD133 protein and release massive amounts of PD‐EVs enriched in CD133. Depletion of CD133 in MDA‐MB‐468 cells did not impair EV secretion, but led to abnormal cell protrusions, impacting the amount of EV released. We found that PD‐EVs from MDA‐MB‐468, but not EVs from CD133 KO cells, promoted tube formation. Moreover, we examined the tissue distribution of PD‐EVs from MDA‐MB‐468 cells. We found that once injected into athymic mice, PD‐EVs fluorescently labelled efficiently distributed to the lungs and liver, whereas EVs from CD133 KO cells did not. By mass spectrometry analysis we identified different proteins in PD‐EV containing CD133 with those in KO cells. Among these proteins, endoglin is known to promote angiogenesis.

Summary/Conclusion : The finding of PD‐EVs secreted by BL‐TNBC cells, particularly those containing CD133, has provided important information about the role of these CD133‐EVs in promoting endothelial cell growth. Our research indicates that CD133‐EVs released by BL1‐TNBC cells play a key role in creating a conducive environment in surrounding tissues, ultimately aiding in metastasis.

Funding : This study was supported by the MarieCurieActions ‐Institut Curie.

A possible role of extracellular vesicles in shifting the endothelial cell population landscape associated with vasectasia in mesenchymal glioblastoma

Fang Cheng Wong 1 , Cristiana Spinelli 1 , Brian Meehan 1 , Lata Adnani 1 , Janusz Rak 1

1 The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada

Introduction : Glioblastoma multiforme (GBM) is a highly aggressive and fatal brain cancer that affects both children and adults. It is characterized by aberrant vascularization and a predictable repertoire of oncogenic mutations. Pursuing the link between oncogenic lesions and vascular anomalies in GBM, we have identified a novel, non‐angiogenic vascular expansion process termed ‘vasectasia.’ This process involves increased circumferential growth of blood vessels facilitated by the transfer of EGFR/EGFRvIII transcript through a subset of extracellular vesicles (EVs). However, the detailed mechanism underlying this regional vascular variation in vasectasia remains to be further investigated. Our current study focuses on elucidating the identity, subtypes and molecular programmes of endothelial and perivascular cells that are involved in GBM‐associated vasectasia.

Methods : A series of well characterized patient‐derived glioma stem cells (GSCs) representing distinct molecular subtypes was employed to study their vascular alterations in the xenograft immune‐deficient NOD scid gamma (NSG) mouse models. Spatial transcriptomics was performed using the Xenium in situ platform with pre‐designed mouse brain panel (247 genes) and a custom gene panel comprising 50 markers specific to endothelial cells and perivascular cells to simultaneously map and profile the molecular landscape of vascular cell populations involved in vasectasia. Normal brains of NSG mice served as controls. Additionally, immunofluorescence was conducted to visualize relevant vascular and EV markers in mouse brain sections, and western blot analysis was employed to elucidate the signalling pathways altered by mesenchymal GSC‐derived EVs isolated by differential ultracentrifugation.

Results : NSG mice intracranially xenografted with mesenchymal GSCs exhibited significantly shorter survival rates compared to those xenografted with proneural GSCs. This was accompanied by increased circumferential vascular growth in the tumour regions, indicative of vasectasia. Spatial mapping of endothelial and perivascular cell subpopulations involved in the vasectasia revealed oncogene‐dependent features of the respective vascular networks. Human brain endothelial cells exposed to mesenchymal GSC‐derived EVs displayed significant changes in key signalling targets downstream of EGFR/EGFRvIII, underscoring the role of EGFR/EGFRvIII in the initiation of vasectasia.

Summary/Conclusion : Brain tumours may trigger several different vascular growth processes triggered by oncogenic EVs, and their comprehensive investigation may enable new therapeutic strategies and diagnostic opportunities.

Funding : This study was supported by the Charles Bruneau Foundation, Canadian Institutes of Health Research.

CLL‐derived EVs shape the fate of microenvironmental cells

Presenter: Orit Uziel

Tel Aviv University, Tel Aviv, Israel

Introduction : We studied here whether CLL cells communicate with their microenvironmental cells (endothelial and B cells) to acquire a survival advantage. We hypothesized that both interactions of CLL cells, either with the endothelial cells or with the normal B cells are mediated by CLL‐derived EVs for their benefit.

Methods : CLL derived EVs were isolated from CLL cells of several untreated patients. EVs were harvested by ultracentrifugation and characterized by NTA, Western blotting and electron microscopy. The engulfment of stained EVs was followed by flow cytometry. Endothelial cells: (1) Underwent phosphoproteome analysis; (2) results were validated by western blotting; (3) secretion of IL‐6 was assessed by ELISA; (4) apoptosis was measured by flow cytometry; (5) CTNNB1 was ectopically expressed; (6) Interaction of all phosphoproteins and CTTNB1 was done by ANAT; (7) Binding of transcription factors was measured by ChIP. B cells: (1) RNA‐seq was performed. (2) Validation of the results: by Q‐RT PCR. (3) SMAD6 was ectopically expressed, and apoptosis was assessed as above.

Results : CLL‐derived EVs were shown to possess the expected 30–150 nm diameter size and shape. These EVs were engulfed by either endothelial or B cells in a dose‐ and time‐dependent manner. The endothelial exposed cells (1) became into IL‐6 producing cells. (2). The IL‐6 enriched medium promoted the phosphorylation of STAT‐3 in fresh CLL cells, decreasing their apoptosis. (3) Twenty‐three proteins were phosphorylated in response to CLL‐ EVs. (4) Network analysis unravelled the central role of phosphor‐β‐catenin. (5) Ectopic expression of β‐catenin increased IL‐6 levels in the culture medium. (6) ChIP analysis revealed that all 3 transcription factors possessed an increased binding to the IL‐6 promoter. In contrast, exposed B cells (1) underwent apoptosis; (2) 24 transcripts were differentially expressed; (3) ectopic expression of SMAD6 in B cells induced apoptosis.

Summary/Conclusion : CLL cells differentially communicate with endothelial and B cells through secreting EVs. Once they are taken up by endothelial cells, they turn them into IL‐6‐producing cells, thus providing the neoplastic cells with a survival advantage. However, B cells get a ‘death message’ sent by the neoplastic EVs to eliminate their potential competitors.

LGALS3BP promotes tumourigenesis by enhancing sPLA2‐X activity to hydrolyse EV membranes and uptake by TAM through the LGALS3BP‐LGALS3 axis

Presenter: Akane Kanamori

Osaka University, Osaka, Japan

Introduction : The Epstein‐Barr virus (EBV) causes several poor prognostic cancers, including lymphoma. Recently, we revealed that tumour‐associated macrophages (TAM) express high levels of secretory phospholipase A2‐X (sPLA2‐X) in EBV‐positive lymphoma. This enzyme modifies the function of tumour‐derived extracellular vesicles (EVs) by hydrolysing their membrane phospholipids. The modified EVs are then incorporated by macrophages, facilitating TAM polarization and activating the cell surface GPCR, promoting tumourigenesis (Kudo et al. 2022, Cell Metabolism).

Methods : To analyse the factors involved in sPLA2‐X activity, proteomic analysis was performed. sPLA2‐X activities were then analysed by LC‐MS/MS to detect degradation products. The roles of LGALS3BP were assessed using western blot and immunostaining.

Results : Interestingly, patient‐derived EVs were much more susceptible to hydrolysis than those from healthy individuals despite having a similar composition of phospholipid. Proteomic analysis of the EVs identified LGALS3BP as a patient‐specific component. LGALS3BP has been reported to be abundant in the serum of several cancer patients; however, the functions were still elusive. LGALS3BP was mainly secreted by TAM, as was sPLA2‐X, and exerted as a ‘protein corona’ that binds to EV membranes rather than a constituent of them. LGALS3BP enhanced the hydrolytic activity of sPLA2‐X by directly binding to the EV surface, possibly acting as a scaffold between them. Although LGALS3BP alone increased the uptake of EVs by target cells, the synergistic activity of LGALS3BP and sPLA2‐X greatly enhanced this uptake, which depended on Galectin‐3 (LGALS3). Finally, the EVs cotreated with LGALS3BP and sPLA2‐X remarkably accelerated lymphomagenesis in vivo.

Summary/Conclusion : These results suggest that LGALS3BP contributes to lymphomagenesis by promoting the hydrolysis of EV membranes through sPLA2‐X activation and enhancing EV uptake to target cells mediated by LGALS3. This results in accelerated formation of the tumour microenvironment through malignant communication between tumour cells and TAM.

Cancer progression by EVs released from gastric cancer culture cells during anticancer drug administration

Akane Sato 1,2 , Kyo Okita 1,2 , Uno Oi 1 , Etsuro Ito 1,2

1 Waseda University, Tokyo, Japan; 2 BioPhenoMA Inc., Tokyo, Japan

Introduction : Gastric cancer is a common disease with high morbidity in East Asia. In clinical practice, 5‐FU is commonly used as the first‐line treatment for advanced gastric cancer. However, many patients develop drug tolerance during treatment. Drug tolerance becomes a barrier to improving treatment efficacy. Tumour‐derived EVs may play a role in the development of 5‐FU tolerance, for example in breast cancer. The expression of GRP78 (glucose‐regulated protein 78 kDa) is increased in tumour tissue of gastric cancer patients, and GRP78 contributes to cancer progression through the release of EVs. Based on these findings, we hypothesized that GRP78 released via EVs induces tumour cells that uptake EVs to acquire stem cell‐like characteristics, leading to the emergence of 5‐FU tolerance.

Methods : Ultrafiltration and polymer precipitation methods were used to isolate EVs from the conditioned medium of human gastric cancer AGS cells treated with 5‐FU. EVs were confirmed through particle size measurement and western blot analysis. The acquisition of cancer stem cell‐like properties in EV‐treated cells was evaluated by cell viability assay and wound healing assay. Trace amounts of GRP78 in EVs were quantified using our ultrasensitive thio‐NAD cycling ELISA.

Results : Using the thio‐NAD cycling ELISA, the limit of detection for GRP78 protein was 0.3 pg/mL. The GRP78 protein concentration in EVs from cells treated with 50 µM 5‐FU was approximately three times higher than in EVs from untreated cells (0 µM 5‐FU). A higher concentration of GRP78 protein in EVs resulted in the acquisition of stem cell‐like properties in tumour cells that accepted the EVs. Cell viability and wound healing assays demonstrated that tumour cells taking up exosomes from 5‐FU‐treated gastric cancer cells exhibited enhanced migration and improved cell viability, indicative of stem cell‐like properties.

Summary/Conclusion : These findings suggest that treatment with 5‐FU leads to the release of EVs rich in GRP78 protein, facilitating the acquisition of cancer stem cell‐like properties in recipient tumour cells. Although the detailed mechanisms remain to be investigated, they may involve the PI3K/Akt signalling pathway.

Investigating Rab27‐dependent extracellular vesicle cargoes and their role in shaping the immune environment of primary tumours

America V Campos‐Gonzalez 1 , Jasmine Peters 1 , Michalis Gounis 2 , Lucy Somerville 1 , Louise Mitchell 1 , Jayanthi Anand 1 , Cassie Clarke 1 , Jim C Norman 1

1 Cancer Research UK Scotland Institute, Glasgow, UK; 2 Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland.

Introduction : Metastatic breast cancer remains a major cause of death among women worldwide, despite progress in treatments. Gaining insight into the mechanisms that drive tumour invasion and treatment response, particularly as they evolve in metastatic environments, is critical for developing better therapies. The Rab family of small GTPases, which play key roles in vesicle trafficking, are essential for processes like transport, signal transduction, and extracellular vesicle (EV) secretion. Recent research highlights that Rab GTPases, especially Rab27, are involved in breast cancer progression and metastasis. Our study has shown that Rab27 regulates the release of mitochondrial DNA (mtDNA)‐containing EVs through a PINK1‐dependent process. This research focuses on the roles of Rab27a, Rab27b, and the mitochondrial kinase PINK1 in sorting mtDNA and other cargo into EVs, and how these mechanisms change as breast cancer metastasizes from the mammary gland to the lungs. Additionally, we investigate how these EV cargoes affect immune cell recruitment to tumours.

Methods : By using CRISPR‐Cas9 to knock out Rab27a or Rab27b in both primary tumour cells and lung micrometastases from the MMTV‐PyMT mouse model.

Results : We discovered that primary tumour cells rely on Rab27 for EV release, while micrometastatic cells use a Rab27‐independent, neutral sphingomyelinase‐2 (nSM2)‐dependent pathway. Furthermore, primary tumour cells release mtDNA‐rich EVs in a Rab27‐dependent manner, while micrometastatic cells produce EVs with low mtDNA content via a Rab27‐independent pathway, as confirmed by qPCR and digital droplet PCR (ddPCR). Importantly, Rab27a was found to play a role in recruiting CD4+ and CD8+ T‐cells to primary tumours, while myeloid cell recruitment was unaffected.

Summary/Conclusion : These findings shed new light on Rab27's involvement in breast cancer metastasis and immune regulation, revealing potential targets for adjuvant therapies that could improve outcomes in patients who are unresponsive to current treatments.

Funding : This study was supported by the CRUK Scotland Institute (CORE Funding).

Extracellular vesicles derived from pro‐inflammatory macrophages induce an inflammatory and invasive phenotype of melanoma cells

Kaisa Mäki‐Mantila 1 , Einari A. Niskanen 1 , Kirsi Kainulainen 1 , Aino Rönkä 1,2 , Niina Aaltonen 1 , Kirsi Rilla 1 , Sanna Pasonen‐Seppänen 1

1 University of Eastern Finland, Kuopio, Finland 2 Kuopio University Hospital, Kuopio, Finland

Introduction : Melanoma progression depends on intercellular communication within the tumour microenvironment (TME), where extracellular vesicles (EVs) have emerged as essential mediators via their molecular cargo. In melanoma, macrophages are enriched in the TME, and their abundance and infiltration into the tumour area are associated with poor prognosis. Tumour‐associated macrophages are roughly divided into proinflammatory (M1) and immunosuppressive (M2) macrophages, both of which secrete EVs to modulate intercellular communication in the TME. Given the crucial role of macrophages in the melanoma progression, we investigated the effects of M1 and M2 macrophage derived EVs on melanoma cells to gain insights into EV‐mediated macrophage‐tumour cell interactions that drive melanoma progression.

Methods : THP‐1 cells were cultured in the presence of PMA to differentiate monocytes into M0 macrophages, which were further polarized to M1 and M2 macrophages with LPS and IFN‐γ or with IL‐4 and IL‐13, respectively. EVs were isolated from the conditioned media using differential ultracentrifugation and characterized with Western blot, TEM, and NTA analysis. The effects of M0‐, M1‐ and M2‐EVs on MV3 melanoma cells were studied at the mRNA level with RNA‐seq and qPCR and at the functional level using invasion assays.

Results : M1‐EVs induced an inflammatory phenotype in melanoma cells by upregulating the expression of several pro‐inflammatory genes such as CXCL8, IL‐6 and IL‐1B. Gene set enrichment analysis revealed that genes related to inflammatory responses and TNFα signalling via NF‐kB are enriched in M1‐EV treated cells. In addition to inflammatory responses, M1‐EVs stimulated invasion of melanoma cells in 3D invasion model. We assume that M1‐EV stimulated invasion is related to inflammation and is induced by the overexpression of inflammatory mediators like CXCL8. Moreover, inhibitor experiments showed that NF‐κB signalling drives M1‐EV‐mediated effects, as IKK inhibitor suppressed M1‐EV‐induced expression of pro‐inflammatory genes.

Summary/Conclusion : M1‐EVs promote the malignancy of melanoma cells by inducing an invasive and inflammatory phenotype via activation of NF‐κB signalling pathway. Cancer‐related inflammation can drive cancer progression through inflammatory factors such as CXCL8, which is known to enhance cell motility. Thus, M1‐EVs may stimulate invasiveness in an inflammation‐related manner. These results emphasize the importance of EV‐mediated interactions between immune and tumour cells in cancer progression.

Melanoma‐derived extracellular vesicles change monocyte phenotype towards a pro‐inflammatory state

Raphaela Rebeca Silveira Assunção, Milena Perez Mak, Roger Chammas, Luciana Nogueira de Sousa Andrade

Universidade de São Paulo, São Paulo, Brazil

Introduction : Melanoma skin cancer is the 17th most incident tumour worldwide and there is still a high rate of non‐response to treatment. Extracellular vesicles (EVs) stand out as a potential biomarker for cancer prognosis; nonetheless, it has been shown that melanoma‐derived EVs influence immune cell response. In this study we aimed to evaluate the impact of EVs from human melanoma cells on proliferation, death and gene expression of monocytes, important players in cancer development.

Methods : EVs from BRAF‐mutated primary and metastatic melanoma cell lines A375 and SKMel‐28, respectively, were isolated by differential ultracentrifugation. Plasma EVs from melanoma patients were isolated by ultracentrifugation and size‐exclusion chromatography columns after informed consent was obtained (ethical committee approval number 51680021.1.0000.0068). EV's mean concentration and size were determined by the nanoparticle tracking system (NTA) and electronic microscopy, and CD9, CD63 or CD81 were detected by western blotting. THP‐1 human monocytes were treated with EVs for 24 and 48 h, and cell proliferation was determined by counting, cell death by flow cytometry after propidium iodide staining and gene expression by RT‐qPCR using specific primers for VEGF‐A, IL‐1b, HLA‐DRA, CCL2 and MCL‐1. Statistical analyses were performed by one way ANOVA followed by Tukey.

Results : EVs (10 10 EVs/mL) from A375 primary melanoma cells inhibited monocyte proliferation and downregulated VEGF‐A and MCL‐1 and upregulated CCL2, HLA‐DRA and IL‐1b genes. Conversely, EVs from SKMel28 metastatic melanoma cells upregulated all mentioned genes without alteration on monocyte proliferation. Concerning circulating EVs from melanoma patients, we noticed two different EVs populations characterized as large and small EVs based on NTA analysis, and their effect on monocyte phenotype is being investigated.

Summary/Conclusion : EVs from melanoma cells modify monocyte phenotype towards a pro‐inflammatory and anti‐tumoural phenotype through the induction of the transcriptional levels of certain inflammatory genes. However, EVs from metastatic disease were also able to induce an increase in VEGF‐A levels, which could favour melanoma progression. Future functional assays will be conducted to better clarify the impact of these EVs on monocyte activity and tumour progression using EVs from metastatic and non‐metastatic melanoma patients.

Funding : This study was supported by the FAPESP (grant number 2023/16355‐4).

Dissecting extracellular vesicle heterogeneity in melanoma

Pragati Lodha 1,2 , Sarah Hofmann 1 , Alperen Acari 1,2 , Jochen Rieck 1 , Shruthi Hemanna 1 , Emma Reynaud 1,2 , Lothar C. Dieterich 1

1 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 2 Faculty of Biosciences, Heidelberg University, Heidelberg, Germany

Introduction : Metastasis is a complex process involving molecular interactions between malignant and multiple host‐derived cell types, locally at the primary tumour site but also shaping pre‐metastatic niches in tumour‐draining lymph nodes (LNs) and in distant organs. Tumour cell‐derived extracellular vesicles (EVs) play an important role in this process. Notably, they are highly enriched in tumour‐draining lymph and are transported by afferent lymphatic vessels to draining LNs, where they interact with resident lymphatic endothelial cells (LECs), promoting their proliferation and inhibiting tumour immunity. However, tumour EVs are very heterogenous, and the recipient cells and progression‐promoting functions of individual EV subsets in the LN microenvironment are currently unknown. In this study, we aimed to molecularly define melanoma EV subsets to identify their individual target cells and functions in pre‐metastatic LNs in melanoma.

Methods : We have established a multi‐parameter flow cytometry‐based approach to probe surface proteins on single EVs derived from melanoma cells, allowing the definition of individual EV subsets by unsupervised clustering according to EV surface markers. In addition, we develop genetic tools to perturb the release of selected EV subsets by melanoma cells through CRISPR‐Cas‐mediated knockout of selected Rab proteins.

Results : Using multi‐parameter flow cytometry, we identified several subsets of EVs released by melanoma cells in vitro. As expected, common EV markers were broadly expressed across those subsets. However, we also identified individual, subset‐specific markers. Furthermore, we established a panel of melanoma cell clones depleted for individual Rab proteins, which may be useful tools to dissect EV subset‐specific functions in vivo.

Summary/Conclusion : Multi‐parameter flow cytometry‐based analysis of EVs is a potentially very powerful tool to characterize EV heterogeneity. Using this approach, we have identified marker proteins of individual EV subsets released by melanoma cells, enabling their enrichment, in vivo tracking, identification of the corresponding recipient cell populations, and, ultimately, their biologic functions in the LN microenvironment. Thereby, our study lays the foundation for future therapeutic targeting of individual EV subsets in the context of melanoma.

The effect of uveal melanoma derived extracellular vesicles on hepatocytes in 3D culture

Emma Symonds 1 , Måns Kadefors 1 , Karin Ekström 1 , Rossella Crescitelli 1 , Roger Olofsson Bagge 1,2

1 Sahlgrenska Center for Cancer Research, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 2 Department of Surgery, Sahlgrenska University Hospital, Region Västra Götaland, Gothenburg, Sweden

Introduction : Uveal melanoma is a rare form of melanoma that arises from melanocytes in the eye. Approximately half of patients with uveal melanoma develop metastases, primarily in the liver (approximately 90% of the patients), with a very poor prognosis. Somatic mutations in the BAP1 gene encoding BRCA1‐associated protein 1 are associated with increased risk of developing metastases. Tumour‐secreted extracellular vesicles (EVs) have been proposed as drivers of pre‐metastatic niche formation. We aimed to investigate if EVs from uveal melanoma cells, with or without BAP1 mutation, would increase the ability of cancer cells to co‐locate with hepatocyte spheroids in vitro.

Methods : Hepatocyte spheroids were developed by seeding THLE2 cells, stained with Calcein‐AM, in ultra‐low attachment culture plates (1500 cells/well) and culturing them for 5 days. EVs were isolated from a BAP1 mutated uveal melanoma cell line (UM22), transfected either with a vector control (BAP1‐mt) or wild‐type BAP1 gene (BAP1‐wt), using differential centrifugation followed by iodixanol density gradient separation. As per the MISEV Guidelines, EVs were characterised by nanoparticle tracking analysis, western blot, and electron microscopy. Hepatocyte spheroids were treated with EVs for 24 h (1.5x107 EVs/well). UM22 BAP1‐mt or UM22 BAP1‐wt cancer cells were then stained with Cell Tracker Deep Red, added to the spheroid cultures (1000 cells/well), and co‐cultured for 24, 48, 72, or 120 h. Spheroids were then fixed and imaged on a Zeiss Celldiscoverer 7 microscope. Images were analysed using ImageJ.

Results : After 24, 48, and 72 h of co‐culture, no cancer cells were observed to interact with the spheroids. After 120 h, cancer cells were seen associating with spheroids treated with BAP1‐mt‐derived EVs: 15.50 ± 8.66 UM22 BAP1‐wt cells and 20.50 ± 8.35 BAP1‐mt cells. Contrastingly, apheroids treated with BAP1‐wt‐derived EVs showed lower cancer cell invasiveness: 7.75 ± 4.79 UM22 BAP1‐wt cells and 12.00 ± 7.02 UM22 BAP1‐mt cells.

Summary/Conclusion : The ability of uveal melanoma cancer cells to interact with hepatocyte spheroids is time‐dependent, requiring at least 120 h for cells to co‐localise with hepatocytes. Treatment of spheroids with BAP1‐mt‐derived EVs enhances the number of uveal melanoma cancer cells interacting with the hepatocyte spheroids.

Investigating the role of monounsaturated fatty acid (MUFA)‐enriched EVs released from micrometastatic mammary cancer cells in cancer progression

Lucy Somerville 1,2 , America V. Campos‐Gonzalez 1 , Engy Shokry 1 , David Sumpton 1 , Michalis Gounis 3 , Cassie Clarke 1 , Jim C. Norman 1

1 Cancer Research UK Scotland Institute, Glasgow, Scotland; 2 School of Cancer Sciences, University of Glasgow, Glasgow, Scotland; 3 Turku Bioscience Centre, University of Turku, Turku, Finland

Introduction : Extracellular vesicles (EVs) have been associated with tumourigenesis and metastasis in breast cancer, through transport of cargo to neighbouring cells in the primary tumour site and distant organs. Recent data from our group has shown that lung micrometastatic mammary cancer cells (YEJG‐M2), which were generated using the PyMT mouse model via syngeneic orthotopic transplantation, have increased proline synthesis, which consequently decreases glutathione levels when compared to primary mammary cancer cells (YEJG‐P). This reduced glutathione alters the Rab27‐dependence of EV release from micrometastatic cells. We proposed that this altered EV release may influence the lipidome of EVs released from micrometastatic cells.

Methods : Lipids from EVs secreted by YEJG‐P and YEJG‐M2 cells were extracted and analysed by liquid‐chromatography mass spectrometry using an untargeted approach. We then deployed saponification to release the acyl chains from the lipid extracts of cells and their EVs as free fatty acids, which were subsequently analysed by gas‐chromatography‐mass spectrometry.

Results : Principal component analysis of the untargeted dataset indicated that EVs released by the micrometastatic YEJG‐M2 cells have a different lipid profile from EVs released by YEJG‐P cells. Statistical analyses indicated that a key contributor to this difference was the fact that several classes of lipid species released from micrometastatic cells had increased levels of C18:1 in their acyl chains. Analysis of the acyl chains only revealed that micrometastatic cells released EVs with significantly increased levels of C18:1‐containing lipids and that this was manifest both as an increase in the quantity of C18:1‐containing lipids and an increased ratio of C18:1/C18:0‐containing lipids. Moreover, both the quantity of C18:1 and the ratio of C18:1/C18:0‐containing lipids were unaltered in cell extracts, indicating that micrometastatic and primary tumour cells do not differ in their MUFA lipid profile but alter the selectivity of sorting of these lipids into EVs.

Summary/Conclusion : EVs released by YEJG‐M2 cells are enriched with C18:1‐containing lipids, and we propose that C18:1‐containing lipids are established to exert a cytoprotective role by opposing Fe 3+ ‐mediated lipid oxidation and ferroptosis, which can occur when glutathione levels are low.

Funding : This study was supported by the CRUK Scotland Institute (McNab Centre for Cancer Innovation).

Effects of in vivo‐like EVs from metastatic cells on the phenotype of recipient cells

Presenter: Kerttu L. Airavaara

University of Helsinki, Helsinki, Finland

Introduction : The contents of EVs resemble that of the parent cell type. Because cell culture conditions affect the phenotype of the cells, the phenotype of EVs will be changed when cells are cultured under physiologically relevant conditions. We recently demonstrated that adaptation to hypoxic conditions takes longer than was previously thought. In this study we build on these findings to demonstrate the effects of EVs from adapted hypoxic metastatic cancer cell lines cultured in 3D on recipient cells. We hypothesise that these physiologically cultured cancer cells will express a different metastatic phenotype compared to cells in normoxic 2D conditions, which can result in different preconditioning of the premetastatic niche.

Methods : Two human ovarian cancer cell lines, one metastatic (SK‐OV‐3) and one not (SK‐OV‐4), were adapted to either normoxic (atmospheric O 2 ) or hypoxic (5% O 2 ) conditions for 2 weeks. After adaptation, the cells were cultured in either 3D or 2D, and EVs were isolated by ultracentrifugation. Because the liver is a common metastatic site for ovarian tumours, EVs from the ovarian cells were then applied to human primary hepatocyte co‐culture spheroids, and the effects, with a focus on premetastatic changes, were examined using qPCR and LC‐MS/MS proteomics.

Results : Our previous results demonstrated an increase in metastatic markers in cell lines in hypoxic conditions. SK‐OV‐3 and SK‐OV‐4 ovarian cancer cell lines could be cultured long‐term under hypoxic conditions and readily formed spheroids also in hypoxic conditions. Different culture conditions affected EV content so that 3D culture combined with hypoxic cultures changed EV content more than these variables alone.

Summary/Conclusion : When culturing cells in hypoxic conditions, sufficient adaptation time is crucial. This is longer than the 72 h typically allowed. Ovarian cancer cell lines could be cultured successfully under these conditions, including in physiologically relevant 3D spheroids. The EVs arising from metastatic cell lines exerted distinct effects on primary hepatocyte co‐culture spheroids, consistent with preconditioning of the premetastatic niche.

Funding : This study was supported by the Research Council of Finland, GeneCellNano flagship project [#337430] and Finnish Cultural Foundation [#00220283].

The role of extracellular vesicles produced by neuroblastoma cells in the regulation of inflammatory pathways

Sina Marie Ceplis 1 , Laia Pagerols Raluy 1 , Konrad Reinshagen 1 , Bente Siebels 1 , Michaela Schweizer 1 , Sara Peggion 1,2

1 University Medical Centre Hamburg‐Eppendorf (UKE), Hamburg, Germany; 2 University Medical Centre Düsseldorf, Düsseldorf, Germany

Introduction : Neuroblastoma (NB) is the most common solid extracranial tumour arising from the developing sympathetic nervous system and accounting for 12%–15% of cancer‐related deaths in children. The amplification of the proto‐oncogene MYCN is associated with poor prognosis and classifies patients straight as high‐risk, therefore representing a clinical challenge. The tumour microenvironment (TME) consists of multiple components, such as stromal cells, immune cells and extracellular vesicles (EVs), known mediators of intercellular interactions. EVs produced by MYCN‐amplified NB cell lines seem to be able to transfer an aggressive phenotype to MYCN non‐amplified cells. Therefore, we presume an EV‐mediated modulation of the inflammatory context within the TME of NB correlating with the MYCN amplification status. Hence, the aim of this study is the protein profiling of EVs from different NB cell lines, thereby decoding a potential MYCN amplification dependency of their inflammatory protein cargo.

Methods : EVs from six stage 4 NB cell lines ( n = 3 MYCN amplified, n = 3 MYCN non‐amplified) were isolated, characterized and tested for sample purity by means of Nanoparticle Tracking Analysis (NTA), Western Blot (WB) and Electron Microscopy (EM). For WB, different EV markers such as CD9, TSG101 and Alix were detected. Finally, protein profiling of both EVs and whole cell lysates was performed with mass spectrometry (MS), and raw data were analysed using ANOVA and linear models.

Results : NTA shows differences in the EV production independent of the MYCN amplification status. Interestingly, the EV, and not the whole cell lysate proteomic analysis, suggests an NB cell line clustering according to their MYCN amplification status. Furthermore, EV MS shows upregulation of several proteins involved in inflammatory processes with a clear differentiation between MYCN‐amplified and non‐amplified cell lines.

Summary/Conclusion : The aforementioned results propose a distinct differential EV production and protein cargo in stage 4 neuroblastoma cell lines according to their MYCN molecular biology. Hence, we observe a differential upregulation of inflammation‐related factors in EVs from MYCN amplified cells compared to those without amplification. Consequently, further research on this emerging topic is needed to shed light onto the tumourigenic mechanisms behind EV‐mediated cell signalling in NB.

Funding : This study was supported by the Else Kröner‐Fresenius‐Stiftung iPrime Scholarship (2021_EKPK.10), UKE Hamburg.

Exploring tumour derived extracellular vesicles from triple negative breast cancer tumour‐on‐a‐chip and 3D models

María Fidel‐Lledó 1 , Vira Sharko 2,3 , Inés Mármol 2,3 , Irene Zúñiga‐Centenero 1 , Diego Baranda‐Martínez‐Abascal 1,4 , Marc Moltó‐Abad 1,4 , Verónica Villagrasa‐Araya 1,4 , Roser Ferrer 1,2,5 , Simó Schwartz Jr. 1,2,5 , Ibane Abasolo 1,4,5,6 , Ignacio Ochoa 2,3,4 , Joaquin Seras‐Franzoso 1,2,7

1 Clinical Biochemistry, Drug Delivery & Therapy, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain; 2 TME (Tissue Microenvironment) Lab, I3A Universidad de Zaragoza, Zaragoza, Spain; 3 Instituto de Investigación Sanitaria de Aragón (IISA), Zaragoza, Spain; 4 CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Madrid, Spain; 5 Clinical Biochemistry Service, Vall d'Hebron University Hospital, Barcelona, Spain; 6 Instituto de Química Avanazada de Cataluña (IQAC), Centro Superior de Investigaciones Científicas, (CSIC), Barcelona, Spain; 7 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain

Introduction : 2D cellular models are still nowadays the workhorse for the study of extracellular vesicles (EVs) in tumour progression. However, there is an urgent need to approach the tissue context in terms of tumour architecture, mechanical stimuli and microenvironment composition. Here, we propose the use of 3D and Organ‐on‐a‐Chip (OoC) models that mimic the tumour physiological niche, for the comprehensive study of the role of tumour cell subpopulations in disease progression. With a particular focus on cancer stem cells (CSCs) and their vesicular secretome.

Methods : The triple negative breast cancer (TNBC) cell line MDA‐MB‐231 was transduced with constructs expressing CD63 fused to the fluorescent reporter mClover3, placed under the control of ALDH1A1 or PGK promoters for selective labelling of EVs secreted by CSCs or bulk cancer cells, respectively. We explored two OoC microfluidic systems: (i) Be‐Double Flow for 2D and (ii) Be‐Transflow for 3D, both with two overlapping compartments separated by a porous membrane, allowing EVs diffusion. 3D cultures included cancer cells (homotypic) or mixed populations of cancer cells and fibroblasts (heterotypic). For migration assays the spheroids were embedded in artificial ECM. Migrating halos were captured through the Thunder Imaging system. EVs quantification was done using the SP‐IRIS/ExoView platform.

Results : EVs derived from CD63‐mClover3+ cells were selectively quantified from both apical and basal microfluidic compartments. EVs secreted by 3D models accounted for a 2‐fold rise in comparison to 2D cultures. Concerning 2D models, bulk cells showed a significant increase in tEVs release compared to CSCs. Exploring more complex setups, substantial amounts of EVs were captured beyond ECM. Moreover, when we populated such ECM with fibroblasts, a significant difference in the tEVs secretion pattern from cancer cell spheroids was observed.

Summary/Conclusion : We described an innovative strategy for the dynamic tracking of the tumour vesicular secretome in complex setups. This approach identifies specific tumour EVs subsets at an early invasion stage, their diffusion through channels that simulate tumour perfusion and the ECM. In addition, it highlights the relevance of shifting to 3D multicellular systems for the transferable study of tEVs.

Funding : This study was supported by the10th Edition Geivex Mobility Fellowships. Project PI22/1301, P‐FIS grant FI23/00223 and co‐funded by the European Union.

Technology based on microfluidic chips for tracking cancer therapy using extracellular vesicles

Eylul Gulsen Yilmaz 1,2 , Gamze Kara‐Magden 3 , Yeseren Saylan 4 , Fatih Inci 1,2

1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey; 3 Department of Chemistry, BUGAMED Biotechnology Industry and Trade Inc., Eskişehir, Turkey; 4 Hacettepe University, Ankara, Turkey

Introduction : Cancer cells encounter diverse fluid shear stresses (FSS) within the tumour microenvironment, but existing in vitro 3D models are not adequate for examining how these forces influence cancer progression and resistance to chemotherapy. To address this gap, new approaches have emerged, including the use of microfluidic systems. These systems are gaining popularity due to their affordability, high‐throughput capabilities, efficient use of resources, and ease of manipulation. Furthermore, they can simulate ECM‐like conditions with biomaterials such as silk fibroin (SF), which is ideal for 3D cell cultures because of its strength, biocompatibility, and biodegradability. Additionally, research has revealed that tumour‐derived extracellular vesicles (EVs) play a significant role in promoting cancer growth, drug resistance, and metastasis.

Methods : In this research, MCF‐7 cells were cultured under static 2D, static 3D, and dynamic environments. EVs were collected from these cultures and isolated using a microfluidic device based on ultrafiltration, featuring 200 and 50 nm filters. The EVs were subsequently characterized using western blot, NTA, and SEM. Then, MCF‐7 cells were treated with the anticancer drugs doxorubicin and docetaxel to examine whether these treatments induce changes in EVs under FSS. Lastly, RT‐qPCR was utilized to analyse variations in miRNA levels.

Results : EVs from the control groups exhibited average sizes of 177 nm (static 2D), 91 nm (static 3D), and 128 nm (dynamic conditions). EVs generated under static environments appeared more aggregated compared to those from dynamic cultures. Increasing doses of Doxorubicin led to a reduction in EV size. SEM images supported these findings, showing consistent effects with both Doxorubicin and Docetaxel treatments. Western blot analysis confirmed successful EV isolation, with a strong CD63 band detected between 30 and 80 kDa, notably around 70 kDa, and a CD81 band observed near 60 kDa. Additionally, miRNA expression was assessed for EVs derived from MCF‐7 cells.

Summary/Conclusion : In summary, we effectively isolated and characterized EVs under various conditions. These EVs hold significant potential as biomarkers for cancer and may serve as a promising tool for early cancer detection.

Funding : The authors gratefully acknowledge support from the Scientific and Technological Research Council of Turkey (TÜBİTAK) 2232 International Fellowship for Outstanding Researchers (Project No: 118C254).

Colorectal cancer‐derived extracellular vesicles affect the immunomodulatory properties of hepatocytes, contributing to liver metastatic colonization

Presenter: Elisa Costanzo

University of Palermo, Palermo, Italy

Introduction : Growing evidence shows a key role of colorectal cancer‐derived small extracellular vesicles (CRC‐sEVs) in shaping the hepatic pre‐metastatic niche (h‐PMN), a supportive and receptive microenvironment for cancer cells colonization. However, the effects of CRC‐sEVs have been widely described on liver non‐parenchymal cells and only marginally on hepatocytes. Nevertheless, our previous study showed CRC‐sEVs’ ability to induce in healthy hepatocytes epithelial‐mesenchymal transition, early event leading to fibrosis, a pivotal feature of h‐PMN. Evasion of immune surveillance is another crucial event defining h‐PMN, mainly caused by the activation of immune check‐point PD‐L1/PD‐1. In the light of a previous study revealing that CRC‐sEVs can carry PD‐L1 and upregulate its expression in macrophages, we hypothesized that CRC‐sEVs could also affect hepatocytes’ immunomodulatory properties, thus supporting the creation of an immunosuppressive microenvironment within h‐PMN.

Methods : The THLE‐2 cell line was used as a model of healthy human hepatocytes and was treated with CRC‐sEVs isolated from the colorectal primary adenocarcinoma cell line SW480.

Results : The study revealed that the treatment with CRC‐sEVs determined in hepatocytes a significant increase of PD‐L1 protein expression, in particular in the nucleus. This unexpected observation has been supported by emerging data describing a nuclear form of PD‐L1 (nPD‐L1) that works as a transcriptional factor. According to that, we observed that the increased nPDL1 in CRC‐sEVs‐treated hepatocytes was associated with the upregulation of its specific targets, GAS6, VISTA, and PD‐L2, proteins involved in the regulation of immune tolerance mechanisms. Interestingly, the upregulation of these regulators of immune response was lost when hepatocytes were pre‐treated with a molecule able to block the nuclear delivery of the sEVs cargo.

Summary/Conclusion : Taken together, these results, beyond revealing CRC‐sEVs ability to actively modulate the immunomodulatory properties of the hepatocytes, could provide new insights to improve our knowledge of the liver metastatic process and to open new avenues for developing early and effective therapeutic strategies.

Elucidating the role of ZEB1 in extracellular vesicle‐mediated crosstalk in the colorectal tumour microenvironment

Van Manh Hung Le, Farzaneh Malekian, Alireza Shamsian, Biljana Gigic, Christoph Kahlert

Heidelberg University Hospital, Heidelberg, Germany

Introduction : Colorectal cancer is the world's second leading cause of cancer‐related deaths, with metastasis to organs like the liver and lungs presenting significant treatment challenges. Epithelial‐to‐mesenchymal transition (EMT), mainly driven by EMT transcription factors (EMT‐TFs), plays a crucial role in initiating metastasis. ZEB1 is a key EMT‐TF that regulates EMT, impacting cell migration, invasion, and various processes in development and cancer progression. Tumour cells not only transform their own phenotype but also modulate local and distant environments to create pro‐tumourigenic niches through extracellular vesicles (EVs). While recent studies have shown that conditioning distant organ microenvironments supports metastasis in pancreatic, breast, and melanoma cancers, the impact of tumour‐derived EVs on the colorectal tumour microenvironment still requires further investigation.

Methods : ZEB1 knockout was performed using a CRISPR/Cas9 plasmid in the HCT116 colorectal cancer cell line. According to MISEV guidelines, EVs were isolated by either sequential ultracentrifugation or ultrafiltration coupled with size exclusion chromatography. EVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy imaging, and immunoblotting. RNA‐sequencing analysis was performed to assess the cellular gene expression landscape after ZEB1 knockout. The protein content of EVs was examined by mass spectrometry to identify differentially expressed proteins associated with EMT, aiming to elucidate their role in modulating the tumour microenvironment.

Results : NTA suggested reduced secretion of EVs from ZEB1 knockout cells compared to parental cells. Preliminary data from EV mass spectrometry revealed alterations in the expression of 58 proteins, and RNA‐sequencing identified 788 differentially expressed genes resulting from ZEB1 knockout.

Summary/Conclusion : Altogether, our exploratory results demonstrate that ZEB1 may play a role in the biogenesis of EVs in HCT116 colorectal cancer cells. Further experiments are required to examine the function of wild‐type and ZEB1 knockout EVs on different cell types in the tumour microenvironment.

Funding : The project is funded by the German Research Foundation (DFG).

EVs from mesenchymal stem cells as a potential therapeutic tool for bladder cancer?

Karolina Pokrzywka, Łukasz Kaźmierski, Małgorzata Maj

Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Toruń, Poland

Introduction : Cancer is still a severe problem in the world. Current cancer therapies focus on destroying tumours, which can lead to functional defects in surrounding tissues. A possible form of regeneration of these tissues is using mesenchymal stem cell (MSCs)‐based therapies. These from adipose tissue deserve special attention because of their non‐invasive methods of isolation and regenerative, immunosuppressive, and anti‐inflammatory potential. However, many features necessary for the healing process also play an essential role in cancer progression. The direction of action of MSCs may depend on the properties of tumour cells remaining after treatment. Recent studies confirmed that soluble mediators released into the environment, particularly extracellular vesicles (EVs), are responsible for the regenerative potential of MSCs. It seems necessary to investigate their safety and the impact of EVs from MSCs on the tumour microenvironment. They may be a valuable tool for oncology therapies if they limit or stop the development of tumours.

Methods : To isolate EVs, we used a conditioned medium from the ASC52telo cell line after 72 h culture. EVs were isolated using a total exosome isolation reagent (invitrogen). The exosome protein content was measured using the spectrophotometric method. To confirm that isolated particles are exosomes exo‐check exosome antibody array was used. Subsequently, four bladder cancer cell lines (5637, HB‐CLS‐1, HT‐1376, T24) were cultured with EVs for 72 h. Then, we assessed the migration and invasion profile of cancer cells using the Cell Invasion and Cell Migration Assay Kit. Their ability to invade and migrate was assessed by incubating the cells on special inserts.

Results : Preliminary studies evaluating the effect of EVs from MSCs on the viability and proliferation of cancer cells have shown that at an appropriate concentration (100 µg/mL), EVs limit the development of cancer cells. In studies on the invasive and migratory phenotype, the effect turned out to be dependent on the cancer line used.

Summary/Conclusion : The obtained results indicate that EVs have a limiting effect on tumour development at concentrations above 100 µg/mL. Their safety was confirmed in vitro on human tumour lines. These results provide a basis for further in vitro analyses and extend the studies to an animal model.

PD‐L1 enrichment on extracellular vesicles is modulated by PTEN and promotes immune evasion in melanoma

Caihong Li, Miriam Zentgraf, Jan Van Deun, Katja Blume, Susanne Rößner, Andreas S. Baur

Department of Dermatology Unikilinikum Erlangen, Friedrich‐Alexander‐Universität (FAU) Erlangen‐Nürnberg, Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany

Introduction : In melanoma, phosphatase and tensin homolog (PTEN) loss frequently occurs and is associated with tumour progression, metastasis, and response to immunotherapy. In certain cancers, PTEN loss enhances the production of programmed death ligand‐1 (PD‐L1)‐enriched extracellular vesicles (EVs) in a PI3K‐dependent manner. Some research had shown that metastatic melanoma released high levels of EVs carrying PD‐L1, which suppressed CD8+ T cell function and mediated resistance to immune checkpoint inhibitor therapy. Hence, exploring the relationship between PTEN‐deficient melanoma and immune response through the release of EVs may offer new perspectives for melanoma treatment.

Methods : Fusing PD‐L1 to a modified oestrogen receptor (mER) ligand‐binding domain generated a tamoxifen‐inducible form. The construct was expressed in melanoma cell lines with PTEN wild type, PTEN mutation, and PTEN loss. The induced mature PD‐L1 in melanoma cell lines was visualized by GFP/RFP analysis using a confocal laser scanning microscope. EVs from supernatants of PD‐L1‐overexpressing cell lines were purified using OptiPrep density gradient and size‐exclusion chromatography (SEC). EV characterization was performed through Western blot and nanoparticle tracking analysis (NTA). The levels of PD‐L1 carried by EVs were analysed by western blot. An MCSP‐CAR expression plasmid was constructed for producing CD8+ MCSP‐specific CAR T cells, targeting/killing melanoma cells.

Results : In the tamoxifen‐inducible system, when the hormone‐binding domain of the mER bound to tamoxifen, the receptor was released from its inhibitory complex, enabling PD‐L1 to exert its function. We observed that after induction, PD‐L1 cleavage/maturation occurred in the perinuclear region, with the active C‐terminal part translocating to the cell membrane while the cleaved‐off N‐terminus remained around the nucleus. The level of EV containing PD‐L1 released from PTEN‐loss melanoma cells was higher than that from PTEN‐wt melanoma cells. The impact of PD‐L1‐enriched EVs produced by melanoma cells with different genotypes on CD8+ T cell‐mediated immune response will be compared.

Summary/Conclusion : PTEN modulates the release of PD‐L1‐carrying EVs, thereby regulating immune evasion in melanoma.

Subtopic Disease/therapy relevant mechanism(s) of action 2

Harnessing the unique lipid composition and small RNA‐delivery capacity of probiotic Pediococcus acidilactici ‐derived extracellular vesicles suppresses spore germination of Clostridioides difficile and opportunistic infection

Wen‐Yu Liu, Bao‐Hong Lee, Chia‐Hsiu Chen, Tang‐Long Shen, You‐Zuo Chen, Wei‐Hsuan Hsu

National Cheng Kung University, Tainan, Taiwan (Republic of China)

Introduction : Clostridioides difficile infection (CDI) presents significant challenges in healthcare due to severe and recurrent infections and the emergence of antibiotic‐resistant strains. Therefore, probiotic‐derived extracellular vesicles (EVs) are considered an alternative therapeutic strategy. This potential is attributed to EVs' ability to remain in the intestine longer than probiotics and be absorbed by both bacteria and host cells, which play crucial roles in modulating the intestinal environment, suggesting that probiotic‐derived EVs could be developed as innovative therapeutic materials.

Methods : This study investigates the therapeutic potential of EVs isolated from probiotic Pediococcus acidilactici for CDI. P. acidilactici ‐derived EVs exhibited a unique lipid composition, specifically cardiolipin, which facilitates the specific targeting of C. difficile. The predictive alignment revealed that P. acidilactici ‐derived EVs carry specific small RNAs that interfere with key virulence genes in C. difficile through sequencing the small RNAs in the P. acidilactici ‐derived EVs and analysing the C. difficile transcriptome.

Results : The proteomic analysis results indicate that the proteins associated with spore germination in C. difficile are inhibited after treatment with P. acidilactici‐ derived EVs. In vivo experiments demonstrated that orally administered P. acidilactici ‐derived EVs effectively reduced the incidence of C. difficile infection in mice.

Summary/Conclusion : This study paves the way for developing P. acidilactici ‐derived EVs as a promising therapeutic approach for CDI.

Funding : This research work and subsidiary spending were mainly supported by the National Science and Technology Council (NSTC) in Taiwan under grant no. NSTC 111‐2636‐B‐006‐014 (Young Scholar Fellowship Program). This research was also supported in part by the Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University (NCKU). The authors gratefully acknowledge the technical services provided by the Bio‐image Core Facility of the National Core Facility Program for Biotechnology, National Science and Technology Council, Taiwan.

Understanding the potential involvement of miRNA regulated genetic pathways in the extracellular vesicles of uveitis patients

Presenter: Inderjeet Kaur

L. V. Prasad Eye Institute, Hyderabad, India

Introduction : Non‐infectious uveitis (NIU) is characterized by a persistent intraocular inflammation of the uveal tract. A complex interplay of genetic and epigenetic factors may explain the dysregulated immune response leading to the development of non‐infectious uveitis. To unravel the complex aetiology underlying uveitis pathogenesis, we investigated the regulation of gene expression for their role in disease development.

Methods : MiRNA sequencing was performed on patients with naïve anterior uveitis, comparing them with non‐inflammatory controls (NI n = 6 in each group). Identified miRNA profiles were further compared among sub‐categories of uveitis: non‐infectious anterior (NIAU n = 4) and non‐infectious posterior (NIPU n = 3) and NI controls ( n = 3) using microarrays. Identified potential miRNAs were validated using qPCR in extended samples from the blood of NIAU and NIPU compared to controls ( n = 15 in each group), as well as in the extracellular vesicles of vitreous humour of NIPU compared to NI controls ( n = 10 in each group). Further, a correlation between miRNAs derived from blood and vitreous derived EVs led to the identification of prominent dysregulated pathways, some of which were further experimentally validated.

Results : A total of 1801 known miRNAs and 1618 novel miRNAs were found to be dysregulated in the study. Notably, six miRNAs showed consistent significant validation in blood samples among anterior and posterior NIU patients. Exosomal miRNA analysis revealed an upregulation for hsa‐miR‐146a, hsa‐miR‐155, hsa‐miR‐30a, hsa‐miR‐29, hsa‐miR‐4677, hsa‐miR‐16‐2, and hsa‐miR‐1227 in NIU. Target prediction analysis revealed 35 dysregulated pathways. A further validation demonstrated the involvement of significantly dysregulated miRNAs in alternative complement pathway activation, indicating its role in the pathogenesis of NIU.

Summary/Conclusion : Our findings discern the regulatory roles of putative miRNAs and their respective target genes in NIU progression. Further studies are warranted in different categories of NIU to assess their role as prognostic markers and major therapeutic targets.

Funding : The authors would like to thank Santen Pharmaceutical Co., Ltd., Japan, and DBT programme support grant BT/PR32404/MED/30/2136/2019 for funding the project. PK would like to acknowledge the Indian Council of Medical Research (ICMR), Government of India (wide letter no: 5/3/8/87/ITR‐F/2020), for supporting her fellowship.

Proteomics insights into small extracellular vesicles medicating endocrine resistance in breast cancer

Shanaya Haque 1 , Morgan Rouse 1 , Francis Li 1 , Purva Nayam 1 , Kaelan Samoranos 1 , Elexa Rallos 1 , William Glembocki 1 , Vanessa Correll 1 , Shimin Chen 1 , Julius Nyalwidhe 1 , Eric Feliberti 1,2 , Lifang Yang 1

1 Macon & Joan Brock Virginia Health Sciences at Old Dominion University, Norfolk, Virginia, USA; 2 Sentara Healthcare, Virginia Beach, Virginia, USA

Introduction : Oestrogen receptor‐positive (ER+) breast cancer represents the most common breast cancer subtype. Tamoxifen and fulvestrant, both directly targeting the ER, have considerably reduced recurrence and mortality from ER+ breast cancer. Unfortunately, acquired resistance to these and other antiestrogens eventually becomes a major challenge, especially in the metastatic setting. Accumulating evidence suggests small extracellular vesicles (sEVs) that are employed by cancer cells for intercellular communication have been involved in mediating endocrine resistance in breast cancer. But the specific sEV molecular cargo and sEV‐mediating intercellular signalling to drive this phenotype remain largely unknown. The aim of this study was to address these issues by conducting an in‐depth characterization of the proteomes of sEVs derived from a breast cancer endocrine resistance model.

Methods : Normal breast epithelium MCF10A, endocrine therapy‐sensitive MCF7 breast cancer cells, and dual tamoxifen‐ and fulvestrant‐resistant MCF7 variant LCC9 cells were cultured. sEVs were isolated from cell supernatants via a differential ultracentrifugation approach. The morphology, size, and purity of isolated sEVs were confirmed by transmission electron microscopy, NanoSight, and western blot. sEV proteins were extracted by the RIPA buffer and quantified by the micro‐BCA protein assay. Label‐free mass spectrometry and bioinformatic analysis were conducted to compare cellular and sEV proteomes across three cell lines.

Results : Compared to MCF10A and MCF7 cells, LCC9 cells have demonstrated a significant reduction in sEV secretion and sEV protein content. These changes are also revealed by unbiased proteomics analysis in which the LCC9 cellular proteome has shown alterations in protein sorting signalling and EV biogenesis pathways. Compared to normal counterparts, sEVs derived from both cancer cells are enriched in proteins that play important roles in cancer growth and metastasis. Further analysis indicated that proteins regulating interferon and PTEN signalling are more upregulated in sEVs derived from drug‐resistant cells than drug‐sensitive versions, suggesting their roles in fostering an endocrine resistance phenotype.

Summary/Conclusion : Our study highlights the mechanistic insights of sEVs in the endocrine resistance in breast cancer. Further investigations are warranted, paving the way to identify novel endocrine resistance biomarkers and druggable therapeutic targets to monitor and reverse this deadly disease.

Funding : This work was supported by Eastern Virginia Medical School Breast Cancer Research funds.

Serum exosome proteomics reveals the therapeutic mechanism of electroacupuncture treatment for rheumatoid arthritis

Presenter: Ningcen Li

Tianjin University of Traditional Chinese Medicine, Tianjin, China

Introduction : Rheumatoid arthritis (RA) is a common autoimmune disease and considered a global public health priority. Previous studies have shown that electroacupuncture (EA) had good therapeutic effects on RA. We here investigated whether EA could alleviate the symptoms of RA through network regulation from the perspective of circulating exosomes.

Methods : EA treatment was administered to Zusanli (ST36) and Huantiao (GB34) in complete Freund's adjuvant (CFA)‐induced arthritis rats. We first evaluated the treatment effects of rats with paw withdrawal latency (PWL), paw swelling and histopathological examination, and observed the influence of regulating serum exosomes on the EA effect. Then label‐free quantitative proteomics and bioinformatics analysis were used to screen biomarkers and differentially expressed proteins of serum exosomes.

Results : The results supported that exosomes and carriers may be involved in the analgesic and anti‐inflammatory effects. EA can alleviate inflammation and pain in CFA‐infected joints in rats, and the therapeutic effect of EA is weakened after using GW4869 to antagonize circulating exosomes. Further use of various methods to infuse the circulating exosomes (Acu‐exo) after EA, it was found that Acu‐exo can simulate the therapeutic effect of EA. Acu‐exo may be an important carrier for EA to exert network regulatory therapeutic effects on RA, and can serve as a targeted nano‐therapeutic drug for RA.

Summary/Conclusion : Our study provides new evidence for the clinical transformation of acupuncture based on exosomes and provides a more comprehensive insight into the pathogenesis of RA and the therapeutic mechanism of EA at the level of serum exosomes.

Funding : This work was supported by the National Natural Science Foundation of China (NSFC) (Nos. 82374563, 82305370, 82004467, 82030125).

The role of extracellular vesicles in driving macrophage recruitment in schwannoma

Sara Veiga 1,2 , Christine Chiasson‐MacKenzie 1,3, Emily Wright 1,3 , Jeremie Vitte 4 , Marco Giovannini 4 , Andrea McClatchey 1,3 *, Shannon Stott 1,2,5 *

1 Krantz Family Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; 2 Department of Surgery, Harvard Medical School, Boston, Massachusetts, USA; 3 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; 4 Department of Head and Neck Surgery, David Geffen School of Medicine at UCLA and Jonsson Comprehensive Cancer Center (JCCC), University of California Los Angeles, Los Angeles, California, USA; 5 Center for Engineering in Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA

Introduction : Schwannomas are rare tumours of the central nervous system and develop in complex microenvironments with multiple cell types. Schwannomas present surprising intrinsic heterogeneity that makes the development of effective therapies challenging. Macrophages are abundantly found in most schwannomas, but not much is known about their role in the disease, or interactions with tumour cells. Extracellular vesicles (EVs) are known means of communication between cells and their roles in tumour and immune interactions have been widely described. In this study we want to investigate how schwannoma intrinsic heterogeneity influences EV cargo and biogenesis and what role vesicles have in macrophage recruitment.

Methods : For this study we used an in vitro model of schwannoma, where Nf2‐deficient Schwann cells are deprived of either neuregulin (−Nrg) or glutamine (−Q), acquiring one of two distinct phenotypic states that mirror intrinsic heterogeneity. EVs were isolated from cell conditions through ultracentrifugation at 100,000 × g for 1 h 30 min. Characterization was done for sizing, using nanoparticle tracking analysis (NTA), and classic EV markers were assessed by western blot. A transwell assay was used to explore if EVs have a role in macrophage recruitment. Isolated EVs as well as the non‐EV fraction and complete cell conditioned media from cells in the two distinct states were placed in the bottom wells, while bone‐marrow derived macrophages were seeded on the top of the porous membrane and incubated for 24 h.

Results : EVs from the two different populations present similar sizes, but −Q cells seem to release more EVs than −Nrg cells. Additionally, nutrient deprivation also seemed to result in different loading of ALIX, but not HSP70, into the EVs. Data from the migration assay show that EVs and cell conditioned media seem to have a similar effect in macrophage recruitment, while almost no cells were seen to migrate on the non‐EV conditions. Furthermore, the distinct states seem to have a different effect on recruitment.

Summary/Conclusion : Our data provide important insight into EV roles in schwannoma, indicating a possible effect in macrophage recruitment into tumours. Our ongoing efforts will continue to define how tumour heterogeneity impacts EVs characteristics and the relevance of these differences to macrophage recruitment in the tumour microenvironment.

Evaluation of extracellular vesicles derived from human osteoarthritic tissues for better diagnosis of osteoarthritis

Jana Matejova, Marko Moravek, Jana Bzdilova, Lucia Slovinska, Livia F. Kolesarova, Marek Lacko, Denisa Harvanova

Pavol Jozef Safarik University in Košice, Košice, Slovakia

Introduction : Osteoarthritis (OA) is a disease of the entire joint, including structural changes in articular cartilage, ligaments, subchondral bone, synovium, and muscles around the joint. Since the pathogenesis of OA is not yet fully understood, it is essential to study the processes occurring within the joint microenvironment. Recent basic research on OA has increasingly focused on the analysis of extracellular vesicles (EVs), which offer deeper insights into the mechanisms of OA. EVs are small membrane particles released by cells that play a significant role in intercellular communication. In OA, EVs released by chondrocytes and other cells in the joint microenvironment can contribute to inflammation and degradation of the extracellular matrix, highlighting their crucial role in disease pathogenesis. Studying OA tissues and their products can offer new insights into the pathogenesis of the disease and help identify potential biomarkers for OA.

Methods : OA tissues, including cartilage (CRT), synovial membrane (SM), and Hoffa's fat pad (HFP), were obtained during total knee replacement, following ethics committee approval and obtaining informed consent from OA patients. Conditioned medium (CM) was prepared from all OA tissues and subsequently characterized using the MILLIPLEX Assays and the MAGPIX Luminex platform. EVs were isolated from CM using a precipitation method and further characterized for size, yield, and expression of surface markers by nanoparticle tracking analysis and flow cytometry.

Results : The highest levels of inflammatory mediators (RANTES, G‐CSF, IL‐1β, IL‐6) were present in CM from SM compared to other tissues. EVs from OA tissues were positive for well‐established surface markers of EVs CD9, CD63 and CD81. SM‐EVs exhibited the highest levels of markers HLA‐DRDPDQ, CD62P, and CD42a epitopes on their surface. (MCSP) marker was significantly more prevalent in CRT‐EVs compared to other sources, while CD44 and CD29 were most present in HFP‐EVs.

Summary/Conclusion : This study proves that the SM plays a major role in the inflammatory process and pathogenesis of OA. The MCSP marker was significantly higher in CRT‐EVs compared to other sources, indicating its potential as a specific biomarker of OA.

Funding : This research was supported by the APVV‐17‐0118 and VEGA 1/0146/24.

CDK6 regulates extracellular vesicles in AML

Belinda S. Maw 1* , Elisabeth Gamper 1* , Nathalie Havranek 1 , Astrid Digruber 2 , Martin Maw 3 , Silvio Kau‐Strebinger 4 , Markus Zojer 1 , Gerwin Heller 5 , Zuzanna Kieron 1 , Jonatan Kendler 1 , Karin Hummel 6 , Ingrid Walter 3 , Waltraud Tschulenk 3 , Monika Ehling‐Schulz 2 , Veronika Sexl 1 , Thorsten Klampfl 1 , Karoline Kollmann 1

1 Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria; 2 Institute of Microbiology, University of Veterinary Medicine, Vienna, Austria; 3 Independent researcher 4 Institute of Morphology, University of Veterinary Medicine, Vienna, Austria; 5 Department of Medicine I, Division of Oncology, Medical University of Vienna, Vienna, Austria; 6 Vetcore facility for proteomics, University of Veterinary Medicine, Vienna, Austria

Introduction : Extracellular vesicles (EVs) are linked to various diseases with implications in the progression, immune activation, biomarkers and therapy. Acute myeloid leukaemia (AML) is a highly aggressive disease with elevated EVs in the serum of patients. One crucial regulator and prognostic biomarker for AML is the cell cycle regulator cyclin‐dependent kinase 6 (CDK6). Palbociclib, a CDK4/6 kinase inhibitor, is already FDA‐approved for breast cancer and in clinical trials for AML; however, the exact mechanisms that are induced by the inhibition of both cell‐cycle kinases and potential combinatorial treatments remain to be elucidated.

Methods : Utilizing transformed HPCLSK cell lines as a novel murine in vitro model of AML, we analysed the effects of CDK4/6 inhibition on cellular states. Transcriptomic changes were measured by RNA‐Seq experiments, and CDK6 interaction partners were identified by mass spectrometry and western blots. To pinpoint the single role of CDK6, we compared CDK4/6 inhibited cells with cells lacking CDK6. We measured EVs by nanoparticle tracking analysis, electron microscopy and mass spectrometry and validated our findings in human AML cell lines.

Results : Human cancer cells are co‐dependent on CDK6 and components of intracellular vesicle pathways. CDK4/6 kinase inhibition in AML results in deregulated expression of genes assigned to intracellular vesicles and vesicle trafficking. Inhibition of CDK4/6 increases EV release of KMT2A‐MLLT3+ AML cells and alters the EV size distribution. We identified several proteins of the endosomal‐exosomal pathways as CDK6 interactors and detected high levels of CDK6 at endosomes, lysosomes and on EVs.

Summary/Conclusion : These data identify a novel function of CDK6 at intracellular and extracellular vesicles and propose an important role of CDK6 as a regulator of intercellular communication in AML.

TKI‐mediated interference with CD49b+ extracellular vesicles is associated with immune reactivation in metastatic renal cell carcinoma patients

Jeannette Salsetta 1 , Elisa D'Angelo 1 , Gianpiero Lupoli 1 , Eriomina Shahaj 2 , Elisabetta Vergani 1 , Agata Cova 1 , Paola Squarcina 1 , Licia Rivoltini 1 , Elena Verzoni 3 , Veronica Huber 1

1 Translational Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2 Genetics and Genomic Medicine Research & Teaching Dept, UCL GOS Institute of Child Health, London, UK; 3 Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Introduction : CD49b (ITGA2), the alpha subunit of α2β1 integrin, is expressed by T and NK cells, fibroblasts and platelets and is involved in adhesion and cell activation. CD49b expression by renal cell carcinoma (RCC) is associated with poor prognosis. As a heterodimer with CD29, CD49b functions as a collagen receptor and is involved in cancer progression and angiogenesis. Pazopanib is a tyrosine kinase inhibitor (TKI) with anti‐angiogenic and immunomodulatory properties (doi: 10.1002/ctm2.434). Here we investigated CD49b expression by plasma extracellular vesicles (EVs) in the context of TKI‐induced immune modulation and antiangiogenic activity in metastatic (m)RCC patients.

Methods : Peripheral blood samples from mRCC patients ( n = 8, INT146/14) were collected at baseline and at 3 and 6 months on‐therapy with Pazopanib. EVs, isolated by differential centrifugation, were evaluated by NTA, microsphere‐assisted flow cytometry (MACSplex) and ELISA for CD49b expression. Statistical analysis was performed with GraphPad Prism.

Results : Circulating CD49b+ EVs originated primarily from T regulatory cells and platelets, as indicated by correlation analysis with cellular immune profiles and blood counts. After 3 months of therapy, we recorded a decrease of plasma EV concentration and of CD49b+EVs with a concomitant decrease of immune suppression‐associated EV markers of Treg (CD2, CD44, CD25, CD44, CD29, CD45, HLA‐DR). This effect was accompanied by increased EVs expressing NK (CD56, CD69, HLA‐DR), T cell (CD2, CD3, CD4, CD8, CD25, CD69) and myeloid (CD14, CD1c, CD11c, CD86, HLA‐DR) markers, suggesting a reactivation of antitumour immunity detectable at the plasma EV level. CD49b+ EV levels correlated with platelet counts, Treg cells and PD‐L1+ monocytes. All of these showed a significant decrease in peripheral blood of patients after 3 months therapy while at the 6‐month time point we measured a recovery close to baseline levels.

Summary/Conclusion : Our findings suggest TKI‐induced decrease of CD49b+ EVs is associated with a reduction of platelets and the immune suppressive components Tregs and PD‐L1+ monocytes, resulting in a systemic immune reactivation. Inhibition of CD49b+ EVs by TKI could affect the local immune response and function of T cells, including Treg. We hypothesize the TKI‐mediated reduction of CD49b+ EVs coincides with the anti‐angiogenic and anti‐tumour activity of TKI via disruption of the CD49b/CD29 axis.

Funding : Italian Association for Cancer Research, AIRC, IG25078.

iPSC‐derived extracellular vesicles mitigate lung injury by regulating macrophage pyroptosis through miR‐223, miR‐18, and miR‐30

Presenter: Pei‐Ling Chi

Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (Republic of China)

Introduction : Uncontrolled lung inflammation is a critical aspect of lung infection‐associated acute lung injury (ALI). Induced pluripotent stem cells (iPSCs) offer a scalable and effective source for therapeutic extracellular vesicles (iPSC‐EVs). However, their mechanisms of action and pathways of communication remain under exploration. This study examines the contents of iPSC‐EVs in reducing inflammation in a mouse model of septic lung injury induced by Vibrio vulnificus .

Methods : Human iPSC‐EVs were administered intraperitoneally to 6‐week‐old male C3H/HeN mice 2 h before subcutaneous V. vulnificus infection. An in vitro model, using RAW 264.7 cells, was employed to investigate how iPSC‐EVs influence macrophage immune responses.

Results : Results demonstrated that iPSC‐EV treatment attenuated V. vulnificus ‐mediated activation of the NLRP3 inflammasome in lung tissue. Additionally, miR‐223, miR‐18, and miR‐30 expression was elevated in the lung tissue of infected mice treated with iPSC‐EVs, relative to untreated controls. In RAW 264.7 cells, miR‐223 suppressed NLRP3 inflammasome expression in V. vulnificus ‐infected macrophages by inhibiting NLRP3 and ASC. Furthermore, miR‐18 and miR‐30 promoted M2 macrophage polarization. Notably, iPSC‐EVs contained higher levels of miR‐223, miR‐18, and miR‐30 than EVs derived from human umbilical cord mesenchymal stem cells (hUC‐MSC‐EVs), suggesting a superior protective effect of iPSC‐EVs against septic lung injury.

Summary/Conclusion : In conclusion, iPSC‐EVs reduce V. vulnificus‐induced septic lung injury through the transfer of miR‐223, miR‐18, and miR‐30, leading to decreased NLRP3 inflammasome activity and an increase in anti‐inflammatory M2 macrophages, thereby supporting inflammation resolution.

Funding : National Science and Technology Council 113‐2314‐B‐075B‐003‐MY2.

Polybrene enhances uptake of Hoffa fat pad‐derived extracellular vesicles into osteoarthritic chondrocytes

Andrea De Luna 1 , Alexander Otahal 1 , Karina Kramer 1 , Markus Neubauer 1 , Stefan Nehrer 1

1 Department for Health Sciences, Medicine and Research, Center for Regenerative Medicine, University for Continuing Education Krems, Krems, Austria

Introduction : As a result of the ageing population, osteoarthritis (OA) became the fifteenth‐highest leading cause of disability, affecting over 7% of the global population. OA is not only a disease affecting joints but also has a major impact on the social life of patients. Current treatment options only lead to alleviation of the symptoms, including pain and inflammation, but not to regeneration of the cartilage and therefore restoration of its mechanical function. Extracellular vesicles (EVs), especially derived from mesenchymal stem cells, represent cell‐free and less restrictive tools for cartilage regeneration compared to conventional cell‐based therapies. It has been shown that only a small percentage of EVs are being taken up by the recipient cell. Therefore, methods to enhance EV uptake and therefore maximize their regenerative effect have to be established. Polybrene (hexadimethrin bromide) is a polycationic polymer that has been used to enhance transduction efficiency of enveloped viral particles via surface charge neutralization. The aim of this study was to determine whether application of polybrene enhances uptake efficiency of HFP‐MSC‐EVs into OA chondrocytes.

Methods : Primary MSCs were isolated from Hoffa's fat pad of knee replacement patients. HFP‐MSC‐EVs were generated in a vertical wheel bioreactor, were enriched via ultrafiltration and were characterised via nanoparticle tracking analysis in scatter and fluorescence mode for marker protein co‐localisation. Patient‐derived osteoarthritic chondrocytes were treated with different concentrations of polybrene, and its influence on cellular metabolic activity was assessed via XTT assay. To analyse the effect of polybrene on the uptake efficiency of EVs into cells, OA chondrocytes were treated with calcein AM labelled HFP‐MSC‐EVs in the presence or absence of polybrene. Confocal microscopy was performed to determine the ratio of internalized HFP‐MSC‐EVs.

Results : Lower concentrations of polybrene did not influence metabolic activity of OA chondrocytes. Supplementation of culture media with polybrene in combination with adding HFP‐MSC‐EVs leads to a four times higher EV uptake rate into OA chondrocytes.

Summary/Conclusion : Polybrene has the potential to enhance EV uptake into OA chondrocytes without influencing cells´ metabolic activity. This approach could be used to further boost the regenerative properties of EVs to treat OA.

Funding : This study was supported by the Gesellschaft für Forschungsförderung NÖ, grant number LSC18‐014.

Development of autism spectrum disorder in preterm infants: implication of bacterial extracellular vesicles

Julia Kuligowski 1,2,3 , Isabel Ten‐Doménech 1,2,3,4 , Guillermo Quintás 5 , Aleix Martí‐Navia 1 , Alba Moreno‐Giménez 6 , Belén Almansa 6 , Ana García‐Blanco 3,6,7

1 Neonatal Research Group, Health Research Institute La Fe (IIS La Fe), Valencia, Spain; 2 Servicio de Análisis de Vesículas Extracelulares (SAVE), Health Research Institute La Fe (IIS La Fe), Valencia, Spain; 3 Primary Care Interventions to Prevent Maternal and Child Chronic Diseases of Perinatal and Developmental Origin Network (RICORS‐SAMID) (RD21/0012/0015), Instituto de Salud Carlos III, Madrid, Spain; 4 Department of Analytical Chemistry, University of Valencia, Burjassot, Spain, 5 Leitat Technological Center, Barcelona, Spain; 6 Mental Health Research Group, Health Research Institute La Fe (IIS La Fe), Valencia, Spain; 7 Division of Psychiatry and Clinical Psychology, University & Polytechnic Hospital La Fe, Valencia, Spain

Introduction : Preterm infants show an elevated prevalence of cases of autism spectrum disorder (ASD) that is up to twelve times higher as compared to term infants. The gut‐brain axis is considered an ambiental factor of major importance in ASD, and bacterial extracellular vesicles (BEVs) might be involved in relevant signalling processes. The aim of this study is to gain insight on the association of the development of ASD and the activity of the gut microbiome as well as BEVs found in preterm infant's faeces samples collected from fourteen patients at 6 months of corrected age in the frame of a prospective, observational clinical study focusing on early screening of ASD.

Methods : Short‐chain fatty acids (SCFAs) and branched‐chain amino acids (BCAAs) were quantified by GC‐MS. The ExoView R200+ platform and customized kits with OmpA and L‐asparaginase antibodies for gram‐negative species were used for capturing and characterization of BEVs in faecal supernatants.

Results : L‐asparaginase‐positive particles were detected at concentrations between 5.6 × 10 2 and 6.7 × 10 3 particles/µL, with a mean particle size of 62 ± 5 nm. The capture of OmpA‐positive BEVs could not be confirmed. BEVs captured via L‐asparaginase binding were also positive for OmpA, with concentrations ranging from 1.4 × 10 2 to 1.5 × 10 3 particles/µL. No differences were observed in particle count, fluorescence intensity, or particle size between BEVs from preterm infants diagnosed with ASD and their controls ( p > 0.05, Wilcoxon rank‐sum test). SCFA and BCAA concentrations were higher in the ASD group, with a significant increase observed for butyric acid ( p < 0.05). The concentration of single L‐asparaginase‐positive particles was negatively correlated with butyric ( r = −0.5) and acetic acid ( r = −0.8) ( p > 0.05, Spearman correlation). The fluorescence intensity of OmpA‐positive particles showed a significant negative correlation with isobutyric and methylbutyric acid.

Summary/Conclusion : The final goal is to establish a predictive model for the evolution of ASD based on BEV and microbiota for preventing its development and enabling an early diagnosis, allowing for improved outcomes and personalized interventions in preterm infants.

Funding : This study was supported by the Instituto de Salud Carlos III [CPII21/00003] and the Generalitat Valenciana [CIAICO/2022/233].

Application of engineered extracellular vesicles in bone defect regeneration

Presenter: Jing Zhang

Huazhong University of Science and Technology, Wuhan, China

Introduction : The challenges of traditional bone grafts and cell therapies in treating critical bone defects have led to increased interest in extracellular vesicles (EVs)‐based therapy. This thesis aims to systematically review the current progress in engineered EVs for bone defect regeneration, focusing on isolation methods, characterization techniques, therapeutic mechanisms, engineering strategies, and their applications in bone regeneration.

Methods : A systematic review of published literature on engineered EVs in bone defect repair was conducted. The review comprehensively analyses various isolation and characterization methods of EVs, therapeutic mechanisms, engineering modification strategies, and their therapeutic applications in bone regeneration.

Results : Current isolation methods mainly include ultracentrifugation, ultrafiltration, size exclusion chromatography, and immunoaffinity capture. Characterization techniques such as electron microscopy, nanoparticle tracking analysis, and dynamic light scattering have been developed to analyse EV properties. EVs exhibit therapeutic potential through immunomodulation, angiogenesis promotion, and enhancement of target cell functions. Surface modification strategies comprise genetic engineering, metabolic engineering, and membrane engineering for pre‐isolation approaches, while post‐isolation methods include membrane fusion, hydrophobic insertion, and chemical conjugation. Cargo loading encompasses endogenous methods through cell transfection and co‐culture, as well as exogenous approaches via electroporation, sonication, and surfactant‐assisted permeation. In applications, bone morphogenetic protein‐2 over‐expressing EVs enhanced osteogenic differentiation through signalling cascade microRNAs, showing superior bone regeneration when combined with various scaffolds. MicroRNA‐engineered EVs, especially those over‐expressing miR‐424, enhanced osteogenic differentiation by activating SMAD1/5/8 phosphorylation. Other bioactive factor‐modified EVs, such as mutant hypoxia‐inducible factor‐1α modified EVs, promoted osteogenesis and angiogenesis in steroid‐induced osteonecrosis, while vascular endothelial growth factor plasmid‐loaded EVs enhanced osteoinduction and vascular remodelling.

Summary/Conclusion : Engineered EVs represent a promising cell‐free therapeutic approach for bone defect regeneration, offering advantages in biocompatibility, tissue targeting, and therapeutic efficacy. Future research should focus on standardizing isolation techniques and elucidating therapeutic mechanisms to facilitate clinical translation. The integration of various engineering strategies with scaffold materials shows particular promise for treating critical‐sized bone defects.

Proteome of blood extracellular vesicles as a readout of the interaction between inflammation and risk of early death

Presenter: Rose W. Gatheru

Kemri‐Wellcome Trust Research Programme, Kilifi, Kenya

Introduction : Globally, the post‐discharge mortality rate observed in children under 5 years poses a great health crisis, especially in low‐ and middle‐income countries. There is clear evidence from emerging data that severe acute malnutrition (SAM) in children under 5 years contributes towards this burden. Hospitalized children with malnutrition are at eight times higher risk of death within 1‐month of discharge than well‐nourished children. Currently, the biological mechanisms underlying the cause of death, which could inform novel interventions to minimize the risk of death in this group of children, are not well understood. Traditional biomarkers and risk factors such as whole plasma and clinical anthropometric measures have low and unspecific predictive value. Recently, studies have revealed that a proportion of the plasma proteome is encapsulated in extracellular vesicles whose content is used in specific inter‐cellular signalling pathways and might not be adequately accessible to trypsin digestion during processing of samples for mass spectrometry analysis. We aim to identify novel proteomic biomarkers in EVs that are associated with post‐discharge mortality in severely malnourished children that will inform therapeutic interventions of post‐discharge mortality.

Methods : This will be accomplished by complementary approach analysis targeting EV‐associated proteins and cytokines and whole plasma using samples obtained with informed consent at discharge from sick, undernourished children who were admitted to a hospital and followed up post‐discharge for 12 months. Quantifying EV‐associated proteins, as well as those in whole plasma, may increase our understanding of the risk of early death post‐discharge and prognostic biomarkers.

Results : We hypothesize a model of ineffective innate and adaptive immune responses contributed to inadequate clearance of infections, hence inflammation. Glucose utilization is thus prioritized to the activated immune cells at the expense of the growth of the recovering infants. Hence, this inflammation‐induced component of starvation may contribute to organ dysfunction, multi‐organ failure and death.

Summary/Conclusion : This is the first study proposing identification of novel biomarkers associated with post‐discharge mortality through characterization EV fractions. The potential biomarkers identified will aid in the management of SAM children that will include specific interventions to reduce inflammation in addition to antimicrobial and nutritional interventions.

Extracellular vesicle‐mediated anti‐tumour immunity in response to radiotherapy

Chisom Uzendu 1 , Abduljalil Mohamed El‐Sibai 2 , Yohan Kim 3 , Roxane R. Lavoie 3 , Haidong Dong 3,4 , Sean Park 4 , Fabrice Lucien 3,4

1 Mayo Clinic Graduate School of Biomedical Sciences, Virology and Gene therapy Track, Mayo Clinic, Rochester, Minnesota, USA; 2 College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; 3 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 4 Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA; 5 Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota, USA

Introduction : Combination of radiotherapy (RT) and immunotherapy has gained popularity to turn up the heat on cold tumours. However, intrinsic resistance is prevalent; hence, better therapeutic strategies are needed to improve the outcome of cancer patients. We recently demonstrated in metastatic prostate cancer that circulating levels of tumour‐derived extracellular vesicles (tdEVs) increased shortly after RT and were associated with reduced overall survival. TdEVs are mediators of antitumour immunity, but their role in response to RT remains largely unknown. Our study elucidates the impact of RT on kinetics and immunomodulatory roles of tdEVs.

Methods : Mouse tumours (B16, MC38, E0771) expressing a bioluminescent EV reporter (palmGRET) were treated with clinically used doses of RT (20 Gy × 1, 8 Gy × 3 fractions). Mock‐treated mice were used as a negative control. TdEV release and organotropism were measured by ex‐vivo imaging at different timepoints post‐RT. Flow cytometry was used to monitor tdEV uptake by immune cells. For in vitro studies, tdEVs were isolated from culture medium by tangential flow filtration or size exclusion chromatography combined with ultrafiltration. Microfluidic resistive pulse sensing (nCS1) and nanoscale flow cytometry (Apogee‐A60‐micro plus) were used to measure tdEV biogenesis. OT‐1 transgenic mice and in vitro co‐culture assays were employed to measure the impact of RT on tdEV‐mediated CD8 T‐cell activation and function. Proteomic analysis of tumour cells and tdEVs was conducted to identify key players of tdEV‐mediated immunosuppression in response to RT.

Results : RT induces tdEV biogenesis (12‐fold change) irrespective of the dose. In tumour‐bearing mice, tdEVs were detected in blood, spleen, lymph nodes, and liver. RT of mouse tumours promotes accumulation of tdEVs in lymph nodes, resulting in increased uptake by immune cells. Co‐culture assays show a reduction in CD8 T‐cell cytotoxic activity in the presence of tdEVs from irradiated cells. Proteomics revealed the immune checkpoint molecule B7‐H3 overexpressed in tdEVs after irradiation (3‐fold change). Genetic deletion of B7‐H3 inhibited the suppressive activity of tdEVs from irradiated cells, resulting in enhanced T‐cell cytotoxicity.

Summary/Conclusion : This study describes tdEVs as a mediator of systemic immunosuppression in response to radiotherapy. We identified B7‐H3 as a potential therapeutic target to boost antitumour immunity in combination with radiotherapy.

Tumour‐secreted extracellular vesicles counteract therapy response by triggering inflammatory mesenchymal stem cell development

Presenter: Hilal Nur Sensoy

Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

Introduction : Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSCs), though their origin and function remain unexplored. We demonstrate that cancer‐secreted extracellular vesicles (EVs) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function.

Methods : The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV‐induced alterations of the MSC transcriptome were analysed and compared with scRNA‐seq data of osteosarcoma and multiple myeloma patient biopsies. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC‐induced resistance in vivo.

Results : We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGF‐β signalling inhibition. Importantly, EV‐induced iMSCs faithfully recapitulate the inflammatory single‐cell RNA signature of stromal cells enriched in multiple myeloma and osteosarcoma patient biopsies. Mechanistically, cancer EVs act through two distinct mechanisms. EV‐associated TGFb induces IL6 production, while the EV‐RNA cargo enhances TLR3‐mediated chemokine production. We reveal that simultaneous blockade of downstream EV‐activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC‐induced resistance.

Summary/Conclusion : Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as physiological triggers of iMSC development and highlight a promising combination strategy to improve therapy response in bone cancer patients.

Hypoxia and acidosis impact the cargo of EVs released by multidrug resistant non‐small cell lung cancer cells

Helena Branco 1,2 , Cristina P. R. Xavier 1,3,4 , Chiara Riganti 5,6 , M. Helena Vasconcelos 1,2

i3S—Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal; 2 Department of Biological Sciences, FFUP—Faculty of Pharmacy, University of Porto, Porto, Portugal; 3 UCIBIO—Applied Molecular Biosciences Unit, Toxicologic Pathology Research Laboratory, University Institute of Health Sciences (1H‐TOXRUN, IUCS‐CESPU), Gandra, Portugal; 4 Associate Laboratory i4HB—Institute for Health and Bioeconomy, University Institute of Health Sciences—CESPU, Gandra, Portugal; 5 Department of Oncology, University of Torino, Torino, Italy; 6 Interdepartmental Research Center for Molecular Biotechnology “G. Tarone”, University of Torino, Torino, Italy

Introduction : Non‐small cell lung cancer (NSCLC) is the world‐leading cause of cancer‐related mortality. Despite recent breakthroughs in NSCLC treatment, the prognosis remains poor, owing to the development of resistance, including multidrug resistance (MDR). Extracellular vesicles (EVs) are key mediators of intercellular communication within the tumour microenvironment (TME). Specifically, growing evidence highlights the role of EVs in the horizontal transfer of drug resistance traits within the TME. Hypoxia and acidosis, defining features of the TME, are associated with increased EV production. Here, we aimed to study the impact of the hypoxic/acidic TME on the cargo of EVs released by MDR NSCLC cells.

Methods : EVs shed by NCI‐H460/R MDR cells were isolated by differential centrifugation. Before EV isolation, cells were kept for 72 h either under normoxia and physiological pH (control), hypoxia (1% oxygen) or acidosis (pH 6.5). Hypoxia induction was confirmed by western blot (WB). EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy and WB. Protein content of EV's cargo was further assessed by mass spectrometry‐based proteomics. The most relevant differentially expressed proteins (DEPs) between control, hypoxia and acidosis cell culture conditions were validated by WB.

Results : Isolated EVs exhibited the typical size range (150–170 nm) and characteristic ‘cup‐shaped’ morphology. EV‐associated protein markers (Hsp70, CD9, CD47, CD81 and Alix) were successfully detected. ApoB, cytochrome c, actinin‐4 and actin were either absent or present at minimal levels in the isolated EVs compared to their corresponding cells. A total of 101 proteins were enriched in EVs from NCI‐H460/R cells released under hypoxia, while 48 proteins were enriched under acidosis, both compared to normoxia/physiological pH. Conversely, 66 and 85 proteins were decreased under hypoxic and acidic conditions, respectively. For both conditions, PANTHER analysis revealed an enrichment in proteins involved in cell metabolism and a decline in proteins involved in JAK/STAT signalling, as well as B and T cell activation. Most relevant DEPs were validated by WB.

Summary/Conclusion : Hypoxia and acidosis significantly impact the cargo of EVs shed by NSCLC MDR cells. This suggests that these EVs may contribute to immunomodulation and have an impact on immunotherapy efficacy. Future work will confirm this hypothesis.

Subtopic EV biomarker development 2

Characterisation of extracellular vesicles in patients with heart failure

I. Fichtner 1 , F. Tafuri 2 , A. Rizzuto 3 , A. Corsini 1 , C. Macchi 1 , I. Giusti 4 , V. Dolo 4 , G. Esposito 5 , C. Perrino 5 , S. Carugo 2,3 , M. Vicenzi 2,3 , M. Ruscica 1,3

1 Department of Pharmacology and Biomolecular Sciences, University of Milan, Milan, Italy; 2 Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy; 3 Department of Cardio‐Thoracic‐Vascular Area, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 University of L'Aquila, L'Aquila, Italy; 5 University of Naples—Federico II, Naples, Italy

Introduction : Heart failure (HF) is a clinical syndrome involving multi‐organ dysfunction as vessels, muscles, kidneys, liver, and haematopoiesis. HF is classified as either reduced (HFrEF) or preserved (HFpEF) based on the ejection fraction (EF) percentage of the left ventricle. Extracellular vesicles (EVs) show potential as a liquid biopsy tool, as their circulation patterns and cargo can indicate the onset and severity of cardiac diseases. We analysed plasma‐derived EVs of 40 HF patients (13 HFpEF and 27 HFrEF) and 21 volunteers (CTR).

Methods : Echocardiographic and clinical data were collected from both groups. Following the MISEV 2023 guidelines, EVs were isolated from plasma using size‐exclusion chromatography and ultracentrifugation, then characterised via NTA, TEM, WB, and FACS analyses. Protein content of EVs was determined by targeted proteomic analysis and by ELISA.

Results : Diagnosis of HF relied on echocardiographic (e.g., %EF, E/é) and biochemical parameters (e.g., NT‐proBNP). EV‐specific protein (CD63, CD9, CD81, Alix and Beta1 integrin) expression and integrity were confirmed by FACS, WB and TEM. Median EV concentration was lower in HF vs CTR (EV/mL/cell count: 2.8 × 10 9 vs. 3.9 × 10 9 ), while the size was larger (mm: 204 vs. 187). Among the different subpopulations analysed, HF showed significantly reduced EVs from cardiomyocytes (CD172a+), endothelial cells (CD62E+) and endothelial progenitor cells (CD309+). Conversely, EVs from lymphocytes T and B (CD8+, CD4+ and CD19+) and lymphatic vessels (CD310+) were all significantly increased. Interestingly, platelet‐derived EVs (CD41a+) decreased along with the total plasma platelet count, while EVs from activated platelet (CD154+/CD41a+) increased in HF patients. Proteomic analysis of EVs from HF showed increased levels of surfactant proteins (SFTPD) and markers of fibrosis (Galectin‐3 and Chitinase‐3‐like‐1 protein). These findings were confirmed through ELISA. When EV subtype analysis was stratified by clinical presentation (HFrEF vs HFpEF), the main differences between patients and CTR were driven by HFrEF.

Summary/Conclusion : EVs isolated from patients with HF displayed a unique fingerprint aligned with the haemodynamic characteristics of the condition. Additionally, the characterization of EVs allows us to distinguish HFpEF from HFrEF, showing different activity of systemic mechanisms (e.g., endothelial and inflammation).

Funding : (1) PRIN ‐ 2022ZPS49L, (2) ANTHEM: “AdvaNced Technologies for Human‐Centred Medicine PNC0000003 – CUP B53C22006670001– Spoke”.

Immune system‐related plasma extracellular vesicles are strong predictors of human longevity

Xin Zhang 1,2 , Sisi Ma 3 , Syeda Iffat Naz 3 , Erik J. Soderblom 4 , Constantin Aliferis 3 , Virginia Byers Kraus 1,2,5

1 Duke Molecular Physiology Institute, Duke University School of Medicine, Duke University, Durham, North Carolina, USA; 2 Department of Orthopaedic Surgery, Duke University School of Medicine, Duke University, Durham, North Carolina, USA; Institute for Health Informatics, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA; 4 Duke Proteomics and Metabolomics Core Facility, Duke University School of Medicine, Duke University, Durham, North Carolina, USA; 5 Department of Medicine, Duke University School of Medicine, Duke University, Durham, North Carolina, USA

Introduction : Extracellular vesicles (EVs) are important in many biological processes, including those related to ageing and longevity. Our causal analyses of adults aged 71 years and older from the prospective Duke Established Populations for Epidemiologic Studies of the Elderly (D‐EPESE) cohort ( n = 1507, PMID: 36182774) identified lymphocytes, physical function and activity, and regular exercise as key contributors to longevity, highlighting the vital roles of immune and muscle systems in extending lifespan. The objective of the present study was to identify circulating extracellular vesicle (EV) biomarkers that indicate longevity.

Methods : The baseline plasma EV proteome ( n = 48 D‐EPESE participants, mean age 77.2 ± 1.7 years; 50% female, 50% Black, 50% < 2‐year survival, 50% ≥ 10‐year survival) was analysed using high‐resolution mass‐spectrometry and flow cytometry. Fresh EDTA blood specimens were centrifuged, and plasma was aliquoted and frozen at −80°C until analysis. After thawing, plasma was centrifuged to remove debris, followed by EV isolation using polymer‐based precipitation and characterization for size, lipid bilayer structure and surface markers. The capacity of EV peptides to predict 10‐year longevity was evaluated in a discovery dataset ( n = 32) and a validation dataset ( n = 16).

Results : Longevity‐associated large EV (LEV) plasma subpopulations, defined by surface markers, were predominantly linked to immune and muscle cells. Of the 7960 identified plasma EV peptides (corresponding to 519 proteins), nearly half were related to the immune system and 10% to muscle. However, among the 756 peptides with higher abundance and 130 with lower abundance in long‐lived versus short‐lived participants, 437 were enriched in the immune system markers, while only 12 were enriched in muscle markers. Final predictive models incorporating 3–5 plasma EV peptides demonstrated high discriminative capacity for longevity, with receiver operating characteristic areas under the curve ranging from 0.91 to 1 in the hold‐out validation dataset.

Summary/Conclusion : Our findings suggest that immune cells produce plasma EVs linked to longevity, shedding light on the key mechanisms that regulate healthy ageing. The EV predictors of longevity indicate that there could be advantages in targeting complement pathways to extend lifespan, potentially using one of the many complement inhibitors currently available or in clinical trials.

Funding : This work was supported by the National Institute on Ageing R56AG060895, R01AG070146, and R01AG054840.

Using extracellular vesicle characterization to discover blood‐based biomarkers for primary and secondary resistance to immune checkpoint inhibition in metastatic melanoma

Presenter: Carmen M. T. Roeper

University Medical Center Hamburg‐Eppendorf, Hamburg, Germany

Introduction : Isolation of extracellular vesicles (EVs) from plasma liquid biopsy samples in malignant melanoma (MM) patients receiving immune checkpoint inhibition (ICI) therapy could enable the identification of novel biomarkers to predict treatment response, detect minimal residual disease, and monitor disease progression—potentially enhancing diagnostic and monitoring precision. Before ICI therapy, the 5‐year survival rate for this tumour type was approximately 5%, but with ICI treatment, survival rates have now risen to nearly 50%.

Methods : EVs were isolated from plasma samples of MM patients (174 samples from 82 patients) undergoing ICI therapy at three key time points: therapy initiation (baseline), 6 months into therapy, and, where applicable, at progression of disease (PD). Samples from 22 healthy controls were also included. Patients were classified into subgroups based on treatment response: Therapy Response, Primary Non‐Response (PD before 6 months), and Secondary Non‐Response (PD after 6 months). EV isolation was achieved using size exclusion chromatography and ultracentrifugation, with nanoparticle tracking analysis (NTA) used to assess particle count and size distribution. Protein concentration was quantified via the Qubit assay, and electron microscopy validated EV isolation. Proteomic profiling of EVs was performed using Liquid Chromatography‐Tandem Mass Spectrometry (LC‐MS/MS), enabling identification and quantification of peptide content.

Results : NTA and electron microscopy confirmed successful isolation of purified EVs. LC‐MS/MS analysis identified 1754 proteins. Biostatistical analysis is currently pending, with findings to be presented at the congress.

Summary/Conclusion : Our goal is to uncover novel biomarkers by isolating EVs from advanced melanoma patients, aiming to improve detection of disease progression and identify emerging resistance to ICI therapy.

Funding : This work was supported by the Fleur Hiege Center for Skin Cancer Research, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany.

Identification of circulating piRNAs biomarkers in extracellular vesicles related to treatment response in patients with idiopathic nephrotic syndrome

Kyung‐Oh Doh 1 , Yun‐Ui Bae 2 , In O. Sun 3 , Soon Hyo Kwon 4

1 Department of Physiology, College of Medicine, Yeungnam University, Daegu, Republic of Korea; 2 Precision Medicine Lung Cancer Center, Konkuk University Medical Center, Seoul, Republic of Korea; 3 Division of Nephrology, Department of Internal Medicine, Presbyterian Medical Center, Jeonju, Republic of Korea. 4 Division of Nephrology, Soonchunhyang University Seoul Hospital, Seoul, Republic of Korea

Introduction : Piwi‐interacting RNAs (piRNAs) are a class of non‐coding RNAs initially discovered in germ cells associated with spermatogenesis. Recent findings indicate that the function of piRNAs extends beyond germ cells. Similar to other small RNAs, piRNAs are present in extracellular vesicles (EVs) and are emerging as biomarkers for various diseases. This study aimed to identify the circulating EV‐piRNA signature to discriminate idiopathic nephrotic syndrome (INS) from idiopathic membranous nephropathy (IMN).

Methods : We prospectively enrolled 60 participants, including individuals with IMN ( n = 19), INS ( n = 21), and healthy volunteers (HVs; n = 20). Circulating EVs were isolated from 1000 µL of serum using the ExoQuick isolation agent. The serum EV‐piRNA profiles of all participants were assessed using RNA sequencing.

Results : No differences in renal function or proteinuria were observed between the IMN and INS groups. The median diameter of the EVs was approximately 180 nm, as measured by dynamic light scattering. Western blotting confirmed the presence of CD9 and the absence of GM‐130, excluding potential contamination. We identified 39 piRNAs that were upregulated and 46 piRNAs that were downregulated in patients with INS compared to HVs. Additionally, 22 piRNAs were upregulated and 23 were downregulated in patients with INS compared to patients with IMN. In patients with IMN compared to HVs, 11 piRNAs were upregulated and 6 were downregulated. Among these, three piRNAs (hsa‐piR‐33064, hsa‐piR‐36365, and hsa‐piR‐54518) showed significant differential expression in patients with INS compared to both HVs and patients with IMN.

Summary/Conclusion : Our study identified EV‐piRNAs associated with INS, suggesting that these circulating EV‐piRNAs could serve as potential biomarkers for the diagnosis of patients with INS.

Funding : This research was supported by the Engineering Research Center Program (2018R1A5A1025511) and the Ministry of Education (NRF‐2020R1I1A3A04037367) through the National Research Foundation of Korea (NRF) and the Soonchunhyang University Research Fund. The biospecimens and data used for this study were provided by the Biobank of Soonchunhyang University Seoul Hospital and the Biobank of Seoul National University Hospital, a member of the Korea Biobank Network (KBN4_A03).

A transcriptomic biomarker for prostate cancer active surveillance in extracellular vesicles from liquid biopsies

May‐Britt Tessem 1 , Morten Beck Rye, 2 Alicia Llorente 3

1 Department of Circulation and Medical Imaging, NTNU, Clinic of Surgery, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway; 2 Department of Clinical and Molecular Medicine, NTNU, Trondheim, Norway; 3 Department of Molecular Cell Biology, Oslo University Hospital, Oslo, Norway

Introduction : Prostate cancer (PCa) is the most common cancer among men worldwide. Many patients have non‐aggressive tumours and do not require radical treatments. Low‐risk PCa patients often undergo active surveillance (AS) with regular check‐ups to monitor disease progression. However, reliable biomarkers are needed to differentiate between aggressive and non‐aggressive PCa types to identify patients suitable for AS. A robust liquid biopsy test would enable more frequent monitoring of patients under AS. Liquid biopsies represent a non‐invasive approach that generally has low risk and causes less discomfort for patients compared to traditional tissue biopsies. For PCa, semen is a particularly valuable biofluid, as a significant portion of the seminal fluid is produced by the prostate gland. This prostate proximity offers a high potential to identify robust biomarkers that can accurately reflect PCa biology. Recently, extracellular vesicles (EVs) have emerged as promising tools for the discovery of biomarkers in liquid biopsies. Their ability to reflect the molecular composition of their cells of origin provides insight into disease states and progression. Therefore, the isolation of seminal extracellular vesicles from semen samples will offer a new avenue for PCa classification.

Methods : Our goal is to identify prostate cancer biomarkers in seminal fluid. In order to do that, we are optimizing protocols for isolating seminal extracellular vesicles. Using samples from healthy donors, we demonstrated that both sequential centrifugation and size exclusion chromatography effectively yield EVs with similar levels of CD63, syntenin, CD9, and CD81.

Results : We obtained approximately 140 µg protein per mL of seminal fluid in the EV pellets.

Summary/Conclusion : Using samples from healthy donors, we demonstrated that both sequential centrifugation and size exclusion chromatography yield extracellular vesicles with comparable levels (slightly higher in SEC) of EV‐related markers such as CD63, syntenin, CD9, and CD81. Our findings not only validate the effectiveness of sequential centrifugation and size exclusion chromatography for seminal fluid EVs isolation but also underscore the potential utility of seminal fluid EVs for downstream analysis in research and clinical settings.

Serum‐derived extracellular vesicles as biomarkers for predicting pregnancy on assisted reproductive technology in patients with endometriosis

Presenter: Ayako Muraoka

Nagoya University Hospital, Nagoya, Japan

Introduction : Endometriosis can cause infertility, and evaluation methods for predicting clinical pregnancy outcomes are desired. Extracellular vesicles (EVs) exist in blood, and small non‐coding RNAs (ncRNAs) in EVs may reflect disease severity. In this study, we investigated small ncRNAs in serum EVs to identify specific biomarkers for predicting clinical pregnancy.

Methods : Serum samples were collected from 48 patients who underwent assisted reproductive technology (ART). EVs were isolated by the serial centrifugation method, which is follows the MISEV2023 principal, and characterized using nanoparticle tracking assays, electron microscopy, and western blotting for EV markers. We performed small RNA sequencing and analysed microRNA (miRNA) profiles in the infertility patients with and without endometriosis to detect pregnancy‐predicting biomarkers.

Results : Candidate miRNAs in serum EVs were selected by comparing patients without endometriosis who became pregnant ( n = 13) with those who did not ( n = 21). A total of 241 miRNAs were detected; however, no trends separated the two groups. Next, EVs from patients with endometriosis were analysed and divided into pregnant ( n = 4) and non‐pregnant ( n = 10) cases. Among the 224 candidate miRNAs, miRNA profiles of pregnant women with endometriosis were separated from those of non‐pregnant women by receiver‐operating characteristics curve analysis (area under the curve > 0.8). Finally, we used small RNA sequencing of the tissue to demonstrate that pregnancy‐predicting miRNAs in serum EVs were produced from endometriosis lesions.

Summary/Conclusion : miRNAs in serum EVs of patients with endometriosis could provide novel noninvasive biomarkers to predict pregnancy and have potential clinical applicability in ART.

Evaluation of EV‐AXL as a potential biomarker for response monitoring in NSCLC

Armin Günther 1,2,3,4 , Dominic Helm 5 , Martin Schneider 5 , Tobias Rubner 6 , Franz Ricklefs 7 , Kendra Maaß 8 , Karsten Richter 9 , Michelle Neßling 9 , Sonja Loges 2,4,10 , Melanie Janning 1,2,4

1 Liquid Biopsy and Single‐Cell Analysis Group, Division of Personalized Medical Oncology, German Cancer Research Center (DKFZ), German Center for Lung Research (DZL), Heidelberg, Germany; 2 DKFZ‐Hector Cancer Institute at the University Medical Center Mannheim, Germany; 3 Faculty of Biosciences, Heidelberg University, Heidelberg, Germany; 4 Department of Personalized Oncology, University Hospital Mannheim, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; 5 Proteomics Core, Facility, German Cancer Research Center (DKFZ) Heidelberg, Heidelberg, Germany; 6 Core Facility Flow Cytometry, German Cancer Research Center (DKFZ) Heidelberg, Germany; 7 Department of Neurosurgery, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 8 Hopp‐Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany; 9 Core Facility Unit Electron Microscopy, German Cancer Research Center (DKFZ) Heidelberg, Heidelberg, Germany; 10 Division of Personalized Medical Oncology, German Cancer Research Center (DKFZ); German Center for Lung Research (DZL), Heidelberg, Germany

Introduction : The expression of the TAM receptor AXL is often correlated with poor clinical response in non‐small‐cell lung cancer (NSCLC) patients. Its activation induces an immune‐evasive tumour microenvironment and mediates cancer drug resistance. Since AXL is associated with lipid rafts in the plasma membrane, we hypothesized that AXL trafficking on small extracellular vesicles (sEVs) may have a functional role in modulating the tumour microenvironment and can be evaluated as a potential EV‐based biomarker for therapy response in NSCLC.

Methods : sEVs from conditioned, serum‐free media of AXL‐high NSCLC cell lines (SK‐LU‐1; NCI‐H1299, and NCI‐H1975) were isolated via differential centrifugation, ultrafiltration and size‐exclusion chromatography (SEC). The expression of EV markers (TSG101, CD81, CD9, and SDCBP), known epithelial/tumour EV proteins (EGFR, EPCAM), cellular contaminants (CANX, GM130) and AXL was tested via Western blot (WB). sEVs were further characterized via electron microscopy, nanoparticle tracking analysis, liquid chromatography–mass spectrometry (LC‐MS), and single particle and anti‐CD9 bead‐based imaging flow cytometry (IFCM). Additionally, sEV‐AXL was evaluated in particle isolates isolated from NSCLC patient's EDTA blood plasma samples.

Results : AXL was detected in the sEV‐enriched SEC fractions of AXL‐high expressing NSCLC cell lines by WB. Compared to the expression in whole cells, AXL was not particularly enriched in sEVs. In contrast, EV markers and epithelial/tumour markers were strongly enriched in sEVs. These findings were confirmed by IFCM indicating a weak AXL+ signal on bead‐bound EVs but no AXL+ signal on single particles, whereas EV markers and epithelial/tumour markers were detected by both, single‐particle and bead‐based IFCM. Additionally, preliminary LC‐MS‐based analysis of sEVs from NCSCL patients’ EDTA blood plasma did not detect AXL expression in sEVs, unlike the detectable expression of EV and epithelial/tumour markers.

Summary/Conclusion : These data indicate that, compared to other transmembrane proteins, AXL is not particularly enriched in sEVs from NSCLC cell lines under normal cell culture conditions. Furthermore, early data from patient samples suggest there is no AXL expression on sEVs isolated from NSCLC blood plasma samples, thus limiting its applicability as a potential sEV biomarker.

Funding : This study was supported by the Hector Foundation II. Margarete Clemens Stiftung. Landesforschungsförderung Hamburg LFF GK10. Graduate school InTechCanDiTh. Deutsches Zentrum für Lungenforschung.

EV‐based identification of Parkinson´s disease in blood and urine

Antri Stefan 1,2* , MartinWolf 1,2 , Rodolphe W. Poupardin 1 , Ulrike Resch 3 , Sarah Hochman 1 , Anna Raninger 1 , Lisa Jakob 1 , Sabine Schmidhuber 4 , Nicole Drexler 5 , Thomas Heuser 5 , Markus Mandler 4 , Achim Schneeberger 4 , Dieter Volc 6 , Dirk Strunk 1,2

1 Paracelcus Medical University, Cell Therapy Institute, Salzburg, Austria; 2 Austrian Red Cross Research (ÖRK), Vienna, Austria; 3 Medical University of Vienna, Vienna, Austria; 4 Tridem Bioscience GmbH & CoKG, Vienna, Austria; 5 Vienna Biocenter Core Facilities, Vienna, Austria; 6 Confraternitaet‐Privatklinik Josefstadt, Vienna, Austria

Introduction : Extracellular vesicles (EVs) are key players in cell‐to‐cell communication in health and disease. Their presence in most tissues and bodily fluids makes them excellent targets for biomarker discovery. We aim to develop an EV‐based biomarker assay that will serve as a tool for vaccine response monitoring in Parkinson's disease (PD) patients. We selected PD‐driving alpha‐synuclein (aSyn) as the primary diagnostic target to be challenged by unsupervised omics for additional biomarker discovery.

Methods : Tangential flow filtration (TFF), size exclusion chromatography (SEC) and ultracentrifugation were applied to enrich EVs from PD patient's urine and blood. We determined EV quantity by tunable resistive pulse sensing and nanoparticle tracking analysis and EV quality by tetraspanin + aSyn expression in enhanced chemiluminescence dot blots, super‐resolution microscopy, cryo‐electron microscopy and enzyme‐linked immunosorbent assay (ELISA). Subtractive biomarker discovery by multi‐omics has been performed in addition. The level of EV‐associated aSyn in patient samples was compared with respective healthy controls ( n = 20 each). Following MISEV 2023 recommendations, the analysis pipeline was applied in bulk material, EV+ and EV‐ fractions.

Results : Various isolation methods resulted in different EV enrichment levels and EV separation from other contaminants. We confirmed EV identity and enrichment by CD9/63/81 dot blots and super‐resolution microscopy. The highest aSyn signals were acquired in patient EV samples isolated by ultracentrifugation compared to SEC for blood samples and TFF for creatinine‐normalized urine samples. Preliminary experiments confirmed the presence of aggregated aSyn in patient EVs by ELISA. Variable aSyn signals were acquired in bulk liquid biopsy material, different SEC fractions, and EV+ and EV‐ preparations. Sensitivity evaluation of our dot blot assay setup using recombinant aSyn revealed a detection limit of ∼10 ng for total and aggregated protein. Bioinformatics analysis of preliminary proteomics data showed differential protein expression within the patient and control groups.

Summary/Conclusion : New theranostic biomarkers from easily accessible non‐invasive sources are important for patient acceptance and study performance. Further proteomics analysis is ongoing for assay validation. Based on low sensitivity in dot blots, RT‐QuIC is planned to investigate the association of EVs and aSyn in PD samples. Once established, the biomarker will be validated as a diagnostic tool for monitoring vaccine response.

Frail older women have a higher concentration of extracellular vesicles derived from platelets

Presenter: Magdalena Sepulveda

University of Talca, Talca, Chile

Introduction : During ageing, several physiological systems are altered, with emerging frailty representing a significant cardiovascular disease risk factor. Among the scales to determine frailty are the Fried Phenotype and Frailty Trail Scale (FTS), but it is also important to evaluate the presence of procoagulant platelets and extracellular vesicles. This study aimed to determine the levels of procoagulant platelets and the concentration of extracellular vesicles in frail and non‐frail older women.

Methods : A blood sample with anticoagulant sodium citrate 3.2% of 50 older women was taken and centrifuged to obtain platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP). PRP was used to analyse the exposure of phosphatidylserine (PS) and intracellular calcium levels. PPP was used for the analysis of extracellular vesicles by ultracentrifugations. The concentration of extracellular vesicles was determined using the Nieuwland's formula. These activation markers were measured using fluorescently labelled antibodies and flow cytometry and expressed as Median Fluorescence Intensity (MFI).

Results : In the studied population, the average age was 73.46 years, and 66% of the women had abdominal obesity. According to the 25‐point cut‐off, 74% were non‐frail and 26% were frail, whereas with the 7‐point cut‐off, 26% were non‐frail and 74% were frail. When the cut‐off score was 25 points for FTS‐5, no significant differences were found in levels of PS and calcium levels, procoagulant platelets and extracellular vesicles. There were significant differences in the concentration of extracellular vesicles positive for Annexin V/P‐Selectin/Mitochondria (AV+/CD62P+/Mitochondria+) ( p value 0.0084), positive for Annexin V/GPIIb/Mitochondria (AV+/CD41a+/Mitochondria+) ( p value 0.0151) and positive for Annexin V/GPIIIa/Mitochondria (AV+/CD61+/Mitochondria+) ( p value 0.0294), where it can be observed that older women with cut‐off ≥ 7 points have a higher concentration of extracellular vesicles.

Summary/Conclusion : A cut‐off score ≥ 7 points for FTS‐5 could represent a novel tool for evaluating the behaviour of platelets in older people, which present procoagulant platelets and higher concentrations of extracellular vesicles than cut‐off < 7 points, which could constitute a cardiovascular risk factor.

Funding : This research was funded by ANID Scholarship grant N° 21230657, ANID/FONDECYT grant N° 1220339 and was also supported by ANID‐Anillo ACT210097 (MIBI: Interdisciplinary Group on Mitochondrial Targeting and Bioenergetics). Also, we thank ANID/FONDEQUIP N° EQM200049 (Flow Cytometry) and ANID/FOVI240035.

Correlation between circular‐RNAs contained in extracellular vesicles with BRCA status in ovarian cancer cells

Giuseppe Cammarata 1 , Giulia di Fazio 2 , Alessia Lo Curto 1 , Giuseppina Poppa 2 , Sandra D'Ascenzo 2 , Francesca Megiorni 3 , Simona Ceccarelli 3 , Vincenza Dolo 2 , Ilaria Giusti 2 , Simona Taverna 1

1 National Research Council, Rome, Italy; 2 University of L'Aquila, L'Aquila, Italy; 3 Sapienza University, Rome, Italy

Ilaria Giusti and Simona Taverna are co‐senior authors .

Introduction : Ovarian cancer (OC) is a common malignancy with high mortality. Highlighting the mechanisms of OC progression and discovery of new biomarkers may improve OC diagnosis and therapy. OC is often caused by germLine mutations in BRCA1 genes. Circular RNAs (circRNAs) are a class of novel non‐coding RNAs that undergoe back‐splicing and form a covalently closed loop. circRNAs have regulative roles in cancer. Extracellular vesicles (EVs) are natural intercellular shuttles for circRNAs. Our study aims to correlate circRNAs shuttled by OC‐EVs with BRCA mutational status.

Methods : Human OC cell lines UWB1.289 (BRCA1‐null), UWB1.289 + BRCA1 (BRCA1‐restored) were cultured in RPMI/HUMEC medium (3% FBS); ES‐2 (BRCA wild type) was cultured in McCoy's 5A medium (10% FBS). EVs were isolated by ultracentrifugation from both UWB cell lines and characterized according to MISEV 2023. To analyse circRNAs in UWB cells and EVs, RNA was extracted with a commercial kit, and complementary DNA was synthesized by reverse transcription. circRNA was evaluated using divergent primers by RT‐qPCR with SYBR Green. GAPDH was used as a reference. Relative changes in circRNA expression were determined by the ΔΔCt method. The effects of UWB‐EVs on ES‐2 cell proliferation and migration were evaluated by MTS/CCK8 and wound healing assay, respectively.

Results : EVs were isolated from both UWB cell lines and characterized: they appear intact and rounded (TEM), with a mean size ranging around 160–190 nm (NTA), and are positive for CD63, CD9, TSG‐101 and negative for CNX (WB). A selection of circRNAs (CDR1AS, HIPK2, HIPK3, FAM120a, ITCH, AFF1, MUC16) was tested on cells and EVs in both UWB cell lines. Among these circRNAs, MUC16 and FAM120a were represented differently in UWB cells and EVs. Notably, UWB‐EVs were able to modulate ES‐2 proliferation and migration.

Summary/Conclusion : Multiple genes with germLine mutations have been implicated in hereditary OC. Our results indicate that, in OC cell lines, circRNAs contained in EVs could be linked to BRCA mutational status and OC progression. This study confirms the potential of circRNAs shuttled by EVs as biomarkers for OC diagnosis and therapeutic targets.

Funding : Project funded from Program PRIN 2022 under grant agreement N°20224XB79P by the European Union, Next‐generation EU, PNRR, M.4‐C2‐1.1.

Human ovarian cancer cell‐culture models (bi‐ and tri‐dimensional) affect the biological properties of extracellular vesicles

Giulia di Fazio 1 , Giuseppina Poppa 1 , Giuseppe Cammarata 2 , Sandra D'Ascenzo 1 , Vincenza Dolo 1 , Simona Taverna 2 , Ilaria Giusti 1 .

1 Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy; 2 Institute of Translational Pharmacology (IFT), National Research Council of Italy (CNR), Palermo, Italy

Giulia di Fazio and Giuseppina Poppa are co‐first authors .

Simona Taverna and Ilaria Giusti are co‐senior authors .

Introduction : Ovarian cancer (OC) is a fatal malignancy in women. Identifying biomarkers for early detection is urgent; Extracellular vesicles (EVs) are a promising biomarker. Although most preliminary in vitro studies conducted on EVs to date have used the two‐dimensional (2D) culture models, for the past few years research has been shifting toward three‐dimensional (3D) models, which are more architecturally similar to in vivo tumours.

Methods : EVs were isolated by differential centrifugation and ultracentrifugation from the ES‐2 (CLS‐Cytion) cell line grown (McCoy's 5A medium with 5% FBS HyClone) as a monolayer or spheroids obtained by using Ultra Low Attachment BIOFLOAT 384‐well plates (faCellitate). EV release from both models was verified by SEM, and they were characterized according to MISEV 2023 guidelines (TEM, NTA, Western blot). The biological effects were tested on human umbilical vein endothelial cells (HUVEC) as a target: proliferation and motility were evaluated, respectively, by CCK8 and the wound healing assay. circ‐RNAs analysis was performed with single assays with divergent primers by real‐time quantitative PCR (RT‐qPCR), using the SYBR Green RT‐PCR Kit.

Results : The protocol set generates three‐dimensional, rounded spheroids with clinically relevant dimensions (> 400 nm). SEM images show the EV release from the cell membrane in 2D and 3D models. EV characterization demonstrated their identity, which is the same for both models: they appear intact and rounded (TEM), with a mean size of 153.6–163.7 nm (NTA), and are positive for CD63, CD9, and TSG‐101 and negative for CNX (WB). 2D and 3D EVs did not stimulate HUVECs proliferation but were able to differentially induce their motility in a dose‐dependent manner. EVs also have a different content of selected circRNAs: EVs released by OC cells growing in 3D model, compared to 2D, are enriched in hsa_circ_ITCH and hsa_circ_CDR1as; conversely, hsa_circ_HipK3 and hsa_circ_MuC‐16 are less represented.

Summary/Conclusion : When approaching the EVs study in search of novel OC biomarkers, the choice of in vitro model should be carefully evaluated since it can affect different functional and, most importantly, molecular tests.

Funding : Project funded from Program PRIN 2022 under grant agreement N°20224XB79P, funded by the European Union, Next‐generation EU, PNRR, M.4‐C2‐1.1.

Extracellular vesicles from 2D and 3D culture models exhibit distinct glycan profiles

Giuseppina Poppa 1 , Giulia di Fazio 1 , Jason P. Webber 2 , Sandra D'Ascenzo 1 , Vincenza Dolo 1 , Ilaria Giusti 1

Giuseppina Poppa and Giulia di Fazio are co‐first authors. Vincenza Dolo and Ilaria Giusti are co‐senior authors.

1 University of L'Aquila, Italy, 2 Swansea University, UK .

Introduction : Glycosylation, a crucial post‐translational protein modification, plays a key role in modulation of protein function, contributing to various stages of tumourigenesis. Studies have shown that tumour‐derived extracellular vesicles (tEVs) have an altered glycoprofile, including distinct glycosylation patterns which may have utility as tumour biomarkers. Current research predominantly relies on monolayer cultures (2D). This study investigates whether tEVs isolated from 3D culture models, which should be more like in vivo conditions, display distinct glycan profiles compared to those isolated from 2D ones.

Methods : The human ovarian (ES‐2) and breast (MDA‐MB‐231, MCF‐7) cancer cell lines were cultured using 2D and 3D culture models. For the 3D cultures, spheroids were formed by the hanging drop method (MDA‐MB‐231) or ultra‐low attachment plates (ES‐2, MCF‐7) and then moved to Petri dishes coated with an anti‐adhesion solution. EVs were isolated from both culture models' supernatants by differential centrifugation. Their characterization involved TEM, NTA, and Western Blot as per MISEV 2023 guidelines. The EVs glycosylation profile was assessed by a lectin‐based affinity approach using an adapted ELISA assay (lectins analysed: PNA, SBA, UEA I, GSL I, GSL II, PSA, WGA, WGA succynilated, DBA‐LCA, PHA‐L).

Results : The presence of EVs in the supernatant from both cell culture models was verified for all cell lines, and the EV identity was confirmed by characterization. The ELISA assay showed significant differences in glycan lectin‐boundedness, indicating distinct enrichment profiles in EVs derived from the different culture models: in 3D‐EVs, compared to 2D‐EVs, glycans recognized by PNA, UEA I, and succinylated WGA in MDA and UEA I, DBA, and GSL II in MCF‐7 decreased by approximately 50%; conversely, an increase of roughly 30% was observed for succinylated WGA, WGA, and PHA‐L in ES‐2. Differences for PNA, UEA I, WGA succinylated, and LCA were also evident when comparing MDA (higher aggressiveness) to MCF‐7 (lower aggressiveness) 3D‐ EVs.

Summary/Conclusion : The data suggests that the different cellular organizations lead to different glycan profiles of isolated EVs, which may exert a variable impact on their evaluation as potential cancer biomarkers and, hypothetically, affect their biological functions.

Funding : This study was supported by the Funded by FFORIC24.23 to IG, Department of Life, Health and Environmental Sciences, University of L'Aquila.

Development of an ultrasensitive method for measuring PD‐L1 on surface of sEVs

Kyo Okita 1,2 , Tetsuo Mioka 2 , Hatsuki Shiga 2 , Akane Sato 1,2 , Etsuro Ito 1,2

1 Waseda University, Tokyo, Japan; 2 BioPhenoMA Inc., Tsukuba, Japan

Introduction : PD‐L1 is a protein that functions as an immune checkpoint. PD‐L1 is highly expressed on the surface of some cancer cells and binds to PD‐1 on T cells to suppress T cell function. In some cancers, increased amounts of PD‐L1 are reported on the surface of small extracellular vesicles (sEVs) in patients' blood. Although PD‐L1 in sEVs has the potential to be a biomarker, the amount of PD‐L1 on sEVs is extremely low, and it is very difficult to measure PD‐L1 on sEVs, especially in healthy individuals. Therefore, we propose an ultrasensitive ELISA method that allows the quantification of extremely low levels of PD‐L1.

Methods : We developed an ultrasensitive ELISA method that combines sandwich ELISA and enzyme cycling. Using this ultrasensitive ELISA, we measured purified PD‐L1 protein, PD‐L1 on the surface of sEVs collected from PD‐L1‐overexpressing cells, and PD‐L1 on the surface of sEVs collected from control sera.

Results : The detection limit for purified PD‐L1 protein was 0.1 pg/mL, demonstrating that our method achieved measurement with an order of magnitude higher sensitivity than conventional methods. When we measured PD‐L1‐positive sEVs, we found that approximately 100 pg/mL of PD‐L1 was present per 100 ng of total protein of PD‐L1‐positive sEVs. In addition, measurements of sEVs in control sera showed that approximately 10 pg/mL of PD‐L1 was present per 1 mg/mL of total protein of sEVs in the sera. Furthermore, measurements of PD‐L1 in control sera from individual donors showed that PD‐L1 levels varied between different control sera.

Summary/Conclusion : These results suggest that it may be possible to measure PD‐L1 on sEVs collected from sera of both healthy individuals and cancer patients, indicating the potential for early cancer diagnosis.

Identifying blood‐based biomarkers for the detection of head and neck squamous cell carcinoma

Yue Su 1,2 , Kekoolani S. Visan 1,2 , Andreas Möller 1,2,3

1 JC STEM Lab for Personalised Cancer Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; 2 Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China; 3 QIMR Berghofer Medical Research Institute, Brisbane, Australia

Yue Su and Kekoolani S. Visan are co‐senior authors .

Introduction : Head‐and‐neck cancer (HNC) is one of the most common cancers worldwide. The majority of HNC cases (90%) are Head and Neck Squamous Cell Carcinoma (HNSCC), with particularly high prevalence in Asia. Diagnosis at advanced stages (stage III or IV), as well as high rates of metastasis and recurrence, largely contributes to the poor survival rate of HNSCC patients. Therefore, there is an urgent need to develop methods for early diagnosis of HNSCC. Small extracellular vesicles (sEVs) are membrane‐enclosed lipid bilayer vesicles of endocytic origin and of less than 200 nm in diameter. They are released by cells and contain subsets of lipids, nucleic acids and proteins specific to their parental cells. Plasma sEVs are mostly derived from blood and vessel cells but are also contain sEV derived from various tissues, including cancer masses. Therefore, plasma sEVs may be an ideal source of specific diagnostic biomarkers of HNSCC, with the added benefits of enabling real‐time monitoring of disease, and their non‐invasive and therefore repeatable nature making them accessible for convenient and efficient collection.

Methods : In this study, sEVs were isolated from plasma samples from cancer‐free healthy controls ( n = 5) and HNSCC patients ( n = 5) using size‐exclusion chromatography. Plasma sEVs were characterised using nanoparticle tracking analysis, western blot assays and transmission electron microscopy. Specific proteins in plasma sEVs of HNSCC patients were analysed and quantified by mass spectrometry.

Results : This approach identified a total of 603 proteins, of which 70 were significantly higher and 86 were lower in HNSCC patients’ blood sEV samples. Validation of the sEV biomarker proteins in a large, independent confirmation cohort of HNSCC patients and matched healthy subjects is being conducted, with initial data showing high reproducibility.

Summary/Conclusion : Overall, this study provides a series of novel, potential biomarkers in plasma sEV for early HNSCC identification and monitoring of therapeutic efficacy.

Next‐generation diagnosis of colorectal cancer using extracellular vesicles from plasma

Rikke Baek 1 , Charlotte Sten 1 , Simon Ladefoged Rasmussen 2,3 , Maiken Mellergaard 3,4 , Ole Thorlacius‐Ussing 2,3 , Henrik Krarup 3,5 , Aase Handberg 3,4 , Malene M. Joergensen 1,3

1 Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark; 2 Department of Gastrointestinal Surgery, Aalborg University Hospital, Aalborg, Denmark; 3 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; 4 Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark; 5 Department of Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark

Introduction : Colorectal cancer (CRC) is one of the most common cancers in the western world. Screening is an efficient method of reducing cancer‐related mortality. Molecular biomarkers for cancer in general and CRC in particular have been proposed, and detection of blood in stool is used for CRC screening. We aimed to test whether extracellular vesicles (EVs) can be used as plasma‐based biomarkers for CRC detection.

Methods : We conducted a cross‐sectional case‐control study of 214 CRC patients and 75 colonoscopy‐verified healthy controls. EDTA‐plasma samples were used, and their content of EVs was extensively phenotyped with the use of the EV Array technology (Joergensen et al. 2013, JEV). The markers were grouped into: EV‐specific, tissue‐specific, inflammation, and cancer in general. We used multivariable logistic regression to calculate the area under the receiver operating characteristics curve (AUC).

Results : Analysis of 84 different EV markers in the plasma samples made it possible to generate EV profile signatures for the two cohorts. Multivariate data analysis was performed and revealed that a four‐marker model after a 10‐fold cross validation gave an AUC of 0.880 (0.837–0.923) with a sensitivity of 0.827 and a specificity of 0.794. The four markers are patent pending and cannot be disclosed.

Summary/Conclusion : Using the EV Array technology, we demonstrate how plasma EVs can serve as a potential diagnostic tool for CRC. The findings will be validated on new samples from the same study (75 patients and 25 controls) along with a new cohort of 100 patients with blood drawn at the time of diagnosis as well as up to 2 years before and compared with 100 healthy controls (Danish Blood Donor Study).

Funding : This study was supported by the Region Nordjylland; ResearchCUBE grant.

Extracellular vesicles to evaluate the mechanisms of action of mesenchymal stromal cells in the premature lung

Mairéad Green 1,2,3 , Ewa Henckel 1,4 , Emanuela Ferretti 5 , Brigitte Lemyre 5 , Maher Shahroor 6 , Shumei Zhong 1 , Chantal Horth 5 , Rebecca Grimwood 5 , Viviane Olson 5 , Dean Fergusson 7 , Bernard Thébaud 1,2,5

1 Regenerative Medicine Program, The Ottawa Hospital Research Institute (OHRI), Ottawa, Ontario, Canada; 2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; 3 Translational and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; 4 Department of Neonatology, Karolinska University Hospital, Stockholm, Stockholm, Sweden; 5 Department of Pediatrics, Division of Neonatology, Children's Hospital of Eastern Ontario (CHEO), Ottawa, Ontario, Canada; 6 Department of Pediatrics, Division of Neonatology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada; 7 Clinical Epidemiology Program, The Ottawa Hospital Research Institute (OHRI), Ottawa, Ontario, Canada

Introduction : Bronchopulmonary dysplasia (BPD), resulting from interrupted lung development, is the most frequent complication of prematurity and lacks effective treatment. Mesenchymal stromal cells (MSCs) improve features of experimental BPD, such as lung function, structure, and inflammation. The HULC‐1 trial recently evaluated the safety and feasibility of these cells for BPD in extremely preterm infants. The mechanisms of action of MSCs in the premature lung are incompletely understood, and we hypothesized that extracellular vesicles (EVs) may reveal evidence of MSC treatment effect.

Methods : Here, we aim to characterize the effect of MSCs on premature lung cells using EVs isolated from tracheal aspirates (TA) and urine (uEV) of preterm infants enrolled in HULC‐1 before and after receiving MSCs. Bead‐based flow cytometry was used to characterize the cellular origin of EVs in both biofluids. Nanoflow cytometry (nFCM) was used to measure TA‐EVs and uEV, and the cellular origin of TA‐EV was determined with antibodies against CD31 (endothelial), CD45 (leukocyte), CD326 (epithelial), CD86 (M1 macrophage) and CD206 (M2 macrophage).

Results : Besides CD9, CD63 and CD81, bead‐based flow cytometry identified HLA‐DRDPDQ, CD24, CD326, and CD44 as highly expressed markers on TA‐EV and uEV, suggesting that the bulk of EVs originate from epithelial cells, granulocytes and macrophages. Using nFCM, we identified a significant, 3‐fold increase in TA‐EV 3‐4 days after MSCs, although no difference was noted in uEV. We found no significant difference in TA‐EVs positive for the surface markers tested, suggesting that the increase of TA‐EVs comes from a different cell type, or from MSCs themselves. When comparing TA‐EV populations to disease severity, increased post‐MSC leukocyte EVs were associated with a less severe chronic lung disease at 40 weeks corrected gestational age. This may reflect cross‐talk between MSCs and resident immune cells, initiating resolution of inflammation.

Summary/Conclusion : We evaluated surface markers present on EVs from both urine and TA of premature infants using two methods. While comparisons pre‐ and post‐MSC did not reveal any changes in EV surface marker expression, TA‐EVs may have implications for monitoring the treatment effect of MSCs, particularly their impact on immune cells.

Funding : This study was supported by the Canada Graduate Scholarship—Master's and by the Clinical Trial Awards of the Stem Cell Network.

Decoding differential gene expression in nasal‐mucus‐derived small extracellular vesicles: insights for respiratory biomarker research

Bhuvanesh Kumar Raju, Vikram Gujar

Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma, USA

Introduction : Respiratory diseases, like chronic rhinosinusitis, allergic rhinitis, and asthma, pose global health concerns, affecting over 500 million people and significantly contributing to the healthcare burden. Traditional diagnostic methods often lack sensitivity and specificity, necessitating the development of novel biomarkers for early and accurate detection of specific disease conditions. Small extracellular vesicles (sEVs), nano‐sized vesicles released by cells, have gained attention as carriers of molecular biomarkers due to their role in cell communication and presence in various biological fluids. This study aims to identify respiratory‐specific biomarkers within sEVs derived from nasal mucus, offering a non‐invasive approach to diagnosing respiratory conditions.

Methods : We received deidentified nasal mucus and serum samples from healthy individuals and extracted the sEVs using precipitation and size exclusion‐based techniques. We characterized these sEVs using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and western blotting. We subsequently employed RNA sequencing to identify potential biomarkers within these vesicles. In addition, we performed single‐cell RNA sequencing for nasal tissue to achieve a high‐resolution view of cellular heterogenicity and to compare cell‐type‐specific biomarkers. We processed the sequencing data using bioinformatic tools, including alignment, normalization, and differential gene expressions, to identify potential respiratory biomarkers.

Results : Our analysis showed a higher level of specific mRNA transcripts, associated with inflammation, cellular stress response, and membrane integrity, which are crucial in respiratory diseases in nasal sEVs compared to serum sEVs. The findings suggest that sEVs from nasal mucus are a rich source of respiratory‐specific biomarkers, which could be utilized to diagnose and monitor inflammation and olfactory dysfunction in respiratory conditions.

Summary/Conclusion : These biomarkers offer a promising avenue for developing non‐invasive diagnostic tools that could improve patient outcomes by enabling earlier and more accurate detection of respiratory diseases. Future research should focus on validating these biomarkers in larger, disease‐specific cohorts.

Funding : This study was supported by the Startup Fund—Oklahoma State University.

Establishing a proteomic analysis of salivary extracellular vesicles to prospectively identify potential biomarkers for type 1 diabetes and multiple sclerosis

Clara Bernardelli, Eduardo Sommella, Piera Selvaggio, Vicky Caponigro, Valentina Citro, Domenica Giannandrea, Ehsan Soleymaninejadian, Carla Martinelli, Monica Miozzo, Pietro Campiglia, Raffaella Chiaramonte, Elena Lesma

University of Milan, Milan, Italy Raffaella Chiaramonte and Elena Lesma are co‐senior authors .

Introduction : Saliva is a complex oral biofluid that has gained considerable attention in biomarker identification due to its non‐invasive and simple collection. Thus, saliva analysis represents a potentially cost‐effective approach for large population screening and follow‐up studies. Among particles secreted in saliva, extracellular vesicles (EVs) are emerging as potential diagnostic and predictive tools. Here we present an effective isolation protocol and proteomic analysis of salivary EVs that could prospectively be used to identify EV‐related biomarkers to monitor autoimmune diseases such as type 1 diabetes (T1D) and multiple sclerosis (MS).

Methods : Saliva specimens were collected with lollipops and saliva rolls from different groups of healthy donors ( n = 30) matched for gender and age (< 18 years, 20–45 years, > 45 years). Saliva samples were collected in the morning, 1 h after the mouthwash and fasting from food and water, and were cleared by differential centrifugations, with the last at 100,000 × g . Ten micrograms of intact EVs were used for proteomic analysis with nLC‐HRMS/MS and subsequent statistical and bioinformatic analyses, including PCA and Gene Ontology, and literature review to identify potential EV‐related disease biomarkers.

Results : Saliva rolls allowed the collection of 3 to 5 mL. Post‐isolation EVs integrity was demonstrated by TEM microscopy. Saliva EVs are positive for CD81 and TSG101. Nanoparticle tracking analysis (NTA) showed no differences in EVs size and number between male and female and across the different ages. Proteomics analysis in label‐free quantification yielded 1225 annotable features in data‐dependent acquisition, while > 2000 annotable and quantifiable proteins were obtained with data‐independent acquisition. The PCA shows a perfect separation between groups and the greatest impact of the age factor on dataset clusterization. Gene ontology revealed the enrichment in components related to EVs and biological functions associated with inflammation and immune response. Accordingly, the literature review showed the presence of potential EV‐associated biomarkers for T1D and MS related to inflammation, complement, lipid metabolism and beta‐cell function regulation.

Summary/Conclusion : Our protocol provides an optimal EVs recovery and a high‐throughput proteomic analysis which promises to be useful for the identification of salivary EV biomarkers to monitor T1D and MS.

Funding : This study was supported by the Project PNC 0000001 D3 4 Health, CUP B53C22006080001, and funded by NextGenerationEU.

TDP43 in plasma extracellular vesicles serves as a biomarker in ALS

Presenter: Yi‐Ya Fang

Prevail Therapeutics, New York, New York, USA

Introduction : There are relatively few validated biomarkers that can be used to identify presymptomatic people who are at risk of developing amyotrophic lateral sclerosis (ALS), diagnose ALS, monitor the disease progression, or assess the efficacy of new therapies. Previously validated biomarkers, such as NfL and GFAP, only reflect downstream effects and do not specifically reflect the molecular pathology of ALS. TDP‐43 is a DNA‐binding protein which has been found to leave the nucleus and form aggregates in ALS patients. However, the ability to use TDP43 as a biomarker of disease is limited because detection of TDP‐43 in the blood and CSF of patients has not been possible. Extracellular vesicles (EVs), which are nano‐sized and lipid bilayer‐bound particles, play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control system for the cellular clearance of ND‐associated proteins.

Methods : In this study, EVs were isolated from human plasma by precipitation, immunoaffinity, centrifugation, and size exclusion columns before applying on TDP‐43 single molecule array (Simoa) assay.

Results : TDP‐43 signal was higher in unlysed EVs compared to lysed EVs, which suggests that TDP‐43 exists mostly in the lumen of EVs rather than the surface of EVs. In addition, we found that TDP‐43 signals in isolated EVs were significantly higher in ALS samples compared to age‐matched healthy samples. By further isolation of neuronally derived EVs using biotinylated L1CAM antibody and streptavidin resin, we observed the same trend in TDP43 signal as total EVs, which was higher TDP‐43 levels in ALS compared to healthy subjects. This isolation method combined with the Simoa assay has shown good reproducibility and linearity, and a freeze‐thaw cycle of isolated EVs did not impact the accuracy of the results.

Summary/Conclusion : In summary, we developed a robust TDP‐43 assay that will enable the potential use of TDP‐43 as an ALS biomarker.

Optimizing tissue EV separation and paired analysis with plasma EVs to identify novel biomarkers for glioblastoma

Presenter: Jiannan Xu

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Glioblastoma (GBM) is the most common malignant primary brain tumour in adults and is associated with a poor prognosis and low survival rate. Early diagnosis and treatment of GBM are hindered by the low sensitivity of imaging methods and the invasive nature of tissue biopsies. Extracellular vesicles (EVs) derived from GBM tissues have the potential to circulate peripherally, reflecting the molecular characteristics of the tumour microenvironment and serving as promising biomarkers for liquid biopsy in GBM. To this end, we optimized the separation method for GBM tissues EVs and conducted a matched proteomic analysis of EVs from both GBM tissue and plasma, aiming to identify novel biomarkers for GBM.

Methods : Human glioblastoma central (GBM‐C) and para‐carcinoma (GBM‐P) tissues EVs separation protocol was developed based on differential centrifugation followed by size exclusion chromatography (SEC) and ultracentrifugation. Additional ultrafiltration was tested to remove intracellular and blood contaminations. Plasma EVs are separated by SEC, followed by ultrafiltration. The morphology, concentration, quantity, and protein profiles of tissue and plasma EVs were further verified by TEM, NTA, western blot, and 4D‐DIA proteomics analysis.

Results : The developed method confirmed the separation of EVs through analyses of size, morphology, and EV related protein markers. The intracellular and blood‐contaminated components were significantly reduced by additional ultrafiltration, while the size and morphology of EVs were not affected. Proteomic profiling identified 2302 differentially expressed proteins (DEPs) and 475 DEPs in tissue and plasma EVs. There are 143 DEPs commonly differentially abundant between tissue and plasma EVs. By integrating DEPs in tissue and paired plasma EVs and conducting KEGG analyses, we identified a unique biomarker panel that was significantly enriched in tumour‐related pathways, such as the PI3K‐Akt signalling pathway and the pathway in cancer. This panel may regulate tumour progression and has the potential to serve as EV‐based biomarkers for GBM.

Summary/Conclusion : We optimized the protocol for the separation of EVs from GBM tissues, achieving high purity and efficiency. Proteomic analyses of EVs from both GBM tissues and paired plasma revealed molecules that may play regulatory roles in the tumour microenvironment and serve as potential markers for GBM progression.

Funding : This study was supported by the Guangzhou Science and Technology Program (2024A04J9986).

Development of a novel pancreatic cancer biomarker based on enzyme activity in circulating extracellular vesicles

Yusuke Yoshioka 1 , Ryosuke Kojima 2 , Sei Furutsuki 2 , Tadahaya Mizuno 2 , Winnie Wong 2 , Yasuteru Urano 2 , Takahiro Ochiya 1

1 Tokyo Medical University, Tokyo, Japan, 2 University of Tokyo, Tokyo, Japan

Introduction : Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, but early detection is unequivocally associated with better long‐term survival. In our previous study, we identified specific extracellular vesicle (EV)‐associated proteins as early detection markers for PDAC through proteomics of patient's serum EVs (Yoshioka, Y. et al. 2022, Cancer Science). While these identified proteins enable the detection of early‐stage pancreatic cancer, their sensitivity remains below 70%, which suggests a risk of missed diagnoses. Therefore, we considered the exploration of new biomarkers for pancreatic cancer. Building on our previous findings, the protein profiles obtained from the serum EVs of PDAC patients revealed a unique set of enzymes, suggesting the potential of EV enzyme activity as a new biomarker. Therefore, to characterize the differential enzyme activity in the EVs of PDAC patients and diabetic patients (a high‐risk group for PDAC), we screened for enzyme activity in serum EVs using a unique fluorescent probe library.

Methods : We purified circulating EV from PDAC patients’ serum and diabetic patients’ serum using ultracentrifugation. To evaluate cancer‐specific enzyme activity in EVs, we used two fluorescent probe libraries targeting exopeptidases and endopeptidases, each comprising 380 probes with various substrate moieties, for a total of 760 probes (Kuriki, Y. et al. 2022, Chemical Science). These libraries consist of fluorogenic substrates for proteases based on peptidyl‐hydroxymethyl rhodamine green (HMRG).

Results : We first established a protocol to monitor the enzymatic activity of EVs using EVs derived from PDAC cell lines. This allowed us to confirm enzymatic activity in cell line‐derived EVs; however, endopeptidase activity in EVs showed a low and unstable signal. We then applied the fluorescent probe library for exopeptidase activity to EVs purified from the sera of 30 pancreatic cancer patients and 27 diabetic patients, obtaining enzyme activity patterns for each sample. As a result, we identified specific probes that could distinguish diabetic patients from PDAC patients.

Summary/Conclusion : We successfully measured enzyme activity in circulating EVs using a fluorescent probe library. Our findings suggest that the enzyme activity in these EVs may exhibit disease‐specific patterns, highlighting their potential as a novel liquid biopsy tool.

Post‐exercise changes in mitochondrial DNA of extracellular vesicles in myalgic encephalomyelitis/chronic fatigue syndrome

Ludovic Giloteaux, Maureen R. Hanson

Cornell University, Ithaca, New York, USA

Introduction : Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating condition characterized by fatigue, cognitive dysfunction, and other symptoms. A hallmark of ME/CFS is post‐exertional malaise (PEM), where exertion leads to prolonged symptom worsening. This highlights the impaired response to physical stress in ME/CFS. Recent studies have focused on the cargo of extracellular vesicles (EVs) in ME/CFS. Our particular interest is mitochondrial DNA (mtDNA) within these EVs. mtDNA can act as damage‐associated molecular patterns (DAMPs), triggering immune signalling and cellular stress. Plasma EVs may help clear out damageing mtDNA, modulating inflammatory responses that worsen ME/CFS symptoms. Investigating EVs and their mtDNA content offers insights into PEM mechanisms and potential biomarkers or therapeutic targets in ME/CFS.

Methods : Twenty‐eight ME/CFS patients and 26 healthy controls completed a cardiopulmonary exercise test (CPET). Blood plasma was collected at baseline, 15 min, and 24 h post‐exercise. Plasma‐derived EVs were isolated using size exclusion chromatography and characterized per MISEV2023 guidelines. DNA was extracted from EVs, and mtDNA was quantified using quantitative real‐time PCR. Group differences and exercise‐induced changes were analysed using linear mixed models, while linear regression assessed associations between mtDNA levels and physiological parameters.

Results : Isolated EVs were positive for CD9 and CD81, with sizes between 100 and 200 nm and typical morphology, showing no group differences. NTA analysis indicated no significant differences in particle numbers between ME/CFS patients and controls at baseline or post‐exercise. Within‐group analysis showed significant EV concentration increases from baseline to 15 min post‐CPET, followed by a decrease during recovery (15 min to 24 h post‐CPET). EV‐mtDNA levels changed dynamically in both groups, rising after CPET and declining during recovery. Notably, EV‐mtDNA levels were significantly correlated with ME/CFS symptoms.

Summary/Conclusion : This study highlights significant post‐exercise changes in EV abundance and mtDNA levels, with differences between ME/CFS patients and controls. These findings enhance the understanding of EVs and their cargo's role in PEM, suggesting the potential of EV‐mtDNA as a biomarker for symptom exacerbation and providing a foundation for future diagnostic and therapeutic approaches for ME/CFS.

Funding : This project was funded by NIH NINDS/OD/NIDA/NHLBI/NHGRI through NINDS U54NS105541 and NIH NIAID U54AI178855 to the Cornell Center for Enervating Neuroimmune Disease.

Comprehensive profiling of endometriosis‐derived extracellular vesicles uncovers novel biomarker candidates

Presenter: Karolina M. Soroczyńska

Medical University of Warsaw, Warsaw, Poland

Introduction : Endometriosis, an enigmatic gynaecological disorder, poses therapeutic challenges with ineffective treatments and a scarcity of early diagnostic biomarkers. Addressing its multifaceted nature necessitates innovative approaches, focusing on diverse biomarkers for precise diagnostics and tailored therapies. EVs in bodily fluids emerge as promising liquid biopsy biomarkers due to their accessibility and diagnostic potential. A comprehensive strategy integrating advanced EV analysis techniques, such as proteomics and single‐vesicle imaging flow cytometry (IFCM), holds promise for identifying new EV‐based biomarkers or therapeutic targets for this heterogeneous condition.

Methods : EVs were isolated from the plasma and peritoneal fluid (PF) of patients with endometriosis and control subjects using size‐exclusion chromatography (SEC), and their isolation was validated through multiple analytical techniques, including Western blot (WB), fluorescence nanoparticle tracking analysis (F‐NTA), and imaging flow cytometry (IFCM). Quantitative proteomics was performed on these samples to identify differentially expressed proteins, which then guided the selection of key proteins for further analysis via high‐throughput IFCM.

Results : A heterogeneous population of EVs, exhibiting typical characteristics of small EVs and containing bona fide EV markers, was identified in plasma and peritoneal fluid (PF) samples from both endometriosis patients and controls. The proteomics analysis of these EVs highlighted specific protein signatures unique to endometriosis patients, revealing distinct molecular profiles with potential as biomarkers for the disease. Some of these identified markers were successfully validated through single EV analyses on the IFCM platform, confirming their relevance for further investigation.

Summary/Conclusion : In summary, our study highlights the potential of EVs as promising liquid biopsy biomarkers for endometriosis. The presence of diverse EV populations, along with identified endometriosis‐specific signatures, suggests promising applications in diagnostics, prognostics, and therapeutics. Further evaluation of these EV signatures is crucial for advancing non‐invasive approaches in manageing this complex gynaecological disorder.

Funding : This research was funded by the National Science Centre under grant 2021/41/B/NZ6/02291.

Purification of extracellular vesicles from canine plasma using a commercially available lipoprotein‐depleting method

Mariana Trigo‐Chapou 1 , Alicia Viloria‐Petit 1, Huiyan Li 2 , Geoffrey Wood 3 , Danny Carreira 1 , Aaron Dhillon 1

1 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada; 2 School of Engineering, College of Engineering and Physical Sciences, University of Guelph, Guelph, Ontario, Canada; 3 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Introduction : Osteosarcoma (OS) is the most common primary bone cancer in dogs. Metastasis to the lungs is the main cause of death in canine OS patients, but there are no biomarkers to detect early metastatic disease. We previously identified OS‐enriched protein cargo with prognostic value via proteomics analysis of EVs released by canine tissue explants. For future clinical application of our findings, a quick and reliable method to purify EVs from dog plasma is necessary. This study tested the SmartSEC DeLipo protocol (SBI) for such purpose.

Methods : Canine blood was collected and processed by the OVC Tumour Bank following standard protocols. To obtain plasma, blood‐containing EDTA tubes were centrifuged at 1500 × g for 10 min. Human plasma and serum were obtained from a commercial source. All samples were stored at −80°C. Three canine plasma samples were first subjected to the SmartSEC DeLipo and to a SEC protocol (qEV 70, IZON), following the manufacturer's instructions. Next, only the SmartSEC DeLipo protocol was used to compare plasma and serum from canine OS patients ( n = 2) to plasma and serum from healthy canine controls ( n = 2) and a healthy human control ( n = 1). EVs were characterized by DLS, western blotting (WB) and TEM. Two previously identified canine OS biomarkers, PSMD14 and PSMA7, were assessed in EV lysates by WB.

Results : The SmartSEC DeLipo protocol resulted in a 6× reduction in 10 nm particles and a 6× enrichment of EV‐compatible particles (> 100 nm) when compared to SEC. EVs obtained with the former method have similar size profiles in plasma versus serum, but different size profiles when comparing serum/plasma from dogs versus human or from dog OS patients versus healthy dogs. EVs were enriched in plasma as compared to serum. Both PSMD14 and PSMA7 were enriched in the plasma of dogs with OS as compared to that of healthy dogs.

Summary/Conclusion : The SmartSEC DeLipo is an effective protocol for the purification of EVs from dog plasma/serum, but plasma improves the EV yield. PSMD14 and PSMA7 can be detected in EV lysates from dog plasma and at higher levels in OS versus healthy dogs. Further studies with larger sample sizes are guaranteed.

Exosomal miRNAs: investigating their potential role in depression

Presenter: Xiao Wang

Lund University, Lund, Sweden

Introduction : Exosomal miRNAs have gained significant attention for their role in gene regulation and the altered expression observed in depression. However, their potential as diagnostic and prognostic markers for depression remains unclear.

Methods : In this study, we used quantitative PCR and Human Panel I miRNA panels (371 miRNAs) to analyse 30 plasma exosome samples, including those from healthy controls, depression patients at baseline, and after psychological treatments.

Results : Our analysis detected 200 of the 371 miRNAs in plasma samples, with 11 showing significant associations with depression. Notably, 7 miRNAs were linked to depression treatment response, including exosomal miR‐146a‐5p, previously associated with both depression and treatment response. Additionally, blood let‐7a‐5p has been reported as a risk factor for major depression.

Summary/Conclusion : These findings lay the groundwork for future molecular studies aimed at elucidating genetic risk mechanisms in depression and advancing therapeutic approaches.

Surface glycans of small extracellular vesicles in vitro and in vivo increases in early and late‐stage breast cancer

Jamie Cooper, Bethy Airstone, Emanuela Carollo, Susan Ann Brooks, Ryan Charles Pink

Oxford Brookes University, Oxford, UK

Introduction : Breast cancer is the most frequently diagnosed cancer and a leading cause of cancer‐related mortality in women globally. Small extracellular vesicles (sEVs) play a crucial role in cell communication and cancer progression. This study aimed to investigate the glycosylation patterns of sEVs derived from breast epithelial cells and plasma samples from breast cancer patients, focusing on the presence of truncated O‐linked glycans, such as the Tn antigen.

Methods : Breast cancer cell lines were investigated for lectin surface binding by confocal microscopy and flow cytometry. The sEVs of these were tested for surface lectin binding and tetraspanin binding using nano‐flow cytometry, single particle interferometry, and direct stochastical optical reconstruction microscopy. Plasma from healthy and stage II‐IV breast cancer patients was tested by nano‐flow cytometry for lectin binding and analysed for the source of lectin+ EVs using 37 colocalised markers by multiplex flow cytometry (MACSPlex).

Results : Quantitative analysis revealed elevated HPA binding in sEVs from metastatic MCF‐7 cells compared to that in non‐metastatic BT‐474 and immortalised normal hTERT‐HME1 cells, suggesting a correlation between lectin binding and metastatic potential. Analysis of sEVs revealed differential glycan presentation with CD81‐positive sEVs from MCF‐7 cells compared to CD63. In patient‐derived plasma sEVs, lectin binding was significantly higher in patients with breast cancer than in healthy individuals, highlighting its potential as a biomarker for cancer detection.

Summary/Conclusion : These findings highlight the complex glycosylation of sEVs and their potential early diagnostic utility in breast cancer for lectin‐positive sEVs.

Funding : J. Cooper and consumables were sponsored by an Oxford Brookes Nigel Groome Studentship.

Characterization of immune cell‐derived extracellular vesicles in sepsis: insights into immune dysregulation and potential diagnostic markers

Presenter: Hsin‐Hwa Tsai

China Medical University Hospital, Taichung, Taiwan

Introduction : Sepsis is a life‐threatening syndrome characterized by multiple organ dysfunction resulting from a dysregulated host response to infection. Despite considerable progress in intensive care and the adoption of goal‐directed therapies, the highly heterogeneous nature of sepsis patients makes it challenging to establish a diagnostic criterion that adequately balances sensitivity and specificity. Emerging evidence highlights the central role of the immune system in the pathophysiology of sepsis. The immune response triggered by invading pathogens often fails to restore homeostasis, resulting in a pathological state marked by both inflammation and immune suppression based on distinct disease stages. As a result, unravelling the complex mechanisms underlying immune dysregulation in sepsis and developing targeted immunotherapies are key priorities in current research. Extracellular vesicles (EVs) have emerged as critical mediators during infections, acting as ‘bridges’ that facilitate intercellular communication. Additionally, exosomes derived from different immune cells may have an impact on immune activation or suppression during sepsis. In this study, we characterized the immune cell origins of EVs in plasma samples from individuals with sepsis, septic shock, and healthy volunteers to identify potential surface markers indicative of sepsis progression.

Methods : Peripheral blood samples were obtained from patients with sepsis and septic shock (sepsis, n = 3; septic shock, n = 7) and from healthy volunteers ( n = 3). EVs were isolated from platelet‐poor plasma using the ExoQuick exosome precipitation solution, quantified with ZetaView technology, and characterized phenotypically via flow cytometry.

Results : Our study found no significant differences in the size and concentration of exosomes between patients with sepsis or septic shock and healthy controls. However, surface protein analysis revealed significant alterations in CD29, CD20, and HLA‐I in the sepsis group compared to healthy individuals. In the septic shock group, markers of antigen‐presenting cells, specifically HLA‐I and HLA‐II, showed significant differences relative to healthy controls.

Summary/Conclusion : EVs originating from different immune cells may influence immune activation or suppression during sepsis. Identifying the cellular origins of these exosomes could offer valuable insights into the intercellular signalling between infected and immune cells, potentially enhancing our understanding of sepsis and guiding the development of new diagnostic and therapeutic approaches.

Flow cytometry techniques for characterizing extracellular vesicles and apoptotic bodies as potential biomarkers in type 1 diabetes and multiple sclerosis

Domenica Giannandrea, Ehsan Soleymaninejadian, Valentina Citro, Natalia Platonova, Clara Bernardelli, Piera Selvaggio, Monica Miozzo, Paola Ciceri, Mario Cozzolino, Lavinia Casati, Elena Lesma, Raffaella Chiaramonte

Università degli Studi di Milano, Milan, Italy Domenica Giannandrea and Ehsan Soleymaninejadian are co‐first authors . Elena Lesma and Raffaella Chiaramonte are co‐senior authors .

Introduction : Extracellular vesicles (EVs) are recognised as valuable biomarkers for disease due to their ability to be released by various cell types into body fluids, including urine, plasma, cerebrospinal fluid, semen, and saliva. The number, size and cargo of circulating EVs correlate with the progression of autoimmune diseases, making them promising diagnostic tools for diseases like multiple sclerosis (MS) and type 1 diabetes (T1D). Specifically, small EVs (sEVs) from platelets, immune, endothelial, and central nervous system (CNS) cells can help the characterization of MS subtypes and provide insights into disease activity and treatment responses. Additionally, exosomes may serve as tissue‐specific biomarkers for diagnosing T1D progression. Apoptotic bodies (ApoBds) are another type of cell‐derived particle that may indicate disease progression. Their presence in circulation reflects apoptotic death in damaged tissues, with studies showing elevated plasma concentrations of ApoBds in neurological patients compared to healthy donors. Flow cytometry is a rapid, minimally invasive technique for analysing these vesicles, requiring only small sample sizes. So, starting from this contest, in this study, flow cytometry strategies were developed to assess the role of sEVs and ApoBds as future biomarkers in MS and T1D.

Methods : For sEV characterization, up to 37 epitopes were analysed on vesicles derived from plasma samples of healthy donors using the MACSPlex Kit. Blood was processed to isolate plasma, which was then ultracentrifuged to obtain sEVs, characterized and quantified by nanoparticle tracking analysis (NTA). For ApoBds characterization, they were isolated in vitro from the OPM2 cell line after bortezomib treatment. Three centrifugation protocols were evaluated, with the most effective being 700 × g for 10 min followed by 3000 × g for 30 min, achieving optimal purity and yield.

Results : The data obtained report the characterization of plasma sEVs for endothelial, immune and platelet markers. Additionally, results showed a significant increase in ApoBds from bortezomib‐treated cells.

Summary/Conclusion : This study establishes a robust methodology for the flow cytometric characterization of sEVs and ApoBds, paving the way for future investigations into their potential as biomarkers for T1D and MS.

Funding : This study was supported by the Project PNC 0000001 D3 4 Health, CUP B53C22006080001, Funded by NextGenerationEU.

Subtopic Cancer biomarkers 2

Circulating levels of prostate‐derived EVs improve the pre‐biopsy diagnosis of clinically significant prostate cancer

Cameron J. Britton 1* , Ekamjit Deol 1* , Jack R. Andrews 2 , Ali Arafa 3 , Yohan Kim 1 , Laureano Rangel Latuche 5 , Phillip J. Schulte 5 , Vidhu B. Joshi 1 , Mohamed E. Ahmed 1 , R. Jeffrey Karnes 1 , Fabrice Lucien 1*#

1 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 2 Department of Urology, Mayo Clinic, Scottsdale, Arizona, USA; 3 Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, USA; 4 Masonic Cancer, University of Minnesota, Minnesota, USA; 5 Department of Quantitative Health Sciences, Mayo Clinic, Rochester, Minnesota, USA

Introduction : It is estimated that 13% of men are diagnosed with prostate cancer (PCa) over their lifetime, with the incidence generally rising with age. Prostate‐Specific Antigen (PSA)‐based screening has led to a reduction in PCa‐specific mortality with the early diagnosis of clinically significant PCa (csPCa). However, PSA suffers from low specificity, leading to unnecessary biopsies in men without cancer and overdiagnosis of indolent PCa. To overcome this, magnetic resonance imaging (MRI) has been used to guide biopsy of suspicious lesions, reducing the number of unnecessary biopsies. Nevertheless, the specific detection of csPCa with MRI does not exceed 40%. Urine molecular biomarkers are attractive non‐invasive tools to improve prostate cancer diagnosis. Prostate specific membrane antigen (PSMA) and Six‐transmembrane epithelial antigen of the prostate 1 (STEAP1) are two prostate‐associated surface proteins found upregulated in csPCa. In this study, we investigated the performance of PSMA‐ and STEAP1‐positive prostate extracellular vesicles (ProstEVs) in improving the diagnosis of csPCa alone and in combination with MRI.

Methods : Blood and urine were prospectively collected from 179 men undergoing MRI and prostate biopsy for suspicion of PCa based on elevated PSA. Circulating levels of PSMA‐ and STEAP1‐positive EVs were measured by standardized and calibrated nanoscale flow cytometry. Diagnostic performance of EVs, including sensitivity and specificity, was measured by multivariable logistic regression models and receiver operator characteristic (ROC) curve analysis. Bootstrapping analysis was performed to reduce overfitting and provide a more accurate assessment of predictive abilities. Performance of models was measured using the DeLong's test.

Results : In the patient cohort, 95/179 (53%) had csPCa; the remainder (47%) had benign disease or indolent PCa. AUC, sensitivity, and specificity were 0.63 (0.58–0.68), 90.07%, and 34.2% for MRI alone; 0.76 (0.68–0.83), 90.1%, and 40.9% for MRI combined with clinical variables (age, digital rectal exam, PSA, race, and family history), 0.81 (0.74–0.87), 90.1%, and 55.4% for MRI + clinical variables + urine ProstEVs.

Summary/Conclusion : This work describes urine levels of prostate EVs as a promising biomarker to augment the current diagnostic algorithm for pre‐biopsy detection of csPCa and reduce unnecessary biopsies in men with benign disease and indolent PCa. Validation in larger prospective patient cohorts is warranted.

Funding : This study was supported by internal funding.

Tumour‐derived FSCN1+EVs serve as potential biomarkers for early detection of hepatocellular carcinoma

Presenter: Shan Lin

Southern Medical University, Guangzhou, People's Republic of China

Introduction : There are limited non‐invasive screening methods in the early stage of hepatocellular carcinoma cancer (HCC). Extracellular vesicles (EVs) have been reported to reflect tumour pathology and serve as a suitable liquid biopsy tool. The potential diagnostic value of plasma‐derived extracellular vesicles for HCC needs to be further studied.

Methods : The TCGA dataset was used to analyse the expression and clinical significance of FSCN1 in HCC. A tissue array was analysed for FSCN1 expression by immunohistochemical staining. Tissues and venous blood were collected from HCC patients ( n = 20) at Nanfang Hospital between 2019 and 2020. The tissue‐derived vesicles were isolated to verify the expression of FSCN1. Plasma‐derived vesicles were isolated by ultracentrifugation, and the proportion of FSCN1+EVs in peripheral blood was detected by nanoflow cytometry. HCCLM3 cells were transfected with lentivirus to overexpress FSCN1 to obtain FSCN1+EVs for in vitro studies.

Results : FSCN1 was significantly elevated in the HCC tissues by bioinformatics and immunohistochemistry detection and was associated with poor HCC prognosis. EVs derived from FSCN1‐upregulated HCC cell lines (FSCN1‐EVs) promoted HCC invasiveness and increased F‐actin expression in vitro. Clinically, FSCN1 was enriched in tumour‐derived extracellular vesicles. Detection of EVs in 20 pairs of tumour tissues and healthy donors at different stages revealed that the abundance of FSCN1+ EVs in plasma could distinguish stage III–IV HCC from stage I–II HCC and healthy donors. FSCN1+ EVs exhibited good performance (AUC 0.8925, 95% CI: 0.7959 to 0.9891) in ROC curve analysis.

Summary/Conclusion : This finding identified FSCN1+ EVs as a potential biomarker for early detection of HCC.

Funding : This study was supported by the Guangdong Province Basic and Applied Basic Research General Project.

Tissue‐derived extracellular vesicles as diagnostic markers in renal cell carcinoma: a comprehensive study on subtype‐specific signatures

Presenter: Xinrui Wu

Renji Hospital Affiliated to Shanghai Jiaotong University, Shanghai, People's Republic of China

Introduction : Tissue‐derived extracellular vesicles (Ti‐EVs) play a significant role in tumour development, yet their potential as diagnostic markers for various pathological types of renal cell carcinoma (RCC) and their specific roles in tumour progression remain unclear.

Methods : In this study, we analysed tissue samples from 25 RCC patients and their matched Ti‐EVs to identify diagnostic panels for different pathological types of RCCs (DPRCCs) using machine learning. To validate our findings, we extracted preoperative and postoperative urinary extracellular vesicles (uEVs) and supernatants from 178 samples. Based on single‐cell RNA sequencing (scRNA‐seq) data from 14 samples, we further explored the cellular origins of these diagnostic panel EVs, validated by extracting Ti‐EVs from primary tumour‐associated macrophages (pTAMs) of 6 low‐grade ccRCC (low‐ccRCC) and 6 high‐grade ccRCC (high‐ccRCC) samples.

Results : We identified NEAT1 and MMP15 in Ti‐EVs as markers associated with papillary RCC (pRCC); DSG2 and AC026888.1 with chromophobe RCC (chRCC); SERPINA1, VEGFA, EGLN3, CPE, and C6orf223 with low‐ccRCC; APOC1, TGFBI, and LINC00887 with high‐ccRCC; and NDUFA4L2 as a diagnostic marker for both low‐ and high‐ccRCC. We achieved an Area Under the Curve (AUC) of 0.922 for low‐ccRCC detection and 0.874 for high‐ccRCC detection in uEVs, demonstrating superior predictive accuracy for low‐ and high‐ccRCC. Additionally, we observed that Ti‐EVs secreted by endothelial cells in pRCC were rich in MMP15, and those from malignant cells (MCs) in chRCC contained DSG2. In low‐ccRCC, pTAM‐derived Ti‐EVs expressed high levels of SERPINA1 and VEGFA, while high‐ccRCC was characterized by APOC1 and TGFBI expression. NDUFA4L2 expression was notably elevated in ccRCC MCs, with higher levels in high‐ccRCC, underscoring its potential role in ccRCC progression.

Summary/Conclusion : In summary, we provide a comprehensive exploration of the entire EV secretion axis from Ti‐EVs to uEVs, presenting new insights for noninvasive RCC diagnostics and unveiling a potential mechanism of ccRCC invasion.

Three‐dimensional matrix stiffness induces tumourigenic profiles in cancer spheroid‐derived extracellular vesicles

Presenter: Maulee Sheth

University of Cincinnati, Cincinnati, Ohio, USA

Introduction : Biophysical cues such as extracellular matrix (ECM) stiffness affect tumour progression in vivo. Physical properties of ECM not only affect intracellular signalling but also impact the tumour microenvironment through secreted extracellular vesicles (EVs). Small EVs (sEVs)‐mediated intercellular communication and horizontal transfer of biomolecular cargo to recipient cells facilitate interplay and rewiring between different components of the TME, promoting tumourigenesis and metastasis. Here, we engineered a 3D spheroid culture system with varying mechanical properties to study the role of EVs in matrix stiffness‐triggered oral squamous cell carcinoma (OSCC) invasiveness.

Methods : EVs were extracted from day 14 culture media of Cal27 spheroids cultured on soft (EVsoft) and stiff (EVstiff) matrices (3 and 12 mg/mL Matrigel, respectively) using a lab‐on‐a‐chip dielectrophoretic device and characterized using nanoparticle tracking analysis (NTA), western blot (CD63, HSP70, and TSG101), imaging flow cytometry, and super‐resolution microscopy. Genomic analysis included directional total RNA and microRNA (miRNA) sequencing. Pathway enrichment was performed for target genes of differentially expressed EV miRNAs (DEMs) found to be differentially expressed in parental spheroids (DEGs).

Results : NTA and western blot indicated a stiffness‐induced increase in the secretion of EVs. miRNA‐seq revealed upregulation of frequently deregulated EV miRNAs such as miR‐1246, ‐122‐5p, and ‐574‐5p, which corroborate with increased tumourigenic behaviour, including modulation of epithelial to mesenchymal transition, metastatic potential, and chemoresistance in OSCC in EVstiff. Long non‐coding RNAs (Set and Anp32b) involved with cancer promotion were also upregulated in EVstiff, while certain mitochondria‐encoded genes, dysregulation of which contributes to progression, were upregulated in EVsoft. Overlay of target genes of EV‐DEMs and spheroid‐DEGs showed 110 and 120 genes corresponding to up‐ and down‐regulated DEMs, respectively. Pathways including MAPK cascade, cell migration and motility, and cell signalling by electrical coupling were enriched in the downregulated DEMs‐upregulated DEGs overlay for EVstiff.

Summary/Conclusion : We report that 3D ECM stiffness alters EV production and composition. Our findings provided the first evidence that parental biomechanical Piezo1 is expressed in spheroid‐derived EVs. Our data suggests that matrix stiffness modulates carcinogenic signatures in EVs in head and neck cancer. References : (1) Sheth et al. 2024, APL Bioengineering; (2) Sharma et al. 2023, Scientific Reports.

Funding : This study was supported by the NSF CAREER ECCS (No. 2046037).

Evolution of Ewing sarcoma: characterization of miRNA signatures in plasma‐derived EVs of Ewing sarcoma before and after neoadjuvant therapy

Presenter: Irene Dirignani

Istituto Ortopedico Rizzoli, Bologna, Italy

Introduction : Ewing sarcoma (EWS), a paediatric bone and soft‐tissue cancer, is characterized by a high level of intra‐tumour heterogeneity and metastatic potential. Extracellular vesicles (EVs) released by tumour cells play a crucial role in tumour progression, metastasis and immune system suppression. MicroRNAs (miRNAs) are small non‐coding RNAs that can be packaged into EVs and influence various cellular processes. This study aims to investigate the expression of 179 miRNAs within EVs in the plasma of Ewing sarcoma patients (with informed consent) before and after neoadjuvant chemotherapy.

Methods : EVs were isolated from 1 mL of frozen plasma sample from 3 healthy donors and 4 Ewing sarcoma patients pre‐ and post‐therapy using Izon qEVs single 35 nm columns. EV‐miRNAs were extracted using the Qiagen miRNeasy Kit and retrotranscribed with the Qiagen miRCURY LNA RT Kit. During reverse transcription, RNA spike‐in templates were added to monitor performance. Real‐time PCR was performed with the Qiagen miRCURY LNA Focus PCR Panel Serum/Plasma. Data analysis was performed with the Qiagen proprietary software GeneGlobe.

Results : Comparative analysis revealed significant differential miRNAs expression between Ewing sarcoma patients and HDs with a fold regulation >2. Compared to HDs, 34 miRNAs were upregulated in patients before therapy, while 11 were downregulated. Similarly, 40 miRNAs were upregulated after therapy, and 19 were downregulated. Interestingly, only 2 miRNAs were differentially expressed (upregulated) in post‐therapy compared to the pre‐therapy samples (miR‐122‐5p, miR‐885‐5p). Of particular interest, miR‐223‐3p was consistently upregulated in Ewing sarcoma patients compared to healthy donors. Analysis of target pathways is underway to elucidate the potential functional implications of these differentially expressed (DE) miRNAs in Ewing sarcoma.

Summary/Conclusion : We identified different DE‐miRNAs in EVs isolated from the plasma of healthy donors and Ewing sarcoma patients, before and after neoadjuvant chemotherapy. Among them, mir‐223‐3p is of interest due to its role in suppressing the immune system and inflammation. Its effect is being investigated in macrophages, which are the main immune population that infiltrate this overall cold tumour. Further cases (2 HDs and 5 patients) are being analysed and will be ready for the congress date.

Funding : This research has received funding from AIRC under IG 2019—ID. 22805—P.I. Katia Scotlandi.

Characterization of extracellular vesicles in breast cancer patients before and after neoadjuvant chemotherapy

Karin Ekström 1 , Nazia Riaz 1 , Karolina Larsson 1 , Rossella Crescitelli 1 , Barbro Linderholm 1 and Roger Olofsson Bagge 1,2

1 University of Gothenburg, Gothenburg, Sweden; 2 Sahlgrenska University Hospital, Gothenburg, Sweden Karin Ekström, Barbro Linderholm and Roger Olofsson Bagge are co‐senior authors .

Introduction : Breast cancer is the most diagnosed cancer and a leading cause of cancer‐related deaths among females worldwide. Extracellular vesicles (EVs) have emerged as potential biomarkers for tumour characteristics and treatment responses in breast cancer. This study investigates EV profiles in luminal B (LumB), human epidermal growth factor receptor‐2 (HER2+), and triple‐negative breast cancer (TNBC) subtypes, focusing on EV quantification and surface marker analyses before and after administration of neoadjuvant chemotherapy (NACT).

Methods : Informed consent was obtained from the study participants. Plasma samples were collected from patients at two time points: before ( n = 58) and after ( n = 30) NACT. EVs were isolated from 1 mL of plasma using qEV1/70 nm SEC columns (Izon). EV morphology was investigated by transmission electron microscopy (TEM). EV concentration and protein measurement were analysed by nanoparticle tracking analysis (NTA) and the Qubit protein assay, respectively. EV surface marker analysis was performed for 37 antigens using the MACSPlex assay. Responders were defined as pathological complete responders, and non‐responders as non‐pathological complete responders.

Results : EVs were successfully isolated from all plasma samples at both time points. TEM analysis confirmed the presence of intact round elements with typical EV size (50‐150 nm) and morphology. NTA analysis showed an average concentration of 2E11 EVs/mL in plasma, with no significant differences between subtypes or time points. However, complete responders to NACT exhibited about 50% lower levels of EVs than non‐responders both before and after NACT. Surface marker analysis detected 30 proteins across all patient groups, including EV‐related proteins (CD9, CD63, and CD81), cancer‐related proteins (CD24, CD146, EpCAM), and immune‐related proteins (CD3, CD8, CD45, CD56, HLA‐ABC, and HLA‐DR/DP/DQ). However, no significant differences were observed between patient groups or time points.

Summary/Conclusion : While EV count did not significantly vary across subtypes or treatments, a lower EV count was observed in responders to NACT. Further investigations are required to elucidate the clinical implications of these findings.

Funding : This study was supported by the Jubileumsklinikens Cancerfond, Sahlgrenska Comprehensive Cancer Centre, Assar Gabrielsson's Foundation, Knut and Alice Wallenberg Foundation, Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden

A novel 3D bone marrow model to study EV‐induced immune alterations in bone cancer

Simone Foderaro 1,2 , Jocelyn Langley 1 , Crescenzo Massaro 1 , Celine de Schrijver 1 , Hilal Nur Sensoy 1 , Jan van Weering 3 , S. Rubina Baglio 1

1 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC, Amsterdam, the Netherlands; 2 Department of Medicine and Surgery, Medical Oncology, Fondazione Policlinico Universitario Campus Bio‐Medico, Università Campus Bio‐Medico Di Roma, Rome, Italy; 3 Department of Human Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam UMC, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands

Introduction : Tumours that originate in or metastasize to the bone are resistant to the most advanced treatments, including immunotherapies, and determine loss of quality of life and increased risk of death. Studies into the dynamics underlying cancer outgrowth in the bone are challenging due to the lack of in vitro or in vivo models that can reproduce the features of the human bone marrow (BM) environment. We recently demonstrated how cancer cell‐secreted extracellular vesicles (EVs) are a major driver of therapy resistance in a xenograft mouse model of osteosarcoma. To investigate the role of EVs in tumour‐induced immune suppression and therapy resistance, we generated a novel 3D bone‐marrow system enabling the study of tumour‐immune cell interactions.

Methods : The 3D BM system was assembled by combining a collagen‐gelatine sponge with a hydrogel derived from plasma from healthy donors. Murine BM was obtained from immunocompetent mice. We stably knocked down (KD) EVs biogenesis components (Rab11b, Rab35 and syntenin) in osteosarcoma cells (K7M2). KD efficacy was assessed by PCR. The impact of the KD on EVs secretion was assessed by NanoLuc‐CD63 reporter assay, western blot and tunable resistive pulse sensing (TRPS). Alterations of BM immune cells were assessed using a high‐dimensional (22‐colour) spectral flow cytometry method developed in our lab.

Results : Our 3D BM system retained cancer and BM cells and enabled tumour‐immune cell interactions. Rab11b, Rab35 and syntenin KD determined a decrease in EVs production. Deep immune profiling revealed tumour‐induced EV‐dependent immune alterations, in particular in the myeloid cell compartment, which differed from those observed in conventional 2D cultures and mimicked in vivo dynamics.

Summary/Conclusion : We developed a robust and easily reproducible 3D BM system for the study of tumour‐induced immune alterations. Our results support a role for EVs in BM immune suppression.

Comprehensive analysis of oncogenic small extracellular vesicles derived from pancreatic ductal adenocarcinoma

Presenter: Marco Falasca

University of Parma, Parma, Italy

Introduction : Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers, with only 12% of patients surviving 5 years post‐diagnosis. Effective early detection remains a critical unmet need in PDAC management. Our study explores the role of small extracellular vesicles (sEVs) in PDAC, as they promise to deliver tumour‐specific biomarkers and therapeutic targets. Tumour‐derived sEVs can carry oncogenic proteins that drive tumour progression, metastasis, immune evasion, and invasion. Identifying specific molecular signatures in these vesicles could yield novel PDAC diagnosis and treatment avenues.

Methods : We examined sEVs from three groups: healthy individuals ( n = 10), PDAC patients ( n = 19), and chronic pancreatitis patients ( n = 3), using the latest MISEV 2023 guidelines. sEVs were isolated via ultracentrifugation and EV‐affinity techniques and characterized by scanning electron microscopy, nanoparticle tracking analysis, immunoblotting, and quantitative proteomics. For proteomic profiling, we utilized data‐independent acquisition mass spectrometry with an orbitrap eclipse tribrid mass spectrometer, followed by nano‐HPLC separation and peptide quantification with DIA‐NN software. Ethical approval was obtained from Royal Perth Hospital and Curtin University ethics committees.

Results : Proteomic analysis identified a distinct panel of 12 proteins in PDAC and chronic pancreatitis sEVs. Among these, SAA2 was exclusively found in PDAC‐derived sEVs, suggesting its potential as a PDAC‐specific biomarker linked to inflammatory processes in the tumour microenvironment. Additionally, detoxification and metabolic enzymes such as GGT1 and ALDH1A1, uniquely enriched in PDAC sEVs, could reflect metabolic adaptations of tumour cells. This protein profile highlights specific pathways in PDAC pathology that are encapsulated within sEVs and may aid in non‐invasive PDAC detection.

Summary/Conclusion : Our findings underscore the promise of sEVs in advancing the field of PDAC diagnostics and therapeutics. The unique molecular profile of PDAC‐derived sEVs, particularly the presence of SAA2 and metabolically active enzymes, presents new possibilities for biomarker development and mechanistic insights into PDAC progression. These insights could inform novel strategies for early detection and intervention, positioning sEVs as a valuable tool in the fight against PDAC.

Funding : This research was funded by a grant from PanKind, The Australian Pancreatic Cancer Foundation.

Tumour‐derived extracellular vesicles are a prognostic marker and a predictor of therapeutic response in metastatic prostate cancer

Jack Andrews 1 , Yohan Kim 2 , Edlira Horjeti 2 , Ali Arafa 3,4 , Heather Gunn 5 , Aurélie De Bruycker 6,7 , Ryan Phillips 8 , Daniel Song 9 , Daniel S. Childs 10 , Oliver A. Sartor 10 , Jacob J. Orme 10 , Aadel A. Chaudhuri 8 , Phuoc Tran 11* , Ana Kiess 9* , Philip Sutera 9* , Carole Mercier 12,13* , Piet Ost 7,12* , Sean S. Park 8* , Fabrice Lucien 2,14*#

1 Department of Urology, Mayo Clinic, Scottsdale, Arizona, USA; 2 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 3 Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, USA; 4 Masonic Cancer, University of Minnesota, Minnesota, USA; 5 Department of Quantitative Health Sciences, Mayo Clinic, Rochester, Minnesota, USA; 6 Department of Radiation Oncology, AZ Groeninge, Kortrijk, Belgium; 7 Department of Human Structure and Repair at Ghent University, Ghent, Belgium; 8 Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota, USA; 9 Department of Radiation Oncology and Molecular Radiation Sciences, John Hopkins University, Baltimore, Maryland, USA; 10 Department of Medical Oncology, Mayo Clinic, Rochester, Minnesota, USA; 11 Department of Radiation Oncology, University of Maryland Medical System, Baltimore, Maryland, USA; 12 Department of Radiation Oncology, Iridium Network, Wilrijk, Antwerp, Belgium; 13 Translational Cancer Research Unit (TCRU), Center for Oncological Research (CORE), University of Antwerp, Edegem, Antwerp, Belgium, 14 Department of Immunology, Mayo Clinic, Rochester, Maryland, USA

Introduction : Oligometastatic prostate cancer (omPCa) is an intermediate disease stage characterized by fewer than three to five metastases on imaging. While systemic therapy has traditionally been the standard treatment, randomized trials (STOMP and ORIOLE) have shown that stereotactic ablative radiotherapy (SABR) can be a safe and potentially curative option, with about 20% of patients achieving durable disease‐free survival. SABR may delay or prevent the need for non‐curative systemic therapy, thus reducing treatment‐related toxicities and the risk of developing castration‐refractory PCa, the most aggressive disease form. Identifying biomarkers that distinguish patients likely to benefit from SABR alone versus those needing systemic therapy is crucial. This study examines the prognostic and predictive value of circulating prostate cancer‐derived extracellular vesicles (EVs) in retrospective analyses of STOMP and ORIOLE cohorts.

Methods : Blood was collected prior to treatment from 173 omPCa patients, 157 receiving SABR and 16 under observation. Frozen platelet‐poor plasma was analysed with IRB approval. Levels of PSMA‐positive EVs were quantified using nanoscale flow cytometry, with measurements performed blinded to clinical data. Each sample was analysed in triplicate. Kaplan–Meier curves and logistic regression assessed the relationship between PSMA+ EVs, serum Prostate‐Specific Antigen (PSA) levels, and clinical outcomes, using optimal cutoff values from a multivariable Cox proportional hazard model.

Results : SABR‐treated patients with low baseline plasma PSMA+EV levels exhibited shorter median radiographic progression‐free survival (rPFS) than those with high levels (26.1 vs. 15.0 months, p = 0.005). Multivariable analysis indicated that low PSMA+ EV levels correlated with a reduced risk of progression (HR = 0.55, 95% CI: 0.34–0.81, p = 0.003). Fifteen percent of patients with low PSA and PSMA+ EV levels experienced a 4.5‐fold reduction in risk of progression (HR = 0.22, 95% CI: 0.09–0.44, p = 0.0001). In the ORIOLE cohort, patients with low PSMA+EV levels benefitted from SABR compared to those in the observation arm (HR = 0.19, 95% CI: 0.065–0.644, p = 0.004), while high PSMA+ EV levels showed no clinical advantage.

Summary/Conclusion : PSMA+EVs serve as a novel prognostic and predictive biomarker for SABR response in omPCa. Combining PSMA+EV and PSA levels can enhance patient selection and inform treatment intensification strategies. A prospective study for EV‐guided therapy in omPCa is ongoing.

Funding : This study was supported by the Erivan K. and Helga Haub Family Fund in Image‐Guided Urology (FL).

Dissecting landscapes of extracellular vesicles and particles associated with oncohistone‐driven paediatric high‐grade gliomas—toward liquid biopsy applications

Nadim Tawil 1 , Laura Montermini 1 , Samia Jahouri 1,2 , Mahsa Jalali 2 , Wajih Jawhar 1,2 , Cathy Russo 1 , Fereshteh shojaeighahrizjani 1 , Brian Meehan 1 , Damien Faury 1 , Dan Ghiran 1 , Claudia Kleinman 2,3 , Nada Jabado 1,2 , Janusz Rak 1,2 *

1 Research Institute of the McGill University Health Centre, Montreal, QC, Canada; 2 McGill University, Montreal, Quebec, Canada; 3 Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada

Introduction : Paediatric high‐grade gliomas (pHGGs) are devastating brain tumours driven by oncogenic alterations of chromatin regulating histones. The diffuse midline glioma (DMG) subtype driven by ‘oncohistone’ H3.3 K27M mutations is associated with a particularly aggressive course and poor surgical and diagnostic access. Therefore, systemic release of tumour‐associated material, including extracellular vesicles and particles (EVPs), may offer new diagnostic opportunities as liquid biopsy analytes, and a reflection of the disease progression, therapeutic responses and relevant complexities of tumour microenvironment. Here we describe the comprehensive profiling of K27M mutant DMG EVPs including their protein and nucleic acid cargo and salient biological properties.

Methods : EVs were isolated from conditioned media of multiple cell lines using ultracentrifugation (UC) and size exclusion chromatography (SEC). EV size distribution and concentration were assessed by nanoparticle tracking analysis (NTA). EV characterization included electron microscopy, western blot analysis, proteomics profiling, super‐resolution microscopy, and ExoView. Digital PCR (ddPCR) was used to detect and quantify DNA and RNA cargo within the EVs, including the presence of the H3 K27M mutation.

Results : A comprehensive characterization of EVPs secreted by K27M mutant DMG cell lines revealed a complex landscape of particle subpopulations. While NTA indicated a preponderance of vesicles with a size range typical of small EVs and western blot analysis confirmed the presence of canonical EV markers (e.g., CD9 and CD63), further proteomic profiling and single EV analysis (nano‐flow cytometry, ExoView) uncovered evidence for multiple particle subsets being released from DMG cells. This heterogeneity was affected by oncohistone mutations, as K27M‐deleted cells exhibited marked differences in EV proteome. Notably, histones were identified as part of the EV‐associated cargo, highlighting their role in tumour biology. Using ddPCR, we detected the H3 K27M mutation within EV‐associated nucleic acids, suggesting the feasibility of using EVPs for diagnostic exploitation of driver mutations in DMG.

Summary/Conclusion : Our study presents the first comprehensive characterization of EVPs released from DMG cells, demonstrating their potential as biomarkers for tumour profiling and possible bioactive components of the tumour microenvironment. Future work will focus on validating these findings in patient samples and further exploring EVs as diagnostic and therapeutic tools in paediatric glioma.

Exosomal galectin‐associated biomarkers of glioblastoma

Oksana Batkivska 1 , Petr Krůpa 2 , Aleš Hejčl, Jan Malý 1 , Olga Janoušková 1

1 Faculty of Science, Centre for Nanomaterials and Biotechnology, Jan Evangelista Purkyně University in Ústí nad Labem, Czech Republic; 2 Department of Neurosurgery, University Hospital Hradec Králové, Czech Republic; 3 Masaryk Hospital, Regional Health Corporation, Ústí nad Labem, Czech Republic

Introduction : Glioblastoma is a highly malignant brain tumour characterized by aggressive growth, invasiveness, and poor prognosis. Its heterogeneity and the selective permeability of the blood‐brain barrier present major obstacles to effective diagnosis and treatment. Among the various molecules transported by exosomes—stable, native, and targeted vesicles—the galectin protein family plays a crucial role in tumourigenesis, including immune modulation, tumour progression, and metastasis. Exosomal miRNAs, such as miR‐1246, miR‐21, and miR‐375, have been identified as regulators of galectin‐associated mRNAs linked to processes in the tumour microenvironment. This study aims to investigate galectin‐associated molecules within exosomes as biomarkers for glioblastoma diagnosis, prognosis, and therapeutic monitoring.

Methods : Plasma exosomes were isolated from glioblastoma patients at baseline (pre‐therapy), 3 months into therapy, and from healthy volunteers using differential ultracentrifugation. Exosome size and concentration were characterized via nanoparticle tracking analysis (NTA), while exosomal markers (TSG101, CD63, and CD81) were confirmed by Western blotting. After RNA extraction from the isolated exosomes, cDNA was synthesized and analysed by qRT‐PCR with selected mRNA primers and miRNA‐specific primers. Direct interactions between miRNAs and mRNAs were validated by luciferase reporter assays.

Results : The levels of exosomal miR‐1246, miR‐21, and miR‐375 were quantified to compare glioblastoma patient groups with healthy controls. Functional assays, including miRNA mimic and inhibitor experiments, explored the regulatory effects of selected miRNAs on LGALS1, LGALS3, LGALS8, and LGALS9 mRNA expression. Protein expression levels of Galectin‐1, ‐3, ‐8, and ‐9 in exosomes were measured, revealing correlations with miRNA and mRNA expression profiles. Notably, galectin levels were significantly elevated in the plasma exosomes of glioblastoma patients compared to those in the control group.

Summary/Conclusion : Plasma exosomal galectin‐associated molecules can effectively distinguish glioblastoma patients from healthy controls, highlighting their potential as non‐invasive, exosome‐based biomarkers for glioblastoma.

Funding : This study was supported by the Czech Health Research Council, NU23‐08‐00307.

Impact of organoid‐derived extracellular vesicles on CAFs and Matrix: correlation with therapeutic resistance in PDAC

Presenter: Christopher Rovera

INSERM U1068, Nice, France

Introduction : Resistance to chemotherapy in pancreatic ductal adenocarcinoma (PDAC) is due to molecular alterations in tumour cells and their microenvironment. This stroma is composed of cancer‐associated fibroblasts (CAFs) and their extracellular matrix, regulating tumour proliferation, invasion and chemoresistance. There is growing evidence that extracellular vesicles (EVs) play a key role in tumour intercellular dialogue and may be effective biomarkers of cancer progression. However, few studies take into account the intra‐tumour and inter‐patient heterogeneity of tumour cells and CAFs.

Methods : We are working with non‐clonal organoids derived from resection or biopsy of primary human PDAC tumours. We already analysed by LC‐MS/MS the proteomic composition of 15 organoid‐derived EVs isolated by ultracentrifugation (1. vesiculome). To study the impact of these EVs on the CAF matrix, we treated CAFs with organoid‐derived EVs and analysed the proteomic composition of the matrix by LC‐MS/MS (2. matrisome). In order to correlate these proteomic data with a chemotherapy response, we are analysing the response of tumour cells to FOLFIRINOX in coculture with organoid EVs on the one hand, or with the matrix of CAFs treated with organoid EVs on the other.

Results : The analysis of the vesiculome clusterize together non‐detected metastasis patients with a poor survival (“borderline patients”) and patients with metastasis, in contrast to resected patients, suggesting that organoid EVs may have predictive value. We are now validating specific signatures with a second cohort of organoids, and we want to validate these signatures in the blood of patients. Interestingly, the analysis of the matrisome clusterize together again the matrices of CAF treated with the borderline patients organoid‐derived EVs and the one from patients with metastasis. Our results suggest that organoid EVs and the matrix of the CAF processing these EVs can be a source of specific signatures related to the clinical picture of patients.

Summary/Conclusion : The results of these experiments will enable us to impose a new clustering on the proteomics data in order to identify and validate vesicular and matrix signatures specific to the response to chemotherapy. Finally, this work could identify new biomarkers and therapeutic targets to prevent PDAC progression.

Funding : This study was supported by the Fondation de France.

Investigation of radiation‐induced extracellular vesicles in head and neck cancer

Presenter: Aoife Gahlawat

National Center for Tumor Diseases NCT, Heidelberg, Germany

Introduction : Radiotherapy has been shown to promote the release of extracellular vesicles (EVs). Here, we aim to investigate the role of radiation‐induced EVs in squamous cell carcinoma of the head and neck (HNSCC) derived from both cell lines and patient plasma, which have been treated with radiotherapy. We aim to understand the interaction of radiation‐induced EVs with the tumour microenvironment as well as their potential as biomarkers in HNSCC.

Methods : We have thoroughly established methods for EV isolation and characterization from both cell line supernatants and patient plasma. We are investigating EV protein and miRNA cargo. We have established live imaging co‐culture systems to understand EV uptake, communication and function in various cell types.

Results : Our preliminary results confirm that EV release is triggered by radiation and that the cargo of radiation‐induced EVs may promote cell invasion and migration. Moreover, we could show that radiation‐induced EVs are taken up more by HNSCC cells which have not been exposed to radiation. We have also identified components of EV cargo which differ in radiation treated cancer patients compared to healthy controls.

Summary/Conclusion : Taken together, our findings contribute to better understanding the role of radiation‐induced EVs in HNSCC. Our results will be further confirmed and developed to support this hypothesis.

Subtopic EV biomarker clinical translation 2

Biomarkers of multiple sclerosis disease progression in circulating astrocyte and neuronal enriched extracellular vesicles

Presenter: Larissa Jank

Johns Hopkins University, Baltimore, Maryland, USA

Introduction : Multiple sclerosis (MS) is a chronic immune‐mediated demyelinating disease of the central nervous system (CNS). While therapies exist for the peripheral inflammation seen in relapsing MS (RMS), progressive MS (PMS) presents challenges due to ongoing glial activation and neurodegeneration. There are no effective treatments for PMS, partly due to a lack of biomarkers for CNS pathogenic processes in MS. Circulating EVs originating from the CNS show promise in providing these biomarkers.

Methods : EVs were isolated from the plasma of 292 MS patients (195 PMS, 97 RMS), using polymer‐precipitation followed by immunoprecipitation of astrocyte‐enriched EVs ([AEVs] GLAST+) and neuronal‐enriched EVs ([NEVs] L1CAM+). We measured complement components in AEVs and synaptopodin in NEVs. Outcomes included Expanded Disability Status Scale (EDSS), the tablet‐based MS performance test (zMSPT), and clinical MRIs. Longitudinal data was collected for up to 3 years (ongoing). Biomarker associations with outcomes were analysed using linear mixed models and logistic regression models adjusted for age, sex, race, disease duration, subtype, EV isolation batch (and individual total follow‐up time). Analyses of retinal layer thickness (OCT) scans are ongoing.

Results : In AEVs, C3 and C3b levels were associated with worse clinical outcomes cross‐sectionally (age‐adjusted EDSS [gARMSS]: C3 β = 3.75, p = 0.044 and zMSPT in RMS C3 β = −1.20, p = 0.028; C3b β = −0.233, p = 0.009) and longitudinally predicted faster brain atrophy in PMS (C3 in the thalamus β = −0.538, p = 0.035 and C3b in cerebral white matter β = −0.043, p = 0.015). C5 and C5a levels were cross‐sectionally associated with worse clinical outcomes (gARMSS: C5a β = 0.235, p = 0.05; zMSPT C5 β = −0.142, p = 0.016, C5a β = −0.115, p = 0.007) and MRI measures (higher WM lesion volume, C5 β = 0.667, p = 0.0007; C5a β = 0.336, p = 0.007 in RMS) but longitudinally predicted EDSS improvement (C5 OR = 2.74, p = 0.031; C5a OR = 2.61, p = 0.038). In NEVs, higher synaptopodin levels longitudinally predicted slower progression clinically (EDSS progression, OR = 0.42, p = 0.021) and on MRI (total brain atrophy, β = 0.669, p = 0.042).

Summary/Conclusion : The EV biomarkers were associated with outcomes in unique patterns, suggesting they represent distinct disease processes: synaptopodin loss is likely linked to synaptic pathology, C5/C5a to acute inflammation, and C3/C3b to chronic compartmentalized inflammation. In the future, these biomarkers may serve as valuable tools for monitoring MS progression and enhance our understanding of underlying mechanisms.

Funding : This work is funded by Genentech (P.A.C., P.B.), the NMSS (L.J., B.D., K.C.F., P.B.), and the NIH (E.M.M., P.A.C.).

Understanding preterm birth: insights from maternal circulating extracellular vesicles

Thea N. Golden 1 , Rita L. Leite 1 , Jerome F. Strauss III 1 , Samuel Parry 1 , and Rebecca A. Simmons 1,2

1 University of Pennsylvania, Philadelphia, Pennsylvania, USA, 2 Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Introduction : Preterm birth (PTB) is a heterogeneous complication that affects over ten percent of pregnancies. To date there are no effective treatments nor the ability to predict who goes on to deliver preterm. The overarching goal of our research is to identify a biomarker obtained in early pregnancy to identify individuals at risk for delivering preterm. Circulating extracellular vesicle (EV) signalling is a key form of maternal‐foetal communication, and PTB is associated with changes in the EV profile. In this project we tested the hypothesis that the aetiology of PTB influences maternal circulating EVs.

Methods : Subjects were enrolled at the time of delivery after obtaining informed consent and included uncomplicated term, spontaneous PTB (sPTB), and medically indicated PTB (mPTB). EVs were isolated from maternal plasma using serial centrifugation, and large (LEVs) were isolated by 15,000 × g and small (SEVs) were isolated by 100,000 × g . The number and size of EVs were quantified by nanoparticle tracking analysis. We quantified the percentage of EVs from trophoblasts (PLAP+), immune cells (CD45+), platelets (CD41a+), mature endothelial cells (CD31+CD34‐), and foetal endothelial cells (CD31+CD34+) using flow cytometry. We also measured the abundance of ND1 by qPCR to quantify mitochondrial DNA in EVs.

Results : Compared to term, the number of LEVs was increased in mPTB, but there were fewer SEVs in sPTB. The size of LEVs was increased in sPTB but not mPTB, while the size of SEVs was unaffected. Also, in sPTB, we observed an increase in platelet and immune cell derived LEVs and maternal endothelial cell derived SEVs. In mPTB, there were more foetal endothelial cell LEVs and fewer mature endothelial cell SEVs. Finally, we observed an increase in the abundance of mitochondrial DNA in SEVs from PTB pregnancies regardless of the type.

Summary/Conclusion : The changes observed at delivery reflect the underlying pathophysiology and identify different cell types affected in sPTB versus mPTB. However, increased mitochondrial DNA release was evident irrespective of the type of preterm birth. Our ongoing research, in a prospectively collected cohort, aims to identify when during pregnancy the size, cargo, and origin of circulating EVs are altered.

Funding : This study was supported by the March of Dimes and NIH grants T32ES019851 and P30ES013508.

Cerebrospinal fluid‐derived small extracellular vesicles (sEVs) more accurately reflect the medulloblastoma proteome than blood plasma sEVs

Presenter: Jamal Ghanam

University Hospital Essen, Essen, Germany

Introduction : Understanding the properties and dynamics of medulloblastoma (MB) progression remains elusive due to a lack of established minimally invasive monitoring techniques. Extracellular vesicles (EVs) carrying disease‐specific signatures have emerged as promising analytes for repetitive liquid biopsies. However, this is hindered by the absence of standardized EV isolation methods and the difficulty of targeting cancer‐specific EVs in body fluids. Here, we evaluate the clinical utility of blood plasma (BP) and cerebrospinal fluid (CSF) small EVs (sEVs), which remains unexplored in MB.

Methods : We employed ultrafiltration and size exclusion chromatography (UF‐SEC) to isolate sEVs from BP and CSF of children with MB. sEVs were characterized according to MISEV2023 guidelines and subjected to liquid chromatography‐tandem mass spectrometry. Gene Ontology analysis and Cytoscape pathway analysis were conducted to investigate associated functions and further identify proteins with datasets from MB tissue, CSF, and common EV markers.

Results : UF‐SEC was optimized to separate sEVs from low volumes of BP and CSF with high recovery rates. We observed a shift in particle number, protein concentration, and CD81‐positive sEVs towards later fractions in CSF‐derived EV preparations, indicating that CSF‐sEVs are notably smaller in size compared to BP‐derived EVs. This was further confirmed by TEM imaging. We analysed the EV proteome to investigate whether the disparities in size and bodily fluid type have a measurable impact on the quantity and quality of EV protein cargo. 210 proteins were identified and grouped into 10 clusters by K‐means clustering, revealing a body‐fluid‐specific protein ontology enrichment. Proteins associated with the extracellular matrix (ECM) and integrin functions were significantly upregulated in CSF‐sEVs. Notably, analysis of MB tissue datasets revealed that ECM and integrin proteins such as ANXA6 were exclusively enriched in CSF‐sEVs.

Summary/Conclusion : This study underscores the importance of tailoring EV isolation methods to specific body fluids and highlights the potential of CSF‐sEVs over BP‐sEVs for MB diagnosis and monitoring. Further research in a larger cohort is needed to confirm these findings and assess their contribution to predicting recurrence, metastasis, outcome, and prognosis.

Multimodal profiling of neuronal and astrocyte EV‐derived miRNAs alongside circulating plasma proteins for differentiating Lewy body disease versus Alzheimer's disease

Presenter: Andrew A. Lin

University of Pennsylvania, Philadelphia, Pennsylvania, USA

Introduction : Alzheimer's disease (AD) and Lewy body disease (LBD) are the two most common causes of neurodegenerative dementia worldwide. Recent studies have made significant advances in developing targeted therapeutics for AD, but patients and healthcare providers continue to face a lack of robust biomarkers for differentiating LBD versus AD. Extracellular vesicles (EVs) have shown considerable promise as sources of biomarkers in neurodegenerative dementia. However, their clinical application has been limited by technical challenges in isolating brain‐derived EVs and a lack of samples with corresponding post‐mortem pathology for confirming patient diagnoses.

Methods : We profiled the miRNA cargoes of neuronal and astrocyte EVs from patient plasma by using a nanofluidic EV isolation platform developed in our group. Specifically, we sequenced the miRNA cargoes of GluR2‐positive (neuronal) and GLAST‐positive (astrocyte) EVs from the plasma of n = 137 dementia patients and clinically‐normal controls with post‐mortem histopathologic confirmation of their diagnoses. We also profiled five plasma protein biomarkers (AB40, AB42, AB42/40, p‐Tau181, p‐Tau231) via commercially‐available digital ELISA as a benchmark comparison. Informed consent was obtained for the collection of all patient plasma samples in this study.

Results : We identified multiple differentially‐expressed EV nucleic acid biomarkers alongside plasma proteins which were statistically robust for comparing LBD versus AD. We show that both GluR2‐positive and GLAST‐positive EVs yield unique miRNAs which provide distinct reservoirs of biological information that build on previously‐reported literature. We also demonstrate concordance of our protein biomarker data with previous literature, and perform computational feature selection to compose proof‐of‐concept biomarker panels for differentiating LBD versus AD. This work offers a set of candidate biomarkers for future studies in neurodegenerative dementia.

Summary/Conclusion : In this work, we validate the hypothesis of whether a multimodal biomarker panel can meaningfully assist in the differential diagnosis of LBD versus AD from patient plasma alongside clinically‐normal controls. This work demonstrates a multimodal and biomarker‐driven proof‐of‐concept approach towards the precise differentiation of neurodegenerative dementia.

Funding : This study was supported by the American Brain Foundation; National Institutes of Health; Robert H. and Clarice Smith and Abigail van Buren Alzheimer's Disease Research Program; Little Family Foundation; Mangurian Foundation; American Academy of Neurology, American Brain Foundation, LBD Center without Walls; Ted Turner and Family Foundation.

Proteomic profiling of small extracellular vesicles (sEVs) in the blood of non‐small cell lung cancer (NSCLC) patients receiving combined chemo‐immunotherapy as initial treatment

Ashish Saxena 1,2 , Serena Lucotti 1 , David Lyden 1,2

1 Weill Cornell Medicine, New York, New York, USA, 2 Sandra and Edward Meyer Cancer Center, New York, New York, USA.

Introduction : The combination of chemotherapy and anti‐PD1 immunotherapy (chemo‐immunotherapy) is a standard treatment for patients with both non‐metastatic NSCLC, who receive this prior to surgery, and those with metastatic disease. sEVs can be found in the blood of patients with NSCLC and contain a variety of proteins which reflect the contents of their cells of origin. These proteins may provide insights into mechanisms of treatment response and resistance to chemo‐immunotherapy and serve as circulating biomarkers for these outcomes. We conducted a pilot study to isolate and characterize circulating sEVs from the blood of patients with metastatic NSCLC and those with non‐metastatic disease who received chemo‐immunotherapy prior to surgery. The protein contents of these sEVs were analysed and compared.

Methods : Peripheral blood from 12 patients with metastatic NSCLC and 5 with non‐metastatic disease was collected at different time points, including prior to receiving any treatment (baseline). sEVs were isolated from plasma by sequential centrifugation at 3000 × g , 12,000 × g , and 100,000 × g . EV proteins were then extracted and analysed by liquid chromatography mass spectrometry (LC‐MS), and levels were compared.

Results : Compared to those with non‐metastatic NSCLC, sEVs isolated at baseline from metastatic patients were enriched for several proteins, including immunoglobulins H, K, and L, and actin subunits. Ten patients received chemo‐immunotherapy as initial treatment and had sufficient clinical follow‐up for analysis. Compared to 5 patients with a good response, the 5 patients who had a poor response to chemo‐immunotherapy had circulating sEVs with higher amounts of the protein reelin (RELN). Among four non‐metastatic patients who had an 80%–100% pathologic response to chemo‐immunotherapy, sEVs isolated after completion of this therapy showed similar protein compositions to baseline, with only increases in ECM1 and F13B and decreases in CD61 and IGLV1‐47 seen.

Summary/Conclusion : Circulating sEV protein contents from NSCLC patients differ between those with metastatic and non‐metastatic disease. Patients with non‐metastatic NSCLC who have a good response to chemo‐immunotherapy do not show much change in their circulating sEV protein content after therapy. Patients with poor response to chemo‐immunotherapy have circulating sEVs containing higher amounts of reelin protein. Reelin gene mutations in NSCLC cells have been associated with enhanced response to immunotherapy.

Serum extracellular vesicles as a source of biomarkers for Parkinson's disease neurodegeneration and rehabilitation monitoring

Alice Gualerzi 1 , Martina Gerli 1 , Gemma Lombardi 2,3 , Silvia Picciolini 1 , Aurora Mangolin 1 , Valentina Mangolini 1,4 , Francesca Rodà 1,5 , Luana Forleo 1,6 , Rita Martino 1 , Stefano Faggiano 1 , Silvia Ramat 7 , Stefano Giuseppe Doronzio 3,8 , Diego Longo 3,8 , Marco Baccini 3 , Francesca Cecchi 3,8 , Marzia Bedoni 1

1 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milano, Italy; 2 Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Università di Firenze, Florence, Italy; 3 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Firenze, Italy; 4 Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Brescia, Italy; 5 Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy; 6 Medical and Surgical Pathophysiology and Transplantation Department, University of Milan, Italy; 7 Parkinson Unit, Azienda Ospedaliera Universitaria Careggi, Firenze, Italy; 8 Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Università di Firenze, Florence, Italy

Introduction : Extracellular vesicles (EVs) are nanoparticles secreted by all body cells and released in biofluids. In Parkinson's disease (PD), EVs seem to represent a vehicle of disease‐related molecules, such as α‐synuclein. Also, blood EV biochemical modifications have been correlated with people with PD (pwPD) clinical profiling. In the present study, we hypothesize EV involvement in both pathogenic mechanisms occurring at PD onset, along with biological processes occurring in the brain during disease progression and therapeutic treatment in pwPD. We expect EVs to be mediators of brain remodelling induced by motor rehabilitation, which is crucial in the treatment of pwPD by ameliorating motor and non‐motor impairments and improving their quality of life.

Methods : After providing informed consent, a cohort of 44 pwPD was recruited. Blood samples, as well as clinical, neuropsychological, and instrumental variables, were collected for each patient before and after a treadmill‐based rehabilitation programme (8 weeks). EVs were isolated from serum by combining size exclusion chromatography and ultracentrifugation methods. Biochemical characterization of EV samples was obtained by Raman spectroscopy to evaluate spectral differences in EV fingerprints before and after rehabilitation. Raman data were subsequently correlated with clinical and motor parameters to evaluate the potential of Raman spectra as predictive biomarkers of rehabilitation efficacy.

Results : EVs were successfully isolated from the serum of pwPD through a reproducible method. No differences in size distribution and protein biomarkers were observed. Raman analysis highlighted significant differences in samples before and after rehabilitation in multiple spectral regions. For both groups, Raman spectra clearly showed peaks attributable to α‐syn and carotenoids with variable intensities, suggesting that motor‐cognitive rehabilitation can induce significant biochemical differences in circulating EVs.

Summary/Conclusion : In conclusion, our data suggest a possible EV contribution in restorative mechanisms induced by intensive rehabilitation and confirm that EVs could provide an accessible and measurable biomarker source for neurodegeneration and rehabilitation‐induced brain remodelling. Thus, EV characterization by Raman spectroscopy can be used as a support tool in the diagnosis and monitoring of PD progression, as well as the effectiveness of rehabilitation programmes, allowing for the personalization of rehabilitation strategies.

Funding : This study was funded by Fondazione Cassa di Risparmio di Firenze.

Diagnostic potential of urinary EVs of tubular and glomerular origin in early diabetic nephropathy

Hanine E. L. Itawi 1 , Morgan Greig 1 , Indika Mallawaarachchi 1 , Jennie Ma 1 , Clarissa Diamantidis 2 , Hayrettin Yavuz 1 , Luca Musante 3 , Hayden Hutson‐Wiley 1 , Mike Solga 1 , Samantha Upson 1 , Michael A. Harding 1 , Julia Scialla 1 , Agnieszka Swiatecka‐Urban 1 , Uta Erdbruegger 1

1 University of Virginia, Charlottesville, Virginia, USA; 2 Duke University, Durham, North Carolina, USA; 3 University of Pennsylvania, Philadelphia, Pennsylvania, USA

Introduction : Urinary extracellular vesicles (uEVs) have been identified as early and sensitive biomarkers in kidney disease; however, their association with diabetic kidney disease (DKD) progression and DKD clinical traits is not yet clear. Characterization of uEVs may confirm the relationship between their glomerular and tubular origin as well as clinical DKD pathophysiological phenotypes.

Methods : In this study, we collected uEVs from 24‐h urine collections in 116 adults with diabetes mellitus enrolled in the Simultaneous Risk Factor Control Using Telehealth (STOP‐DKD). To determine uEVs count, size and cargo, we characterized them by spectral flow cytometry (SFC), SP‐IRIS (Exoview R100) for small sample volumes and nanoparticles tracking analysis. We used markers for CD26/DPP4 (PT S1) + , CD35/CR1 (podocyte) + , CD10/MME/NEP (PT S1, S2, S3) + , and tetraspanin, CD9 (EV marker). We used linear regression to assess the cross‐sectional association of SFC data, kidney function and degree of microalbuminuria (UACR). SP‐IRIS analysis was performed on a cohort of 10 healthy and 10 early DKD patients by testing CD63/CD9 and DPP4.

Results : Participants were 63.65 ± 9.20 years of age; 50.4% were female. Baseline eGFR was 81 ± 22 mL/min/1.73m 2 and median UACR was 20.26 (IQR 8.38 to 92.97 mg/g). Higher counts of uEVs detected by SFC carrying DPP4 and MME were significantly associated with higher eGFR after adjustment for sex, race, age, log UACR and total urine creatinine. No markers demonstrated significant association with log UACR, and size was not considered further. SP‐IRIS analysed DPP4 and MME subpopulations captured by the CD63 and CD9 probes for exploration of small sample sizes. DPP4/Total Ratio was significantly higher in healthy patients when captured by CD63 ( p = 0.0176), and results of SP‐IRIS demonstrate that sample size can be microscaled.

Summary/Conclusion : These results suggest that subgroups of tubular derived uEVs detected by SFC are associated with a significant change in eGFR in a cohort of early DKD. Tubular markers detected by SP‐IRIS are decreased in DKD when compared to HC. Our data shows a significant promise for the uEVs signature detected by single EV analysis. Further investigation is necessary to fully understand if these tubular derived uEVs derive from healthy tubular cells or are expressed more during tubular stress in early DKD.

Funding : This study was supported by the American Diabetes Association funding: 7‐22‐ICTSPM‐19.

Extracellular nanovesicles in perfusion liquids as biomarkers of kidney graft function recovery

Marylou Pichonnière, Thomas Duret, Pierre Boulard, Mohammed I. Y. Elmallah, Audrey Héraud, Justine Gainche, Lucie Maigret, Matthias Büchler, Sebastien Roger, Philippe Gatault

University of Tours, Tours, France Sebastien Roger and Philippe Gatault are co‐senior authors .

Introduction : Kidney transplantation is the most efficient treatment for end‐stage chronic renal disease. To face difficulties of grafts’ shortage, kidneys are taken from deceased donors with expanded criteria (advanced age donors, acute renal failure). In 21% of kidney transplants, recipient patients present a delayed graft function (DGF), clinically defined by the need for dialysis the first week post‐operation. DGF can decrease short‐ and long‐term graft survival. It's mostly associated with ischaemia‐reperfusion injuries. Grafts from expanded criteria donors are placed in perfusion machines with preservation liquid. Extracellular vesicles are important intercellular communication messengers. Small extracellular vesicles (sEV) are a class of EV with a diameter of lower than 200 nm. In stress and ischemia conditions, several sEV are released in the extracellular environment. The purpose of this project is to evaluate the potential role of sEV in perfusion liquids and their profiles as biomarkers of graft function recovery.

Methods : Perfusion liquids are collected from the Nephrology department, University Hospital of Tours. sEV are isolated by differential ultracentrifugation. Size and quantity are determined with nanoparticle tracking analysis (NTA) and visualized with transmission electronic microscopy. The distribution of CD9, CD63 or CD81 populations was evaluated by fluo‐NTA. Proteins are analysed by western blot. Integration in monocyte derived‐dendritic cells (moDC) and HK‐2 (proximal tubular epithelial kidney human cells) is evaluated by flow cytometry (FCM) from PKH67‐labelled EV. The modulation of phenotype, activity is analysed with FCM and multiplexing assay.

Results : Thirty‐five perfusion liquids were analysed, with 8 associated with DGF (23%). They contain a median quantity of 8.1 × 10 10 EV/mL, with a median diameter of 162 nm. sEVs released include CD9, CD81 or CD63 populations in variable quantities and express conventional markers (tetraspanins, synthenin‐1, flotilin‐1). They also expressed HSP‐27, HSP‐90, P2X4, and P2X7 proteins. sEVs were time‐dependently integrated in MoDC and HK‐2 (maximum uptake at 4 and 6 h, respectively). The incorporation of sEV and their content seems to modulate immature moDC to a mature phenotype.

Summary/Conclusion : sEV are released by kidneys during cold perfusion. The identification of protein content remains essential to procure information regarding the state and quality of the organ and may be predictive of the transplantation’ success and function recovery.

Osteosarcoma biomarker discovery in plasma extracellular vesicles using a targeted proteomics approach

Snehadri Sinha 1,2 , Virinder Kaur Sarhadi 1,2 , Md Mahamudul Alam 1,2 , Pia Siljander 1,3 , Riitta Seppänen‐Kaijansinkko 1.2 , Tuula Salo 1,2,4

1 University of Helsinki, Helsinki, Finland; 2 HUS Helsinki University Hospital, Helsinki, Finland; 3 Finnish Red Cross Blood Service, Finland, Helsinki, Finland; 4 Univeristy of Oulu and Oulu University Hospital, Oulu, Finland

Introduction : Osteosarcoma (OS) is an aggressive form of bone cancer that primarily affects adolescents and older adults. Diagnosis typically involves imaging tests and biopsy procedures, which can be invasive and cause discomfort for patients. Currently, there are no established biomarkers for OS, although high levels of alkaline phosphatase and lactate dehydrogenase in blood may suggest an advanced stage of the disease. Liquid biopsies, such as plasma‐derived extracellular vesicles (EVs), offer a less invasive alternative for diagnosis. Here, we conducted a case‐control study using plasma from OS patients and controls, and screened plasma EVs for candidate biomarkers and evaluated their diagnostic utility. Additionally, we tested two common EV isolation methods to determine the optimal choice for proteomic analysis.

Methods : Plasma samples from OS patients and control individuals were obtained from the Helsinki biobank, while OS cell lines were derived from patient biopsies or commercial sources. EVs were isolated from plasma and cell culture conditioned media using either ultracentrifugation (UC) or size exclusion chromatography (SEC), and the efficacy of each method was assessed based on EV purity and yield. EVs were characterised by nanoparticle tracking analysis, electron microscopy, and Western blotting for established EV markers. Next, the EVs will be analysed with a proximity extension assay to quantify selected cytokines and immune surveillance‐related molecules. Comparisons between plasma EVs and cell‐derived EVs will also be performed. Lastly, the expression of biomarkers of interest will be validated using additional protein assays.

Results : Both UC and SEC methods isolated an adequate amount of EVs from plasma for proteomic analysis. However, UC was more effective than the commercial SEC columns at separating EVs from other plasma components, particularly lipoproteins. The UC‐isolated EVs were therefore considered more suitable for proteomic analysis. The results from proteomics are currently pending.

Summary/Conclusion : The proteomics data will allow us to identify biomarkers of interest and to evaluate the suitability of small plasma volumes for EV‐based diagnostics. UC allows better separation of EVs from plasma components than SEC. This study advances the understanding of EVs as potential non‐invasive biomarkers, with implications for improving OS diagnostic accuracy and patient outcomes.

Effective discrimination and relapse prediction of DLBCL‐lymphoma by protease activity and/or proteomics in plasma extracellular vesicles (pEV)

Presenter: Rebecca E. Baur

Uniklinikum Erlangen, Erlangen, Germany

Introduction : Diffuse large B‐cell lymphoma (DLBCL) is the most common type of non‐Hodgkin lymphoma, often having a poor outcome due to tumour relapse and treatment failure. The International Prognostic Index (IPI) has been used to classify risk groups; however, it has low to intermediate reliability. CAR T cells have emerged as a new and effective treatment for patients with treatment failure but need a production time of several weeks. Taken together, a much better risk stratification in DLBCL patients has a high clinical need.

Methods : Plasma EV from different lymphoma entities (Hodgkin's lymphoma (HL: n = 12), indolent lymphoma (IL: n = 30) and DLBCL ( n = 61) and healthy donors ( n = 55) (informed consent was obtained) were isolated via dual‐mode chromatography (DMC) and analysed for the activity of 11 different proteases (patent in preparation). Plasma EV‐associated protease activity (PAA) was determined using a FRET‐peptide‐based fluorescence assay. Proteomic content was analysed using a commercial proximity extension assay (Olink Proteomics) analysing 3000 factors. Machine learning algorithms were employed to build classifiers.

Results : We found that among different lymphomas protease activities (PA) in DLBCL‐Lymphoma were most significant and discriminated the cancer from healthy individuals with an AUC of 0.93‐1.0. Confirming these results, proteomics on pEV biomarkers separated both populations with an AUC of 1.0. PA decreased rapidly upon successful treatment and increased before tumour relapse. Persistent high/increasing vs. decreasing PA correlated with the clinical outcome. Finally, high PA at diagnosis was significantly associated with treatment failure in the first 2 years.

Summary/Conclusion : In summary, pEV protease activity alone or supported by Olink proteomics analysis may offer a simple, fast and convenient tool in clinical practice to detect and monitor DLBCL Lymphome and potentially other lymphomas. The assay could be used, for example, to prepare a lifesaving CAR T‐cell therapy in a timely fashion.

Funding : This study was supported by the Bundesministerium für Bildung und Forschung (BMBF), Bayerisches Staatsministerium für Wirtschaft (Bayern Innovativ) and the Hahn Schickard Institute in Freiburg.

Non‐coding RNAs (miRNAs) regulate expression of genes implicated in Diabetic Retinopathy pathogenesis

Presenter: Sarmeela Sharma

LV Prasad Eye Institute, Hyderabad, India

Introduction : Diabetic retinopathy (DR) is an ocular complication of diabetes with higher prevalence among the developing countries. Management of this disease only targets the end‐stage symptoms, thereby making the vision loss irreversible in DR subjects. Early diagnosis and better prognosis of this disease could help overcome this problem in the future.

Methods : To understand the regulation of genes involved in DR, global expression profiling for regulatory molecules miRNA was performed in DR blood samples ( n = 3 each from NPDR, PDR and healthy controls) using microarray. The identified miRNAs were subjected to pathway analysis. Expression of significant miRNAs was further explored in extracellular vesicles (EVs) isolated from DR serum and vitreous samples.

Results : A total of 1486 differentially expressed ncRNA genes were identified in this study. Among those, 890 genes were downregulated, while the remaining 596 were upregulated. The miRNAs found dysregulated were implicated in DR‐associated pathways: cytoskeleton remodelling, oxidative stress, angiogenesis, autophagy, apoptosis, lipid metabolism, inflammation, cellular adhesion, proliferation and vascular homeostasis. A concurrent negative regulation of the genes regulated by these miRNAs was also observed in DR blood samples. Among the significant pathways, extracellular vesicle related processes were enriched. The significant miRNAs were also found differentially expressed in the EVs derived from systemic (serum) and ocular fluids (vitreous).

Summary/Conclusion : Vitreous/serum derived EV cargo packed significant information on the type of miRNAs involved in the DR pathogenesis. The specific signature of these EVs could also be explored as a predictive marker for DR development or progression among diabetic individuals.

Funding : This study was supported by the ICMR.

Characterization of human immunodeficiency virus (HIV) proteins secreted in extracellular vesicles (EVs) during infection for use in at‐home diagnostics

Casey Scott‐Weathers, Kaitlyn King, Gary Baisa, Kimberly Luke

Intuitive Biosciences, San Francisco, California, USA

Introduction : Early diagnosis of HIV is key for early treatment and preventing spread to others. There is only one FDA‐approved at‐home test that relies on detection of both antibodies and HIV proteins. Antigen/antibody tests rely on detection of the p24 capsid protein, a marker of early infection, and antibodies against the viral envelope protein. These Ag/Ab tests can take up to 90 days post‐exposure to detect infection. To develop diagnostics that can be performed at home and earlier than 90 days post‐exposure, we investigated EVs for HIV biomarkers. Using infected cell lines and plasma from infected individuals, we confirmed the abundance of HIV proteins in EVs and developed an EV‐capture and cargo detection platform in a lateral flow assay.

Methods : EVs were enriched using SEC fractionation and concentrated using 10 or 100 kDa MWCO spin columns or using silicon carbide resin (Norgen). EVs were characterized by NTA or TRPS and protein content was quantified using the BCA method. EV marker TSG101 and HIV proteins were examined by ELISA or western analysis. Co‐localization of HIV proteins with tetraspanins was performed using super‐resolution microscopy. Last, EV‐p24 was detected on a prototype two‐phase lateral flow test using a visual signal via coloured latex beads. Exempt human specimens were used from a prior IRB‐approved collection.

Results : EVs enriched from HIV+ plasma showed increased levels of HIV proteins by ELISA or Western protein analysis when compared to HIV‐ plasma. The HIV proteins p24, Tat, Nef and gp120 were detected in EVs enriched from HIV+ subjects by Western analysis. HIV proteins p24 and Nef were further evaluated by super‐resolution microscopy and were co‐localized with tetraspanins with increased detection after EV permeabilization. Nef and p24 in seroconversion panels were detected earlier than in Ag/Ab tests. Using EVs from HIV+ cell lines, the LOD of p24+ from EVs spiked into normal human plasma was less than 7.5 × 10 4 EVs by two‐phase lateral flow.

Summary/Conclusion : We demonstrate that EVs from HIV+ plasma contain HIV protein biomarkers for diagnostics. Detection of p24 and Nef can improve the early detection of HIV in an at‐home lateral flow test, allowing earlier testing and diagnosis.

Funding : This study was funded by the NIH/NIAID Contract 75N93023C00007.

A novel multiple marker microarray analyser and methodology to predict major obstetric syndromes using surface markers of circulating extracellular vesicles (EVs) from maternal plasma

Malene Møller Jørgensen 1,2 , Rikke Bæk 1 , Jenni K. Sloth 1 , Rami Sammour 3 , Adi Sharabi‐Nov 4 , Manu Vatish 5 , Hamutal Meiri 6 , Marei Sammar 7

1 Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark; 2 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; 3 Department of Obstetrics and Gynecology, Bnai‐Zion University Medical Center, Maternal and Fetal Medicine Unit, Haifa, Israel; 4 Departments of Statistics, Tel Hai Academic College, and Ziv Medical Center, Safed, Israel; 5 Nuffield Department of Women's & Reproductive Health, University of Oxford, Oxford, United Kingdom; 6 TeleMarpe Ltd, Tel Aviv, Israel; 7 Prof. Ephraim Katzir Department of Biotechnology Engineering, Braude College of Engineering, Karmiel, Israel

Introduction : Placental‐derived extracellular vesicles (EVs) are nano‐organelles that facilitate intercellular communication between the feto‐placental unit and the mother. We evaluated a novel multiple microarray analyser for identifying surface markers on plasma EVs that predict preterm delivery (PTD) and preeclampsia (PE) compared to term delivery (TD) controls.

Methods : In this prospective exploratory cohort study pregnant women between 24 and 40 gestational weeks with PTD ( n = 16), PE ( n = 19), and matched TD controls ( n = 15) were recruited from Bnai Zion Medical Center, Haifa, Israel. Plasma samples were tested using a multiple microarray analyser. Glass slides with 17 antibodies to EV surface receptors (selected from 85 candidates) were incubated with raw plasma samples, detected by biotinylated secondary antibodies specific to EVs or placental EVs (PEVs), and labelled with cyanine 5–streptavidin. PBS and whole human IgG served as controls. The fluorescent signal ratio to negative controls was log 2 transformed and analysed for sensitivity and specificity using the area under the receiver operating characteristics curves (AUROC). The best pair ratios of general EVs/PEVs were used for univariate analysis, and the top pairs were combined for multivariate analysis. Results were validated by comparison with EVs purified using standard procedures.

Results : Heatmaps differentiated surface profiles of PE, PTD, and TD receptors on total EVs and PEVs. Similar results were obtained with purified EVs and EVs from raw plasma. Univariate analyses identified markers predicting PTD and PE over TD controls with AUC > 0.6 and sensitivity > 50% at 80% specificity. Combining the best markers in a multivariate model, PE prediction over TD had an AUC of 0.89 (95% CI: 0.72–1.0) with 90% sensitivity and 90% specificity, marked by inflammation (TNF‐RII), relaxation (PP13), and immune‐modulation (LFA1) receptors. PTD prediction over TD had an AUC of 0.97 (0.94–1.0), 84% sensitivity, and 90% specificity, marked by cell adhesion (ICAM), immune suppression, and general EV markers (CD81, CD82, and Alix). PE prediction over PTD had an AUC of 0.91 (0.79–0.99) with 80% sensitivity and 90% specificity with markers for complement activation (C1q) and autoimmunity markers.

Summary/Conclusion : The new, robust EV multi‐array analyser and methodology offer a simple, fast diagnostic tool that reveals novel surface markers for major obstetric syndromes.

Circulating tumour DNA and extracellular vesicle‐associated DNA as biomarkers for cancer detection: a systematic review and meta‐analysis

Tad Wu 1,2 , Kyle Dickinson 1 , Ian Schiller 1 , Thupten Tsering 1,2 , Yunxi Chen 1,2 , Amélie Nadeau 1,2 , Nandini Dendukuri 1 , Venkata Ramana Agnihotram 1 , Julia V. Burnier 1,2

1 Research Institute of the McGill University Health Centre, Montreal, Canada; 2 McGill University, Montreal, Canada

Introduction : Liquid biopsy is an emerging tool that allows detecting and monitoring tumours through analysing various analytes in biofluids like blood. An important component of liquid biopsy is circulating tumour (ct) DNA, which is a powerful biomarker and is already in use clinically. Recently, it has been shown that cancer cells can also release DNA via extracellular vesicles (EVs). This EV‐associated DNA (EV‐DNA) has therefore emerged as another promising biomarker for several tumour types. However, its clinical utility remains under investigation. In this meta‐analysis, we compared the sensitivity and specificity of EV‐DNA and ctDNA for detecting cancer‐related mutations.

Methods : Using our previously created extracellular vesicle‐associated DNA database (EV‐ADD), which compiles all published literature on EV‐DNA, we included studies that compared the sensitivity and specificity of detecting cancer‐specific mutations in EV‐DNA and ctDNA in human biofluids. Then, we systematically extracted relevant data from the included studies. The QUADAS‐2 tool was used for the quality assessment of the included studies. For each individual study, a Wilson score 95% confidence interval was computed for both the sensitivity and specificity of EV‐DNA and ctDNA. The reference standard was the presence of mutations in the patients’ tumour tissue. Summary estimates for sensitivity and specificity and 95% credible intervals were derived using a Bayesian bivariate model. The EV‐ADD metric was used to classify studies according to their compliance with MISEV guidelines. Statistical significance of the difference in sensitivity or specificity was examined using the posterior probability of the difference exceeding 0 being less than 2.5% or greater than 97.5%.

Results : After applying our inclusion criteria, we identified and analysed 11 studies involving 902 patients. Summary estimates showed a sensitivity of 62.4% (95% credible interval: 44.0%–78.5%) and specificity of 93.7% (95% credible interval: 88.6%–96.8%) for EV‐DNA, compared to 50.4% (95% credible interval: 32.5%–69.4%) and 88.4% (95% credible interval: 79.2%–94.9%) for ctDNA. Additionally, studies with higher EV‐ADD scores, reflecting adherence to MISEV guidelines, demonstrated higher sensitivity for both EV‐DNA and ctDNA.

Summary/Conclusion : This analysis underscores the potential of EV‐DNA as a complementary biomarker to ctDNA in cancer detection. Future research should focus on larger patient cohorts and expanding to other biofluids.

Exploring chemokine receptor‐positive extracellular vesicles as novel cancer biomarkers using label‐free surface plasmon resonance technology

Kaat Verleye, Sam Noppen, Julie Jacobs, Dominique Schols

Department of Microbiology, Immunology and Transplantation, Molecular Structural and Translational Virology Research Group, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

Introduction : Several chemokine receptors are often overexpressed in cancer cells, and this expression can be used as a disease biomarker. The overexpression also translates to an increased protein amount on extracellular vesicle (EV) surfaces, and thus the analysis of EVs can become an important tool in cancer diagnostics. Therefore, we developed a label‐free, real‐time binding assay to detect chemokine receptor‐positive EVs using surface plasmon resonance (SPR) with a focus on CXCR4, which is overexpressed in many different types of cancer. CXCR4 also plays an important role in the development of T‐cell acute lymphoblastic leukaemia (T‐ALL) and is also overexpressed on the membrane of T‐ALL cancer cells.

Methods : EVs from different human cancer cell lines (U87, MOLT4, Raji, THP‐1, MDA‐MB‐231, MCF‐7 and SK‐BR‐3) are isolated using size exclusion chromatography. The EVs are characterized using nanoparticle tracking analysis (NTA) and SPR. SPR is used to detect the EV biomarkers (CD9, CD63 and CD81) and to detect the expression of the chemokine receptor CXCR4. An in vivo mouse model for T‐ALL is set up using an IV injection of MOLT‐4/luc2 cells. This mouse model will be used to analyse the CXCR4 expression on plasma EVs by using SPR, fluorescent NTA, super‐resolution microscopy (SRM) and Western blot.

Results : SPR detected a difference in EV biomarkers (CD9, CD63, CD81) between the EVs isolated from the different in vitro cell types. SPR also detected the expression of CXCR4 on EVs from both endogenously expressing cells and CXCR4‐transfected cells. The overexpression of recombinant CXCR4 in U87 cells translated to an increased CXCR4 expression on the EVs. MOLT4 EVs showed the highest endogenous expression of CXCR4, and the parental cells were used in the in vivo T‐ALL model. SPR was able to detect CXCR4+ EVs in crude plasma from T‐ALL mice. The SPR results were confirmed by fluorescent NTA, western blot and SRM.

Summary/Conclusion : Our bioassay detected cancer cell‐derived EVs expressing a high amount of CXCR4. In addition, CXCR4 was detected endogenously on EVs from different cancer cell types and on EVs in plasma of a T‐ALL mouse model, indicating it could become an important novel EV biomarker for T‐ALL disease progression.

Hyaluronan coated‐extracellular vesicles (EVs): an emerging multifunctional platform for cancer biomarker identification

Presenter: Debashish Paul

Shiv Nadar Institution of Eminence, Dadri, Uttar Pradesh, India

Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed nanopouches, produced by all cells and abundant in various body fluids. These vesicles mediate the exchange of diverse biomolecules, including nucleic acids, proteins, carbohydrates, and metabolites, between parent and recipient cells.

Methods : This study employed high‐resolution atomic force microscopy (AFM) and spectroscopy (AFS) to explore differences between EVs from colon cancer cells and normal colon epithelial cells.

Results : Cancer‐derived EVs were more abundant in cell media. They exhibited a higher surface density of hyaluronan (HA), a carbohydrate polymer linked to tumour malignancy, highlighting their potential as colon cancer biomarkers. Contour length measurements using single‐molecule force spectroscopy (SMFS) revealed that these cancer EVs are decorated with low molecular weight HA (LMW‐HA < 200 kDa) and are intrinsically elastic, likely due to the HA and its associated water network on the EV surface.

Summary/Conclusion : In addition, AFM‐based techniques such as electrostatic force microscopy (EFM), scanning kelvin probe microscopy (SKPM), and conductive probe force microscopy (CPFM) were used to map the surface electrical properties of HA‐coated cancer EVs, showing promise for non‐invasive liquid biopsy screening.

Effects of cancer‐derived extracellular vesicles on the tumour microenvironment: the functional significance and potential use in non‐invasive cancer diagnostics

Presenter: Agnese Brokāne

Latvian Biomedical Research and Study Centre, Riga, Latvia

Introduction : The tumour microenvironment (TME) is a complex network of cancer, stromal, and immune cells, as well as extracellular matrix components, that collectively influence cancer progression. Extracellular vesicles (EVs) mediate intercellular communication within the TME, promoting tumour growth by modulating recipient cell functions. We hypothesized that these effects could be detected in vitro and serve as functional biomarkers in precision medicine. Here, we examine the impact of cancer‐derived EVs on TME cell gene expression profiles, aiming to identify cancer‐associated changes and their implications for non‐invasive cancer diagnostics.

Methods : SW620 CRC cells were cultured in a hollow fibre bioreactor; EVs were harvested daily and isolated by SEC. EVs were also isolated from the plasma of CRC patients and healthy donors (HD), with informed consent obtained, followed by their characterization. Recipient cells, including fibroblasts (HS68, WI38), monocytes (U937, THP1), and CRC cells (SW480), were treated with SW620 EVs and plasma EVs from CRC patients and HD. EV uptake was assessed via microscopy and flow cytometry, and RNA sequencing followed by qRT‐PCR was used to assess gene expression changes.

Results : HS68 cells showed the strongest transcriptional response to SW620 EVs, with 247 genes upregulated and 174 downregulated in the EV treated cells, impacting pathways related to metabolism, proliferation, and extracellular matrix remodelling. qRT‐PCR validation of genes MMP10, MMP11, RGS9, and STC1 corroborated their increased expression in SW620 EV treated cells and their expression levels in HS68 cells were EV dose dependent. Furthermore, high expression of each gene in tumour tissues also correlated with poor CRC patient survival. Next, a total of nine candidate genes were selected for validation in an independent patient cohort based on their upregulation in cells treated with cancer patient plasma EVs versus HD EVs and controls, and their association with a cancer‐associated fibroblast phenotype. Most of the genes were induced by both CRC and HD EVs, while RACGAP1 showed significantly stronger induction by CRC than HD EVs.

Summary/Conclusion : This study shows proof of principle for detecting cancer‐derived EV‐induced changes in gene expression in vitro, thus paving a way for the development of novel functional biomarkers for CRC detection and prognosis.

Funding : This study was supported by Project No. VPP‐EM‐BIOMEDICINA‐2022/1‐0001.

Multiparametric and integrated characterization of plasma‐derived extracellular vesicles: a highly sensitive liquid biopsy platform for cancer

Ghazal Narimanfar 1 , Filippo Maltoni 1 , Dorian Forte 1 , Roberto Maria Pellegrino 2 , Simone Tiberi 1 , Martina Barone 3 , Filippo Branzanti 3 , Hillary Catellani 4 , Alessia Tieghi 4 , Carla Emiliani 2 , Francesca Palandri 3 , Lucia Catani 1,3

1 University of Bologna, Bologna, Italy; 2 University of Perugia, Perugia, Italy; 3 IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Bologna, Italy, 4 Azienda Unitа Sanitaria Locale‐IRCCS di Reggio Emilia, Reggio Emilia, Italy

Introduction : The inflammatory microenvironment supports the malignant clone in myeloproliferative neoplasms (MPNs) like polycythemia vera (PV) and essential thrombocythemia (ET), making it critical to explore therapeutic strategies. Extracellular vesicles (EVs), carrying proteins, lipids, and nucleic acids, play a role in intercellular signals and also in the regulation of immunity and inflammation. This study aims to provide an integrated and sensitive liquid biopsy platform of circulating EVs, offering new insights into MPN pathology and potential therapeutic targets.

Methods : We combined lipidomic (liquid chromatography/mass spectrometry), transcriptomic, and surface protein profiling (flow cytometry) to analyse EVs isolated from plasma of PV ( n = 10) and ET ( n = 10) patients and sex/age‐matched healthy donors (HD = 10) by size‐exclusion chromatography.

Results : Comparing the mRNA cargo of PV and HD EVs, we found the overexpression of haemostasis/coagulation and platelet activation (GPIBB, ITGA2B, GP1BA, CD226), inflammation (LCN2) and tumour‐associated (MYL9, MYBL2, CMTM5, TSC22D1, LCN2) transcripts. We also report how the transcriptome of circulating EVs captures PV‐specific features: NF‐E2, GFI1B, TIMP1, all overexpressed, play a role in erythrocytosis, development and maturation of erythrocytes and megakaryocytes, and bone marrow fibrosis, respectively. When we compared the mRNA cargo of ET and HD EVs, we found that most of the transcripts mirrored the PV counterparts, and no significant differences were observed between the transcriptomes of ET and PV EVs. Consistently, surface protein profiling revealed significant enrichment of immune/inflammatory (CD8, CD29), EV (CD9, CD63, CD81), and platelet‐associated markers (CD41a, CD42b, CD62P) in PV EVs versus those from HD. Elevated expression of CD9, CD29, and CD63 was also observed in ET‐EVs. Lipid class analysis revealed that PV EVs were significantly enriched in ether phospholipids (plasmenyl‐linked phosphatidylcholine (PC)‐P and ether‐linked PC‐O) in comparison with the HD counterparts. PC‐P typically signifies a protective or adaptive response to oxidative stress, inflammation, or metabolic demands. Conversely, no significant differences were observed between ET and HD EVs in lipid‐class cargo.

Summary/Conclusion : Our work demonstrates that circulating EVs in MPN patients are enriched for tumour‐ and biology‐related molecules with the potential to contribute to the inflammatory microenvironment and proves the feasibility of comprehensive circulating EV profiling to study MPN.

High concentrations of peripheral blood leukocyte‐derived extracellular vesicles predict favourable clinical outcomes in pancreatic cancer patients

Presenter: Domenico De Bellis

University “G. d'Annunzio” of Chieti‐Pescara, Chieti‐Pescara, Italy

Introduction : Pancreatic cancer (PC) is one of the most aggressive malignancies, ranking among the leading causes of cancer‐related mortality globally. Despite advances in understanding tumour biology, PC's asymptomatic progression and poor response to therapy result in limited survival outcomes. Therefore, identifying robust biomarkers is crucial to improving diagnostic accuracy, prognostication, and therapeutic management. Leukocyte‐derived extracellular vesicles (LEVs), which mediate intercellular communication, have shown potential as indicators of immune response and disease progression. This study investigates whether circulating EVs derived from leukocytes serve as predictive biomarkers for clinical outcomes in PC patients, potentially guiding personalized treatment approaches.

Methods : This prospective study included 56 patients with histologically confirmed PC and 48 age‐ and sex‐matched healthy controls. Blood samples were collected, and a patented, advanced flow cytometry protocol was applied to quantify leukocyte‐derived (CD45+) and immune checkpoint molecule‐expressing PD‐L1+ EVs. The relationship between EVs levels and clinical outcomes, including overall survival (OS), progression‐free survival (PFS), and disease control, was evaluated, focusing particularly on patients with advanced and unresectable disease undergoing chemotherapy.

Results : Analysis revealed that PC patients exhibited significantly elevated levels of total EVs, CD45+ EVs, and PD‐L1+ EVs compared to healthy controls ( p < 0.05). Elevated baseline levels of CD45+ EVs were strongly associated with favourable survival outcomes, independently predicting improved OS (HR = 0.17; p = 0.02) and PFS in advanced PC cases. Notably, patients whose CD45+ EV levels remained stable or decreased during chemotherapy achieved higher rates of disease control, whereas increases in EV levels were indicative of progressive disease. These findings highlight the dynamic role of leukocyte‐derived EVs in monitoring therapeutic efficacy and disease progression.

Summary/Conclusion : The study underscores the clinical utility of blood‐derived CD45+ EVs as a novel, non‐invasive biomarker for prognostic and predictive purposes in pancreatic cancer. By providing real‐time insights into immune status and treatment response, leukocyte‐derived EVs could enhance decision‐making in clinical oncology, supporting tailored treatment regimens for patients with PC. This investigation opens avenues for further research on EV‐based liquid biopsies.

Novel plasma‐derived extracellular vesicle biomarkers for treatment response in small cell lung cancer identified by proximity extension assay

Shona Pedersen 1 , Hubert Krzyslak 2 , Weronika M. Szejniuk 3,4,5 , Ursula G. Falkmer 3,4,5 , Ahmad Hassanein 1 , Søren R. Kristensen 2,5

1 College of Medicine, QU Health, Qatar University, Doha, Qatar, 2 Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark; 3 Department of Oncology, Aalborg University Hospital, Aalborg, Denmark; 4 Clinical Cancer Research Centre, Aalborg University Hospital, Aalborg, Denmark; 5 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark

Introduction : Small cell lung cancer (SCLC) is characterized by its aggressive nature and poor prognosis, often due to late diagnosis and chemo‐resistant recurrence. This study aims to identify novel therapeutic protein markers associated with immuno‐oncological processes in circulating blood extracellular vesicles (EVs) to improve SCLC management and patient outcomes.

Methods : Plasma samples were collected from 50 SCLC patients before and after two cycles of chemotherapy between February 2022 and May 2024. Plasma‐derived large EVs (PLEVs) were isolated using double high‐speed centrifugation and confirmed by western blotting according to the MISEV 2023 guidelines. The Olink PEA Immuno‐Oncology panel was used to analyse 92 proteins in PLEV lysates. Statistical analyses and functional enrichment studies were performed to identify biomarkers of treatment response and associated biological processes.

Results : Three predictive models (OPLS‐DA, random forest, and XGBoost) were employed to assess changes in protein levels. The OPLS‐DA model identified five proteins with optimal performance, achieving 100% accuracy in the original data and 89.2% accuracy in cross‐validation. Functional enrichment analysis revealed that impaired complement activation, altered extracellular matrix organization, and integrin‐mediated signalling may contribute to understanding the clinical implications for SCLC treatment guidance.

Summary/Conclusion : This pioneering study demonstrates the potential of circulating EVs as a promising source of novel protein biomarkers associated with SCLC pathogenesis and remission. The identified proteins and biological processes may have significant clinical implications for guiding chemotherapy treatment in SCLC patients, potentially improving management strategies and patient outcomes.

Funding : This study was supported by the Qatar University and Aalborg University.

Digital profiling of single microglia extracellular vesicle phenotypes for understanding the innate immune system and monitoring brain cancer

Presenter: Xueming Niu

The University of Queensland, Brisbane, Australia

Introduction : An abnormal inflammatory microenvironment is considered a hallmark of high‐grade brain cancer. Microglia are the predominant resident immune cell population in the brain and play a key role in regulating tumour progression and response to treatment in patients. Extracellular vesicles (EVs) derived from microglia carry molecular signals that reflect the inflammatory state of the brain. In our study, we developed a single‐molecule resolution nanotechnology platform to capture microglia‐EVs from the blood of glioblastoma (GBM) patients and profile alterations in the phenotype of microglia during therapy and at recurrence.

Methods : We developed a digital platform that capitalizes on a gold‐topped silicon nanopillar array integrated with superplasmonic Au–Ag alloy nanoboxes for multiplex readouts. Microglia EVs were captured on the nanopillar and detected by surface‐enhanced Raman scattering (SERS) nanoboxes targeting an inflammatory marker panel. Counting EV‐activated nanopillars generated a digital readout of single EV molecular profiles. The inflammatory biomarker panel was first validated on the platform using EVs derived from pro‐ and anti‐inflammatory murine and human microglia cells. Subsequently, we applied this platform to profile EVs in a preclinical mouse model and in GBM patients to detect tumour recurrence and track therapeutic response.

Results : Based on our in vitro experiments, we demonstrated that the nanopillar chip was able to capture single microglia‐derived EVs and analyse the pro‐ or anti‐inflammatory phenotypes of microglia EVs. As a result of clinical samples, our platform also enabled longitudinal monitoring of changes in the brain's inflammatory microenvironment. We found that the upregulation of inflammatory markers in microglia EVs, which indicated more significant immune responses, correlated with therapeutic resistance and the possibility of tumour recurrence. These findings highlight our platform's potential to provide real‐time monitoring of GBM‐related inflammatory microenvironmental changes through profiling microglia‐EVs.

Summary/Conclusion : Microglia‐EVs offer promising insights into the GBM microenvironment, helping us understand tumour progression. Our nanopillar platform provides a real‐time and precise strategy for monitoring tumour evolution by analysing EVs at single‐molecule resolution. This platform has the potential to become a tool in personalized patient management, allowing for the optimization of therapeutic strategies that enhance treatment outcomes.

Identification of predictive miRNA biomarkers from neonatal CSF extracellular vesicles in post‐haemorrhagic ventricular dilatation via machine learning approaches

Priska Bauerstätter 1 , Gabriel A. Vignolle 1 , Silvia Schönthaler 1 , Lucia Ciglar 1 , Christa Nöhammer 1 , Katharina Goeral 2 , Klemens Vierlinger 1

1 Center for Health & Bioresources, Competence Unit Molecular Diagnostics, AIT Austrian Institute of Technology GmbH, Vienna, Austria; 2 Division of Neonatology, Department of Pediatrics and Adolescent Medicine, Medical University of Vienna—Comprehensive Center for Pediatrics, Intensive Care and Neuropediatrics, Vienna, Austria Priska Bauerstätter and Gabriel A. Vignolle are co‐first authors and contributed equally .

Introduction : Neonatal post‐haemorrhagic ventricular dilatation (PHVD), a serious complication of intraventricular haemorrhage (IVH), is linked to significant neurodevelopmental impairments. Early biomarkers are critical for timely prognosis and intervention. At the onset of PHVD cerebrospinal fluid (CSF) is drained to reduce the intracranial pressure; we aim to use this abundantly available biofluid to identify miRNA biomarkers from isolated extracellular vesicles (EVs).

Methods : We isolated EVs from 190 CSF samples from 43 neonates diagnosed with PHVD. High‐throughput sequencing was used to profile EV‐associated miRNAs, generating a novel dataset of miRNA signatures. Machine learning (ML) and artificial intelligence (AI) algorithms were applied to analyse these miRNA profiles and predict neurodevelopmental outcomes at 2 and 3 years of age.

Results : We were able to detect 248 miRNAs consistently in 80% of the samples. Our ML models identified key miRNAs with strong predictive power for cognitive and motor outcomes. These miRNAs showed potential as early, minimally invasive biomarkers for neurodevelopmental prognosis. The identified biomarkers provided insights into the pathophysiology of PHVD and may inform the development of targeted therapies.

Summary/Conclusion : This study highlights the potential of EV‐associated miRNAs as predictive biomarkers for neurodevelopmental outcomes in neonates with PHVD. Further validation in larger cohorts could establish these miRNAs as valuable clinical tools for early diagnosis and prognosis, offering new avenues for mitigating long‐term neurodevelopmental deficits.

Funding : This research was funded by the Vienna Science and Technology Fund/WWTF: LS20‐030 PIMIENTO—PrecIsion Medicine in IntravENTricular haemorrhage for Outcome prediction.

Unravelling the significance of exosomal immune checkpoint protein PVR/CD155 in lung carcinoma progression

Presenter: Maureen Cambier

Molecular Angiogenesis Laboratory, Leuven, Belgium

Introduction : Immunotherapy has revolutionized cancer treatment, but understanding the resistance to this treatment is crucial for improving outcomes. In this study, we investigate the role of extracellular vesicles (EVs) in immune escape through immune checkpoint protein (ICP) expression, focusing on the PVR/CD155 axis. We developed a new methodology that identifies several ICPs on the surface of EVs in single‐liquid biopsies.

Methods : We analysed the expression of several ICPs identified through literature screening and TCGA data analysis. Plasma samples from healthy individuals ( n = 24) and cancer patients ( n = 25) undergoing immunotherapy were collected. EVs were isolated and characterized using differential ultracentrifugation. A longitudinal study with responders ( n = 3) and non‐responders ( n = 3) was also performed before treatment and after 3 and 6 months. ICPs on EV surfaces were identified via MAGPIX (Luminex) analysis. Knockdowns were generated with siRNA and CRISPR‐Cas9 and studied with functional assays.

Results : Analysis of TCGA data revealed high levels of PVR/CD155 in lung cancer. EV‐associated PVR/CD155 levels were elevated in patient samples. Furthermore, its expression was even more abundant during immunotherapy in non‐responders, suggesting an emergence of resistance. We studied ICP expression in several human and mouse models and discovered an enrichment of PVR in EVs. Knockdown of PVR/CD155 cells presented reduced migration and proliferation rates in human and mouse models.

Summary/Conclusion : Our study introduces a novel method for characterizing circulating ICPs‐EVs in lung cancer patients, with implications for patient monitoring and therapeutic target discovery. Further investigation of PVR/CD155+EV in coculture systems with immune cells will deepen understanding of its role in immunotherapy resistance.

Funding : This work is supported by ULiège, the Léon Fredericq Fundation, and the Walloon Region.

GCC2‐containing small extracellular vesicles as a biomarker for early‐stage lung adenocarcinoma diagnosis and prognosis: a multicentre retrospective study

Presenter: Byeong Hyeon Choi

Korea University, Republic of Korea

Introduction : Early diagnosis and accurate prognosis are crucial for initiating effective therapeutic interventions and improving lung adenocarcinoma survival rates. Emerging evidence suggests that tumour‐derived small extracellular vesicle (sEV) cargo could serve as a cancer‐specific biomarker. A previous study identified GRIP and coiled‐coil domain‐containing 2 (GCC2)‐enriched sEV (sEV‐GCC2) as a promising diagnostic biomarker for lung adenocarcinoma. However, the diagnostic, predictive, and tumourigenic roles of sEV‐GCC2 remain unclear. This retrospective multicentre study aimed to evaluate and validate the diagnostic and prognostic values and tumourigenicity of sEV‐GCC2 in early‐stage lung adenocarcinoma.

Methods : We evaluated plasma samples from surgically treated patients with lung adenocarcinoma and controls, which were retrospectively obtained from five institutions. sEVs were isolated using size‐exclusion chromatography, and sEV‐GCC2 was quantified using enzyme‐linked immunosorbent assays. Immunohistochemical staining (IHC) was performed to validate the expression of GCC2 in tumour sites. Kaplan–Meier and Cox regression analyses were performed for survival analyses. GCC2 knockdown cell lines were used to assess the implications of GCC2 in sEVs in vitro and in vivo for lung cancer progression and lymph node metastasis.

Results : Four hundred seventy plasma samples (150 controls, 320 patients) were analysed. The mean follow‐up duration was 34.7 ± 24.0 months. Patients had significantly higher sEV‐GCC2 levels than controls, with an area under the curve (AUC) of 0.856 (95% confidence interval [CI], 0.820–0.886; p < 0.0001). For patients with TisN0–T1miN0 disease vs. controls, the AUC was 0.802 (95% CI, 0.734–0.859). Multivariate analysis showed significant associations between sEV‐GCC2 concentration and pathological TNM stages and tumour location in the left lower lobe. IHC revealed significantly higher GCC2 expression in lung adenocarcinoma tissues than in controls ( p < 0.001). Higher sEV‐GCC2 concentrations were linked to greater recurrence rates and lower recurrence‐free survival, even in stages 0–IA1. Additionally, sEV‐GCC2 increased cancer cell proliferation, tumour growth, and lymph node metastasis, but not GCC2‐deficient sEV.

Summary/Conclusion : We verified sEV‐GCC2 as a diagnostic and prognostic biomarker of early‐stage lung adenocarcinoma. sEV‐GCC2 could be clinically significant for better pathological evaluation and a key molecule in lung adenocarcinoma progression.

Funding : This study was supported by the Korea Health Industry Development Institute: RS‐2024‐00436472. Ministry of Science and ICT: RS‐2024‐00459242. Korea Medical Device Development Fund: 1711138151, KMDF_PR_20200901_0094_02.

Natural killer cell‐derived extracellular vesicles contain meta‐clusters of cytotoxicity‐ and immunoregulatory‐related proteins, highlighting their potential as cancer immunotherapeutics

Frederic St‐Denis‐Bissonnette 1,2 , Melanie Kirkby 1,2 , Emily Fekete 1 , Gauri Muradia 1 , Sarah Cummings 1 , Angela Wang 1 , Dylan Burger 2,4 , Xu Zhang 1,2 , Lisheng Wang 2,4 , Jessie R. Lavoie 1,2

1 Health Canada, Ottawa, Canada; 2 University of Ottawa, Ottawa, Canada; 3 Ottawa Hospital Research Institute, Ottawa, Canada

Introduction : Natural killer cells have emerged as critical players in anti‐tumour immunity, with their extracellular vesicles (NK‐EVs) directly contributing to this activity. NK‐EVs are rich in bioactive molecules, including various proteins potentially involved in their potent anti‐tumour and immunomodulatory properties. However, NK‐EVs’ mechanism of action (MoA) in cancer immunity remains unclear. A deep understanding of their proteomic composition could enhance therapeutic insights and inform the development of more effective treatments. This study aims to elucidate NK‐EVs’ MoA in the context of triple‐negative breast cancer (TNBC), using a proteomic‐based approach.

Methods : Clinical‐grade NK‐EVs were produced from the NK92‐MI cell line using a biomanufacturing workflow following the MISEV guidelines (published in JEV in 2023). The NK‐EVs' proteome was assessed using bottom‐up mass spectrometry. In silico proteomic analyses results of identified key proteins in cancer immunity were validated through western blotting and various bioassays targeting cell death pathways. Additionally, the functional role of the unexplored NK‐EV corona (extraluminal surface moiety) was investigated.

Results : In silico analysis identified several protein clusters from NK‐EVs involving apoptosis, immunomodulation, and various cell signalling pathways. A prominent meta‐cluster related to cytotoxicity was uncovered, and functional bioassays confirmed that NK‐EV treatment of TNBC cells leads to rapid activation of apoptosis, necrosis, and caspase‐3/7/8/9 pathways within min. Notably, disruption of the NK‐EV corona by proteinase treatment, as evidenced by a substantial decrease in protein‐to‐particle ratio, significantly impaired NK‐EV‐induced cytotoxicity against TNBC cells ( p < 0.05), highlighting an important role of the EV protein corona in the bioactivity of NK‐EVs.

Summary/Conclusion : This study provides a robust characterization of clinical‐grade NK‐EVs’ proteome, deciphering their protein composition and ability to induce rapid cancer cell death through apoptosis and necrosis pathways. This is the first study to demonstrate that the integrity of the NK‐EVs’ protein corona is critical for their cytotoxic function, as its disruption markedly reduced the NK‐EVs’ bioactivity against cancer cells. These findings offer valuable insights into NK‐EVs' MoA and support their potential as a novel therapeutic for TNBC and other hard‐to‐treat cancers.

miRNA profiling of marine fish olive flounder ( Paralichthys olivaceus ) Plasma derived exosomes in response to viral haemorrhagic septicaemia virus

Chamilani Nikapitiya, E. H. T. Thulshan Jayathilaka, Mawalle Kankanamge Hasitha Madhawa Dias, Hyun‐Jin Shin, and Mahanama De Zoysa

College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea

Introduction : EVs have been divided into medium/large EVs (>200 nm) and small EVs (< 200 nm) by the minimal information for studies of extracellular vehicles (EVs) 2018/2023 guidelines. World Organization for Animal Health (OIE) has listed viral haemorrhagic septicaemia (VHS) as an important contagious viral disease that influences wild or farmed fish worldwide. The etiological agent of VHS is the viral hemorrhagic septicemia virus (VHSV). Understanding the molecular mechanisms involved in VHSV infection is important for identifying new therapeutic targets against the infection. This study aimed to characterize (physicochemical and molecular level) the plasma‐derived exosomes from olive flounder (Paralichthys olivaceus) upon VHSV challenge and phosphate buffered saline (PBS) injection (VHSV‐Exo and PBS‐Exo, respectively) to find the VHSV effect on the exosomes content.

Methods : The exosomes were isolated using the differential ultracentrifugation method and characterized from PBS injected and VHSV challenged olive flounder plasma. Total RNA was isolated, small RNA sequencing libraries were constructed and libraries were sequenced. To profile the miRNAs expression patterns of VHSV‐Exo and PBS‐Exo, first the expression levels of miRNAs were normalized and then differentially expressed (DE) miRNAs were analysed using DESeq2. DE miRNAs were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses for functional and pathway enrichment analysis.

Results : Nanoparticle tracking analysis revealed a lower particle size and higher concentration of VHSV‐Exo compared to the PBS‐Exo group. Both comprised exosome markers (CD9, CD81, and CD63). We identified 48 known miRNAs and the highly abundant miRNA in both groups was pol‐miR‐21‐5p/hhi‐miR‐21. Among 48, one miRNA (hhi‐miR‐1788) was specific to the VHSV‐Exo group. Furthermore, six miRNAs were upregulated while seven were downregulated (fold change > 1.2 or < 0.83; q value < 0.05). Enrichment analysis indicated that VHSV mainly regulates transcription, cell‐cell adhesion, cell‐matrix adhesion, and cytokine‐mediated signalling pathways.

Summary/Conclusion : Our findings indicated that VHSV infection influenced the miRNA expression profile, and identified DE miRNAs could be considered for the diagnosis of VHSV infection in fish. Further validation of these miRNAs assist in developing novel engineered therapeutics together with exosomes as drug delivery carriers in the future.

Funding : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) 2021R1A2C1004431 and RS‐2024‐00347938.

Extracellular vesicles carry diagnostic and prognostic capabilities in liver cancer patients undergoing hepatectomy

Ulrike Resch 1,2 , Luisa M. Schmidt 2 , Hubert Hackl 3 , Silvio Kau‐Strebinger 4 , David Pereyra 5 , Jonas Santol 5,6 ,Marcus Krueger 2 , Patrick Starlinger 5,6 , Alice Assinger 1

1 Department of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria; 2 CECAD Cluster of Excellence for Ageing Research, Institute for Genetics, University of Cologne, Cologne, Germany; 3 Institute of Bioinformatics, Biocenter, Medical University of Innsbruck, Innsbruck, Austria; 4 Department of Biomedical Sciences and Pathobiology, Center of Pathobiology, Institute of Morphology, Vetmeduni, Vienna, Austria; 5 Division of Visceral Surgery, Department of General Surgery, Medical University of Vienna, Vienna, Austria; 6 Division of Hepatobiliary and Pancreas Surgery, Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA

Introduction : Post‐hepatectomy liver failure (PHLF) remains a significant risk for patients undergoing partial hepatectomy (PHx). Reliable prognostic markers and treatments to enhance liver regeneration are lacking. Plasma extracellular vesicles (EVs) can reflect systemic and tissue‐wide proteostasis and stress, potentially aiding liver regeneration. However, their role in PHLF is still unknown. Therefore, we employed an unbiased longitudinal proteomic analysis of 18k and 100k EV subpopulations and EV depleted plasma to identify etiologic EV‐enclosed factors promoting liver regeneration.

Methods : The study included a subset of 14 HCC patients submitted to PHx (ClincalTrials.gov, NCT01921985 , NCT01700231 ) with 7 cases with PHLF and 7 healthy controls (noPHLF). CTAD‐plasma was collected before (PRE) and after PHx (POD1, POD5) and centrifuged at 18k and 100k to purify EV‐subpopulations. EV‐size, numbers, shape and cellular origin (CD31, CD45, CD61, CD235, ANXA5/calcein) were examined using NTA, TEM and FACS. EVs and EV‐depleted plasma were processed for mass spectrometry following the SP3‐on‐beads digestion protocol and analysed using an EvoSep One‐60SPD‐timsTOF‐pro 2 setting operating in dia‐PASEF mode.

Results : EV‐quantities in plasma were reduced after PHx, but the proportion of apoptotic particles appeared increased in the PHLF cohort. Up to three‐quarters of EVs were CD61‐positive, followed by CD45‐positive and minor fractions of CD31 and CD235. TEM analysis indicated increased heterogeneity in 18k as compared to 100k EVs, while aggregates were comparably present. In MS‐analyses we identified 1062 proteins (1030, 806, and 785 proteins in 18k EVs, 100k EVs and depleted plasma) with 682/64% proteins present in all sample types and 159/15% detected in 18k EVs only. Sampling‐time independent comparisons of PHLF versus noPHLF cohorts revealed that 18k EVs discriminate PHLF from noPHLF to a much better extent than 100k EV or EV depleted plasma with minor overlaps in significantly different proteins. The detailed analysis of sampling time dependent differences in patient cohorts in 18k and 100, as well as EV depleted plasma, will be presented.

Summary/Conclusion : The study highlights significant differences in the protein composition of EVs in regard to EV‐isolation procedures, patients with PHLF or without PHLF and sampling time. between PHLF patients and those without PHLF. allow for the development of new prognostic markers and therapies.

The innovative targeting ligand production method using cancer sEVs for the personalized therapy

Presenter: Maryam Firouzi

OUHSC, Oklahoma City, Oklahoma, USA

Introduction : Despite advancements in tumour‐targeting agents, meta‐analyses reveal that less than 15% effectively reach tumours, leading to high off‐target side effects. This highlights the need for new tumour‐targeting ligands to improve efficacy. We developed an innovative method for producing tumour‐targeting antibodies using cancer‐derived small extracellular vesicles (sEVs). Since sEVs retain their parent cells' lipid bilayer structures and functions, antibodies against these vesicles can effectively target the originating cancer cells. This approach could yield antibody‐decorated targeting systems with enhanced tumour‐targeting efficiency, potentially overcoming limitations.

Methods : The sEVs from ovarian cancer cells (Ovcar‐8) were isolated and injected into mice. Once the specificity of the plasma collected for the Ovcar‐8 cells was confirmed, hybridoma cells were generated for antibody production. Among various monoclonal antibodies (mAbs) produced by different hybridoma cells, the highest Ovcar‐8 targeted mAb was isolated and purified. The selected mAb was decorated to the Paclitaxel‐encapsulated CD8+ T cell exosome (AB‐TSEV/P) surfaces in the next step. Then, the formulation was injected into the Ovcar‐8‐bearing mice, and the changes in tumour volume and body weight were measured for 80 days. An in vivo biodistribution study was conducted using IR‐780 dye‐loaded AB‐TSEV (AB‐TSEV/IR).

Results : The size of Ovcar‐8‐derived sEVs and CD8+ T cell sEVs was 150–200 nm. The plasma from mice after injection of sEVs showed specificity to Ovcar‐8 cells through multifold cell killing efficacy over non‐mother cancer and non‐cancer cells. Based on our results from immunocytochemistry analysis, the selected monoclonal antibody had a significantly higher affinity to Ovcar‐8 cells than non‐cancerous cells. mAb‐decorated formulation (AB‐TSEV/P) significantly decreased tumour growth compared to formulations without mAb. Also, an in vivo biodistribution study showed that the mAb‐decorated formulation preferred a higher accumulation in the tumour compared to the control.

Summary/Conclusion : We developed a new strategy for antibody production using cancer cell sEVs. This antibody could target their parent cells. Thus, the targeted formulation decorated with this antibody will be able to significantly arrive at the tumour, overcoming the current suboptimal dose of cancer therapy. Although we only used ovarian cancer sEVs for this study, considering the potential of this approach, this technology can be applied to different cancers and other diseases.

Subtopic Cancer biomarkers 2

Immunoregulatory role of extracellular vesicles in advanced non‐small cell lung cancer

Patrizia Ghidotti 1 , Claudia Proto 2 , Marta Brambilla 2 , Francesca Pontis 1 , Mattia Boeri 1 , Benedetta Bussolati 3 , Cristina Grange 3 , Daniela Boselli 4 , Annalisa Radeghieri 5 , Miriam Romano 5 , Rossella Crescitelli 6 , Gabriella Sozzi 1 , Elena Jachetti 7 , Orazio Fortunato 1

1 Epigenomics and Biomarkers of Solid Tumors Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2 Thoracic Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 3 Department of Medical Sciences, University of Torino, Turin, Italy; 4 FRACTAL Facility, San Raffaele Hospital, Milan, Italy; 5 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; 6 Sahlgrenska Center for Cancer Research, University of Gothenburg, Gothenburg, Sweden; 7 Molecular Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Introduction : Tumour PD‐L1 is the only predictive biomarker used in clinical practice for the use of immune checkpoint inhibitors (ICIs) in NSCLC. Extracellular vesicles (EVs) are membrane‐limited particles described as biomarkers for cancer progression and as modulators of anticancer immune response. In this study, we aimed to evaluate EVs of advanced NSCLC patients with low PD‐L1 expression to find biomarkers for combination therapy (chemotherapy plus ICIs) and to investigate their role in the anti‐tumour immune response.

Methods : Plasma EVs were isolated using ultracentrifuge and sucrose density gradient from responder (R), stable disease (SD) and non‐responder (NR) advanced NSCLC patients ( n = 40) with PDL1 < 50%, treated with combination therapy. EV characterization was performed following MISEV guidelines. Tetraspanin expression was assessed via super resolution microscopy and nano‐scale flow cytometry. miRNA cargo was evaluated by using the miRCURY LNA miRNA Panel and digital PCR. Proteomic analysis on EVs was done by Tymora Analytical. EV subpopulations were sorted through FACS or immune‐capture beads.

Results : Super‐resolution microscopy revealed a significant increase in CD9+ EVs in NR samples. Instead, in nano‐scale flow cytometry analysis, R‐EVs resulted enriched for CD81+EpCAM+ and CD63+EpCAM+ events. We found 6 miRNAs differentially represented between the three groups of patients. Among them, miR‐142‐3p was the most expressed in R patients both at baseline and after the start of the treatment. Via sucrose density gradient, we confirmed that this miRNA was mainly detected in fractions containing small‐EVs. Moreover, miR‐142‐3p levels were modulated during T cells activation. The transfection of T cells with a miR‐142‐3p mimic slightly increased IFN‐γ production in both CD4 and CD8 T cells. Proteomic analysis found 13 proteins up‐regulated in R‐EVs and another 13 proteins in NR‐EVs at baseline. Instead, 113 proteins changed after combination therapy. We successfully isolated EV subpopulations based on CD9 and CD45 expression.

Summary/Conclusion : Our preliminary results showed that R‐, SD‐ and NR‐EVs differed in terms of tetraspanins, miRNAs and protein content, suggesting a potential use as biomarkers for therapy response. Further studies are needed to elucidate the role of EV subpopulations in immune response modulation in lung cancer.

Funding : The study was supported by grants from the Italian Ministry of Health (GR‐2019‐12369047).

Subtopic EV clinical trials 2

First‐in‐man clinical pilot study showing the safety and tolerability of intravenous injection of ER2001 (a self‐assembled HTT‐specific siRNA) in patients with early manifest HD

Presenter: Lishan Lin

Sun Yat‐Sen University, Guangzhou, People's Republic of China

Introduction : ER2001 is a genetic circuit (plasmid) encoding both a neuronee‐targeting rabies virus glycoprotein (RVG) tag and an HTT siRNA. This circuit is able to reprogram liver cells to transcribe and self‐assemble HTT siRNA into RVG‐tagged exosomes after intravenous administration. The RVG‐guided HTT siRNA is further delivered through the exosome‐circulating system to the cortex and striatum. The study is to evaluate the safety and tolerability of ER2001 in patients with early manifest HD, including the pharmacokinetics (PK) of ER2001 in plasma and the exposure of ER2001 in cerebrospinal fluid (CSF).

Methods : This open label, single centre pilot study evaluates a single dose of ER2001 followed by multiple doses in eligible patients with early manifest HD. The primary endpoints were incidence of adverse events and abnormal clinical Laboratory results. Participants received a total of 8 intravenous injections of ER2001, and the treatment period of 14 weeks was followed by an observation phase of 84 days after the last injection.

Results : A total of 10 HD patients, including 5 males and 5 females, aged between 27 and 51 years, were enrolled in this study. Up to now, there were a total of 27 adverse effects, and none of the AEs was related to ER2001. A significant reduction in plasma mHTT from baseline was detected in HD patients. The PK, PD, and clinical outcomes will be updated once available.

Summary/Conclusion : Intravenous injection of ER2001 is safe and feasible in patients with early manifest HD, thus warranting further evaluation in randomized clinical trials.

Early‐stage lung cancer liquid biopsy based on automated EV‐SERS‐AI system: a retrospective and prospective multi‐ethnic clinical trial

Presenter: On Shim

EXoPERT, Seoul, Republic of Korea

Introduction : Lung cancer, with one of the highest mortality rates among cancers, remains an area where liquid biopsy techniques are underutilized. While extracellular vesicles; EVs in blood have distinctive features across various tumours, including lung cancer, utilizing these features for diagnosis poses challenges due to biomarker variability and tumour heterogeneity. Here, we present a lung cancer liquid biopsy system based on AI analysis of Raman spectroscopic signals of plasma EVs, validated through a clinical trial using prospective and retrospective multi‐ethnic samples.

Methods : EVs were isolated from plasma using the dual‐size exclusion chromatography (SEC) method. Raman signals were detected through the surface‐enhanced Raman spectroscopy (SERS) technique. To reflect the heterogenicity of EVs, signals were detected at 100 different spots per sample. The EV isolation and detection were implemented using our automated isolation and detection equipment and rigorously performed through an external lab. Blood samples were retrospectively and prospectively collected from 11 institutions in Korea and 4 institutions in the United States. Our CNN‐based algorithm has three parts: EV signal filtering, cancer signal classification, and lung cancer progression score refinement. The final cancer score integrates the results from the 100 signals. To train and validate the algorithm, we used over 690k SERS signals from 1853 Korean subjects and 244 subjects from the US clinical study. Finally, we validated the clinical performance.

Results : The algorithm prediction result of our 135 hold‐out test set achieved a sensitivity of 75% and a specificity of 95%. The sensitivity by cancer stage reaches 69% and 82% for early‐stage and advanced stage, respectively. The false‐positive rate for other cancer types—prostate, ovarian, and liver cancer, is 20%.

Summary/Conclusion : We implemented and validated a novel liquid biopsy system for lung cancer, encompassing a series of processes from EV isolation to signal detection. We evaluated clinical performance on multi‐ethnic samples, demonstrating high predictive accuracy for early lung cancer detection. These results highlight the potential for this system as a new screening approach.

Funding : This work was supported by the Korea Medical Device Development Fund grant funded by the Korean government (Project Number: 1711195429, RS‐2020‐ KD000094 ).

Exozirkel—The circadian rhythm of proteomic liquid biopsy markers from extracellular vesicles

Presenter: Giorgia Campione

German Cancer Research Center (DKFZ), Heidelberg, Germany

Introduction : In the field of oncological diagnostics, there are increasing efforts to reduce invasive tissue sampling and replace it with ‘liquid biopsy’ (LB) procedures. In the currently established LB procedures, nucleic acid‐based techniques are primarily used. Yet, the predictive power of these methods is limited. To fully exploit the potential of LB, protein‐based methods need to be established. Unlike free plasma proteins, EVs are specific to their (tumour) cells of origin and therefore are of central importance for protein‐based LB. However, EVs vary greatly intra‐ and interindividually, which currently limits their diagnostic utility. In cell culture models, diurnal dynamics have been described as one possible reason for this variability. The aim of this project is to investigate the extent to which the protein loading, plasma concentration, and characteristics of EVs in peripheral blood follow circadian rhythm in healthy individuals and patients with systemic tumour diseases.

Methods : We recruited 15 patients from University Medical Centre Mannheim (UMM) and 12 healthy volunteers. EVs were isolated from peripheral blood samples taken every 3 h over 24 h using ‘top‐down’ Iodixanol DGs followed by SEC. The particle number, concentration and protein load were measured, and the EV‐derived proteome was subjected to mass spectrometric analysis. Concurrently, various parameters were determined from saliva samples to validate interindividual circadian fluctuations and control non‐circadian confounding factors. Our clinical study was approved by the ethics committee of UMM (2022‐46). Written informed consent was obtained. Statistical analysis is performed using ANOVA or multiple contrast testing.

Results : In healthy volunteers, EV particle concentration and protein loading of EVs remained constant during the day, with a trend towards less interindividual variance in the morning. Circadian proteomic signatures were assessed in healthy individuals and cancer patients.

Summary/Conclusion : The aim of the study is to determine whether the circadian rhythm of EVs indicates particularly suitable times of day for liquid biopsy, thereby improving the clinical application of this diagnostic method. By understanding which specific EV components fluctuate throughout the day, numerous scientifically interesting approaches arise, which are of significant interest for the entire field of EV research.

Preclinical development of EV application in the inner ear for trauma reduction

Susanne Sasse 1 , Odett Kaiser 1 , Jennifer Harre 1 , Mario Gimona 2,3 , Eva Rohde 2 , Daniela Auer 2 , Stefan Rund 2 , Tanja Schally 2 , Carina Kals 2 , Christina Folie 2 , Elisabeth Bayer 2 , Hinrich Staecker 4 , Athanasia Warnecke 1

1 Hannover Medical School, Hannover, Germany; 2 Paracelsus Medical University Salzburg, Salzburg, Austria; 3 Paris Lodron University, Salzburg, Austria; 4 University of Kansas School of Medicine, Kansas City, Kansas, USA Susanne Sasse and Odett Kaiser are co‐first authors .

Introduction : Patients with profound hearing loss undergoing cochlear implantation often lose their residual hearing due to implantation trauma, which impairs hearing performance. So far, there are no approved drugs available for this purpose. Extracellular vesicles (EV) derived from human umbilical cord mesenchymal stromal cells have been tested as novel drug candidates exerting immunomodulatory and/or neuroprotective effects in the inner ear after implantation trauma. To prepare a clinical trial, the safety and biodistribution of EV in different cochlear implantation trauma animal models were investigated.

Methods : Following a series of initial in vitro and in vivo experiments supporting the biological concept, preclinical safety was evaluated in NMRI mice after a single intracochlear application of various doses of EV under GLP compliance. Safety and efficacy were investigated in guinea pigs after cochlear implantation trauma, and long‐term safety was tested in guinea pigs after EV application. Biodistribution of DiD‐labelled EV was evaluated initially in guinea pigs and was extended under GLP‐like conditions in NMRI mice. All data were compiled in various documents for clinical trial application.

Results : Applied EV was well tolerated, and no abnormalities or drug‐related clinical symptoms were observed at any time in either mice or guinea pigs. Application improved hearing after implantation trauma in the guinea pig cochlear implant model. Six months after the EV application, hearing thresholds remained at a similar level to the control group. Regardless of the species, DiD‐labelled EVs could be tracked depending on their duration of action and detected at the applied cochleae (hair cells, supporting cells and spiral ganglion neuronees) and in the kidney, indicating a physiological degradation.

Summary/Conclusion : Preclinical testing of a single EV application in the inner ear for trauma reduction did not raise any specific safety concerns in any of the described animal models. Submission of the study via the Clinical Trials Information System (CTIS) resulted in the approval of a Phase I/II clinical trial (ESCRT) for safety and efficacy in October 2024.

Funding : This work was funded by MED‐EL, the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy—EXC 2177/1 (Project ID 390895286) and the Project “ExtraNeu” from the State of Salzburg, Austria.

Clinical trial ESCRT: study design of first intracochlear application of vesicle‐enriched secretome fractions

Odett Kaiser 1 , Susanne Sasse 1 , Jennifer Harre 1 , Mario Gimona 2,3 , Eva Rohde 2 , Daniela Auer 2 , Tanja Schally 2 , Stefan Rund 2 , Carina Kals 2 , Christina Folie 2 , Elisabeth Bayer 2 , Hinrich Staecker 3 , Nils Kristian Prenzler 1 , Athanasia Warnecke 1

1 Hannover Medical School, Hannover, Germany; 2 Paracelsus Medical University (PMU), Salzburg, Austria; 3 Paris‐Lodron University Salzburg, Salzburg, Austria; 4 University of Kansas School of Medicine, Kansas City, Kansas, USA

Introduction : Following cochlear implantation, immune response can lead to loss of residual hearing and impaired hearing performance. So far, there are no pharmacological therapies to compensate for this. A new medicinal product derived from mesenchymal stromal cells, vesicle‐enriched secretome fractions (VSF), comprises extracellular vesicles (EV), particles and soluble factors. Preclinically, VSF demonstrated immunomodulatory and/or neuroprotective effects and was evaluated for safety. Clinically, VSF has been used in a first named patient approach to demonstrate the feasibility of intracochlear application during cochlear implantation. A first‐in‐human phase I/II clinical trial named ESCRT has been submitted to the Central European system for clinical trials.

Methods : In this first‐in‐human open‐label monocentric phase I/IIa clinical trial, a single dose (30 µL of 0.5–1 × 10 11 particles/mL) of VSF is applied to the inner ear during cochlear implant surgery as adjuvant treatment prior to insertion of the electrode array. Eleven patients (aged ≥ 18) with profound hearing loss receiving cochlear implantation are included. The number and severity of adverse events (AEs) are documented, and immune response, auditory nerve function, and speech perception are evaluated. A sentinel approach will be included. Study duration per subject is approximately 6 (±1) months, with the entire trial scheduled to last approximately 21 months.

Results : The submission of ESCRT via the Clinical Trials Information System (CTIS) was approved in October 2024 and was initiated immediately. Safety as a primary endpoint, based on the detection and documentation of severe and serious AEs, will be recorded using a sentinel approach based on the results of the first three patients.

Summary/Conclusion : The process of taking a new drug candidate from the idea to the bench to a clinical trial is challenging, time‐consuming and expensive. Many unexpected hurdles have to be overcome on the way to approval. Thus, this clinical trial of the first intracochlear approach of EV application can serve as an exemplary pathway to achieve the broader goal of developing precision medicines with the potential to improve and preserve hearing after cochlear implantation trauma.

Funding : This work was funded by MED‐EL (Austria), by Deutsche Forschungsgemeinschaft (DFG; EXC 2177/1, Project ID 390895286), and by the Project “ExtraNeu” from the State of Salzburg, Austria.

Recent developments in clinical trials on the use of extracellular vesicles in cancer

Yasemen Kesimoğlu 1,2 , Mehmet Hikmet Üçışık 3

1 Yeditepe University, Istanbul, Türkiye; 2 İstanbul Topkapı University, Istanbul, Türkiye; 3 Istanbul Medipol University, Istanbul, Türkiye

Introduction : Cardiovascular diseases are the leading cause of death in the world, followed by cancer. Among the deaths caused by cancer, lung, colon, stomach, breast, oesophagus, pancreas, liver and prostate are the most common, respectively. Extracellular vesicles are structures with a lipid double layer, which are synthesised by cells and contain components such as protein, RNA and DNA. Extracellular vesicles can be isolated from different cell sources such as mammals, plants, and bacteria and from different samples such as urine, blood, and saliva. Extracellular vesicles can also be used as biomarkers, vaccines, vesicular therapy, or drug delivery systems. In this study, the purpose of using extracellular vesicles in cancer research studies registered at clinical.trials.gov was investigated.

Methods : The clinical trials website was searched using the keywords ‘cancer,’ ‘exosome,’ ‘extracellular vesicle.’ A total of 213 clinical trials were identified. Trials were identified by their clinicaltrials.gov ID to avoid re‐analysis. After removing trials that didn't fit topic of the trial, there were 194 clinical trials. The type of cancer, purpose of using extracellular vesicles, source, material analysed, activity status of trial, and location of trial were analysed.

Results : Of the cancer clinical trials, 19.1% were conducted in lung cancer, 14.4% in prostate cancer, and 12.4% in breast cancer. Of patient samples, 72% were blood, and 15% were urine. Of extracellular vesicles isolated from different sources, 56.7% were investigated for biomarkers, 33.5% for drug efficacy, 1.5% for drug delivery studies. RNA was analysed in 33.5%, protein in 30.4% of the extracellular vesicle studies. In 26.8% of clinical trials, information about the material to be studied was not provided. When analysing the current status of clinical trials, 32.5% were recruiting, 21% were completed status. When analysing the location of the trials, 34% were conducted in USA, 23.2% in China.

Summary/Conclusion : While lung cancer was the most commonly studied cancer, blood samples were most commonly used for extracellular vesicle isolation. While isolated extracellular vesicles were most often studied as biomarkers, RNA and protein analyses were most often preferred for extracellular vesicle samples. While one third of clinical research is still ongoing, the location of studies shows that one third of them are in the United States.

Genetically engineered HLA‐G targeted exosome and safety study

Presenter: Kai‐Wen Kan

China Medical University, Taichung, Taiwan (Republic of China)

Introduction : HLA‐G functions not only as an immune checkpoint but also as a neoantigen that is highly overexpressed in many cancer types. While chemotherapy agents and oligonucleotides are widely used in cancer treatment, their lack of specific binding to target cancer cells increases the risk of off‐target toxicity and secondary pharmacodynamic effects, with in vivo stability posing a challenge for sustained therapeutic activity. In past decades, nanoparticles like liposomes have been explored for drug delivery; however, issues with immunogenicity, non‐targeted tissue accumulation, and limited targeting capabilities remain significant challenges in the development of synthetic nanoparticles for drug delivery.

Methods : The HLA‐G targeted exosome is a genetically engineered exosome produced from HEK293T cells, modified with an anti‐HLA‐G nanobody to specifically target HLA‐G‐expressing cancer cells, while showing no toxicity in normal cells. In a 36‐day, multiple‐dose toxicity study in ICR mice, weekly intravenous administration was evaluated, focusing on body weight, food intake, clinical observations, organ weights, and immune response.

Results : Through intravenous injection, HLA‐G targeted exosome accumulated in the tumour area within 30 min, and no HLA‐G targeted exosome related adverse events were reported in the GLP‐toxicology study. The above non‐clinical studies demonstrate a high safety margin for HLA‐G targeted exosomes, making them an excellent platform for a series of drug developments. During the study period, no mortality was noted in the study. No test article‐related finding was noted in the results of clinical observation, CNS evaluation and ophthalmologic examination. No irritation was noted in the injection site after dosing. No test article‐related findings were noted in the results of body weights, body weight gains, food consumptions, organ weights and clinical pathology evaluation. At the gross necropsy, no test article‐related finding was noted in all animals. Upon the histopathology evaluation, no test article‐related histopathologic finding was noted.

Summary/Conclusion : In summary, SOB100 is an HLA‐G‐targeted exosome designed for drug delivery, supporting the development of a range of anti‐cancer therapies. Human safety data from the Phase I study is expected to be available in 2025.

Subtopic EV therapeutics development 2

Extracellular vesicles (EVs) from human cerebral endothelial cells grown in oxygen‐glucose deprivation conditions exhibit potent anti‐cancer activity against a wide array of human cancer cell lines with no effect on benign cells

Presenter: Dilip Moonka

Henry Ford Health, Detroit, Michigan, USA

Introduction : After observing a patient with advanced hepatocellular carcinoma (HCC) experience complete resolution of the HCC after experiencing an ischaemic stroke, we hypothesized that EVs from stroke patients could have anti‐neoplastic activity. We evaluated the anti‐cancer activity of stroke‐EVs as well as the anti‐cancer activity of EVs from human cerebral endothelial cells (hCEC) exposed to oxygen‐glucose deprivation to mimic stroke conditions in tissue culture.

Methods : All EVs in all experiments were isolated by ultracentrifugation. Plasma EVs from three patients experiencing ischaemic stroke (stroke‐EVs) were tested at 108 particles/mL against HCC cell lines, HepG2 and Hep3B, for five days using the MTT cell viability assay. Each assay consisted of five duplicate wells. EVs from healthy patients were also evaluated, and the benign liver cell line, THLE‐2, was used as a non‐cancerous control. EVs obtained from hCEC cells exposed to oxygen‐glucose deprivation (OGD‐EVs), to simulate stroke conditions, were tested against all three lines and an array of other human cancer cell lines.

Results : Plasma EVs from healthy controls significantly increased viability of all three cell lines compared to untreated cells: HepG2 (113%–171%: p < 0.001), Hep3B (165%–170%: p < 0.001), THLE‐2 (151%–160%: p < 0.001). Stroke‐EVs from three stroke patients inhibited HepG2 viability by 89%, 90% and 75% ( p < 0.001) compared to untreated cells. The stroke‐EVs inhibited Hep3B viability by 75%, 81% and 72% ( p < 0.001). Stroke‐EVs did not inhibit THLE‐2 cells (+15%, +5.4%, −2%). OGD‐EVs (108 p/mL) from hCEC cells generated in exosome free media inhibited HepG2 cells by 73.1% ( p < 0.001) and Hep3B cells by 76.5%. EVs from hCEC cells not exposed to OGD had no effect on any cells, benign or malignant, and no EVs inhibited THLE‐2 cells. At 10 8 particles/mL, OGD‐EVs inhibited pancreas cells (Panc‐1, 62%), lung cells (A549, 74%), breast cells (MDA‐MB‐468, 84%; MDA‐MB‐231, 77%), prostate cells (PC3, 78%), colon cells (HCT‐116, 64%), and ovarian cells (A2780, 65%; OVCAR‐3, 64%). All differences were significant at p < 0.001. The OGD‐EVs did not affect the multi‐resistant ovarian cell line, A2780‐cis, and the benign fibroblast cell line NIH‐3T3.

Summary/Conclusion : Conclusions: OGD‐EVs from HCEC cells represent a potential therapeutic option for patients with HCC and other malignancies.

Extracellular vesicle‐based drug delivery of cabazitaxel for metastatic prostate cancer treatment

Mughilan Selvarajah 1,2 , Krizia Sagini 1,2 , Beatriz Martín‐Gracia 1,2 , Simona Migliano 1,2 , Andreas Åslund 3 , Tore Skotland 1,2 , Kirsten Sandvig 1,2,4 , Tore‐Geir Iversen 1,2 , Alicia Llorente 1,2,5

1 Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 2 Centre for Cancer Cell Programming, Faculty of Medicine, University of Oslo, Oslo, Norway; 3 Department of Biotechnology and Nanomedicine, SINTEF AS, Trondheim, Norway; 4 Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway; 5 Department of Mechanical, Electrical and Chemical Engineering, Oslo Metropolitan University, Oslo, Norway

Introduction : Cabazitaxel (CBZ) is one of the chemotherapeutic drugs used for the treatment of metastatic prostate cancer (PCa). However, with conventional chemotherapy, only a small fraction of the drug reaches the cancer cells. Nanocarriers, such as extracellular vesicles (EVs), could potentially be used to increase the clinical efficiency of CBZ. This study aims to develop a platform for delivery of CBZ to metastatic PCa cells using EVs. The current focus of the project has been on establishing (1) an endogenous method for loading EVs with CBZ and (2) methods for assessing CBZ‐loading into EVs.

Methods : The endogenous loading method consists of incubating PCa cells (PC‐3 cell line) with CBZ in different conditions followed by differential ultracentrifugation of the conditioned media to isolate EVs. Then, cell viability was measured using the CellTiterGlo‐assay and a protein synthesis assay, and EV secretion was measured by nanoparticle tracking analysis (NTA) and Western blot (WB) detection of EV markers. Furthermore, the CBZ loading efficiency of the endogenous method was assessed by treating recipient PC‐3 cells with isolated EVs. The recipient cells were analysed by measuring cell viability using a protein synthesis assay and cell proliferation using live‐cell imaging. Quantification of CBZ in EVs was performed by mass spectrometry (MS) analysis.

Results : NTA showed that EV secretion was significantly reduced when the PC‐3 cells were treated with free CBZ, but not when treated with CBZ‐encapsulated poly(ethylbutyl cyanoacrylate) (PEBCA) nanoparticles. Furthermore, analysis of several EV markers by WB suggested that different EV subtypes are affected differently by CBZ. Additionally, when unloaded EVs were incubated with recipient PC‐3 cells, cell viability and cell proliferation were affected. Preliminary results showed that CBZ‐EVs affect recipient PC‐3 cells differently than unloaded EVs. MS analysis to quantify CBZ loading into EVs is ongoing.

Summary/Conclusion : In this study, important aspects for endogenous loading of EVs with CBZ have been investigated. This includes optimal CBZ treatment conditions and the effects of CBZ on donor cell viability and EV secretion. Experiments analysing the loading efficiency of CBZ in EVs and the effect of CBZ‐EV treatment in recipient cells are ongoing.

Funding : The project is funded by the South‐Eastern Norway Regional Health Authority.

Mesenchymal stem cell‐derived extracellular vesicles promote angiogenesis

Vojtěch Šprincl 1,2 , Kristýna Šintáková 1,2 , Nataliya Romanyuk 1

1 Czech Academy of Science, Prague, Czech Republic; 2 Charles University, Prague, Czech Republic

Introduction : Acute ischaemic stroke is a leading cause of mortality and disability worldwide. The brain affected by ischaemia undergoes complex pathophysiological changes, which are further exacerbated by oxidative stress during reperfusion, leading to disruption of the blood‐brain barrier (BBB). Research has indicated that angiogenesis plays a critical role in neuroregeneration after reperfusion. Mesenchymal stem cells (MSCs) have been widely explored for their regenerative capabilities across various conditions, including angiogenesis. Their protective effects are largely believed to stem from their ability to create a supportive environment. Extracellular vesicles (EVs) have been proposed as key mediators of these properties. We aim to investigate the angiogenic properties of MSCs‐derived EVs in in vitro models of angiogenesis and the BBB.

Methods : We isolated EVs from the conditioned media of human adipose tissue‐derived MSCs using sucrose cushion ultracentrifugation. The EVs were characterized by size using multi‐angle dynamic light scattering (MADLS) and by the presence of specific protein markers (CD9, Alix, TSG101) via immunoblotting. To assess their therapeutic potential, we conducted a transwell uptake assay, scratch assay, and tube formation assay to evaluate their effects on endothelial cells (HAEC). To further study the protective properties of MSC‐EVs we have created and characterized a static in vitro model of the BBB.

Results : MADLS revealed that the MSC‐EVs have a diameter of 85 ± 40 nm. Immunoblotting confirmed the presence of EV markers CD9, Alix, and TSG101. MSC‐EVs were effectively incorporated into endothelial cells, promoting wound closure and significantly increasing total tube length in vitro. The BBB model was characterized by immunocytochemistry (Claudin‐5, Occludin, ZO‐1), transendothelial electrical resistance, and the Lucifer yellow assay.

Summary/Conclusion : We successfully isolated and characterized EVs from MSC cultures and demonstrated their angiogenic potential in endothelial cells using three different in vitro assays. These findings suggest that MSC‐derived EVs could be a promising tool for supporting neuroregeneration after stroke. Future studies will focus on evaluating these effects in a static in vitro model of the BBB to explore their therapeutic potential in more complex systems.

Funding : This study was supported by the GAUK 409222, NU22‐08‐00124, OP JAK CZ.02.01.01/00/22_008/0004562.

Development of an extracellular vesicle (EV)‐based formulation for the targeted delivery of doxorubicin to tumour tissue

Elisabetta Crippa 1 , Gloria Raineri 1 , Alessandro Villa 1 , Giangiacomo Beretta 2 , Paolo Ciana 1

1 Department of Health Sciences, University of Milan, Milan, Italy; 2 Department of Environmental Science and Policy, University of Milan, Milan, Italy

Introduction : Drug delivery systems aim to overcome biological barriers, target cells precisely, and stabilize transported molecules in the bloodstream. While many lipid‐ and polymer‐based nanoparticles have been developed, few are in clinical use. This study introduces extracellular vesicles (EVs) as a novel drug delivery system. Our recent research shows that EVs from cancer patient plasma or tumour cell lines selectively target neoplastic tissues and can cross the blood‐brain barrier (BBB) to reach central nervous system tumours—unique traits of cancer‐derived EVs. This study aims to use EVs from the MCF7 breast adenocarcinoma cell line as a delivery system for the clinically approved chemotherapeutic doxorubicin.

Methods : MCF7 cells were cultured in RPMI medium with 10% FBS. EVs were isolated from the conditioned medium by ultracentrifugation (100,000 × g , 2 h, 4°C, Beckman Coulter). Particle concentration and size were assessed by nanoparticle tracking analysis (Nanosight LM14) and flow cytometry (Cytek Aurora). EVs markers (TSG101, CD63) were confirmed by immunoblotting. EVs structure was visualized using cryo‐electron microscopy (JEOL). Doxorubicin was loaded into EVs (EVs‐Doxo) via passive incubation (overnight, 4°C) and purified from free drug by ultracentrifugation. The pellet was resuspended in PBS, and encapsulated drug levels were quantified using HPLC‐VIS (LC‐940 Varian). Delivery efficiency was tested by MTT assay, and biodistribution was analysed in C57BL/6 mice.

Results : In this study, 67 µg of doxorubicin was successfully encapsulated in 6E08 EVs without altering their size distribution profile. At the same concentration, the EVs‐Doxo formulation demonstrated a greater cytotoxic effect compared to free doxorubicin, as shown by MTT assays. Importantly, the selectivity for tumour tissue, a characteristic of EVs derived from neoplastic cells, was maintained after doxorubicin encapsulation. This finding highlights the promising potential of adapting this protocol to utilize patient‐derived EVs as a personalized, autologous delivery system for doxorubicin.

Summary/Conclusion : This proof‐of‐concept study conducted on EVs from a human cell line enables the adaptation of the loading protocol for EVs derived from the plasma of cancer patients, with the future goal of applying this protocol as the basis for personalized chemotherapy.

Funding : AIRC (IG 2020‐ID.24914); European Community—NextGenerationEU (PNRR M4C2‐Investment 1.4‐CN00000041‐23 PNRR_CN3RNA_SPOKE8 e SPOKE9). P.C. is the holder of these funds.

Polygonum cuspidatum‐derived nanoparticles promote the healing of deep second‐degree burns by activating the Nrf2‐Keap1 pathway through microRNA delivery

Presenter: Qi Xiu

Southern Medical University, Guangzhou, People's Republic of China

Introduction : The therapeutic potential of herbs has been extensively studied, primarily focusing on small molecule metabolites. However, research on cross‐kingdom interactions between plants and humans, particularly involving plant nucleic acids, remains limited. Emerging evidence suggests that plant‐derived nanoparticles play a crucial role in regulating human health and diseases through interkingdom crosstalk. In this study, we demonstrate for the first time that polygonum cuspidatum‐derived nanoparticles (PDNs) effectively scavenge reactive oxygen species (ROS) and reverse oxidative stress in HUVECs by transferring incorporated miRNAs, thereby activating the Nrf2‐Keap1 pathway.

Methods : Firstly, polygonum cuspidatum‐derived nanoparticles (PDNs) were isolated from the plant's juice, with other disruptive components removed. The physical characteristics of PDNs were analysed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS), confirming their nano‐sized bilayer membrane structure. Secondly, PDNs were incubated with various skin‐derived cell lines to assess their biosafety, pro‐migration, and anti‐oxidation properties. Subsequently, PDNs were applied to the wound areas of C57/6J mice with 10 mm 2 deep second‐degree burns. Thirdly, multi‐omics analysis and experimental validation were performed to elucidate the specific mechanisms involved.

Results : First, PDNs are slightly negatively charged spherical structures with a diameter of 150 nm, capable of stable preservation at 4°C, −20°C, and −80°C for over a month. Second, in vitro experiments showed that PDNs have significant antioxidant effects, promote the migration of vascular endothelial and keratinized epithelial cells, and induce M0 macrophages to differentiate into anti‐inflammatory M2 macrophages. In vivo, PDNs significantly accelerated wound healing by reversing oxidative stress and promoting angiogenesis. Thirdly, seven miRNAs from PDNs were identified as targets of human Keap1 mRNA, leading to the activation of the Nrf2‐Keap1 antioxidant pathway.

Summary/Conclusion : PDNs, enriched with plant‐derived biomacromolecules, can enter the human body and effectively modulate human cell signalling pathways through cross‐kingdom interactions. Specifically, PDNs activate the Nrf 2‐Keap1 pathway by delivering multiple miRNAs to HUVECs, enhancing their antioxidant capacity and promoting rapid healing at the wound site.

Funding : This work was supported by grants from the National Science Fund for Distinguished Young Scholars (82025024), National Natural Science Foundation project of China (82472387), and the Guangzhou Municipal Science and Technology Program (SL2024B03J00976).

A scalable 4D scaffold‐based bioreactor platform for sEV research, development, and GMP manufacturing

Roland Hubaux 1 , David Sergeant 2 , Michael Francotte 3 , Benoît Champluvier 1 , Jean‐Paul Prieels 1,3 , Frédéric Tonglet 1,4

1 ConvEyXO SA, Charleroi, Belgium; 2 IPRATECH SA, Gosselies, Belgium; 3 XomeXBio SA, Charleroi, Belgium; 4 Exo‐Harvest SA, Charleroi, Belgium

Introduction : Small extracellular vesicles (sEVs) have emerged as powerful therapeutic tools, but scalable GMP‐compliant production and COGS are challenging. Exo‐Harvest and Ipratech developed a 4D bioreactor platform that allows the integration of a scaffold like the one developed exclusively for ConvEyXO by Antleron. This platform has been validated with the ConvEyXO process and addresses sEV availability and cost of goods sold. It enables innovative parallel small‐scale production, especially for adherent cells, while supporting scalability for industrialization.

Methods : The electronic and control software is based on the CYTOSYS platform (IPRATECH). The platform has been equipped with custom scaffolds of controlled, geometry offering a homogeneous environment optimizing adherent cell growth and enhancing sEV yield. The design and manufacturing of the materials have been adapted at different scales to ensure full scalability and applicability across various cell types and development stages. Quantity indicator controllers with specific sensors regulate the critical parameters (pH, temperature, oxygen, media changes and perfusion). Adherent HEK293 cells have been used during the optimisation. ConvEyXO's immortalized cell line from Wharton Jelly derived mesenchymal stem cells (MSCs) was used to validate the platform. The downstream purification and characterisation of the sEVs were performed following the standard protocols from ConvEyXO, including sEV size distribution by MRPS, quantification of protein, cholesterol, and residual DNA, assessment of tetraspanin marker positivity (CD9, CD63, CD81), and absence of GM130. The therapeutic potential of the sEVs was assessed in vitro on macrophages (mouse cell line and human primary cells).

Results : Three bioreactor systems were developed: the first supports R&D using parallel bioreactors (6 or 12 units) for screening, cell line engineering, media optimization, and process development. The second, with advanced sensors and actuators, fine‐tunes perfusion and feeding strategies. The third system, designed for GMP‐compliant large‐scale manufacturing, leverages custom scaffolds and continuous perfusion to achieve high cell density and consistent, high‐purity sEVs production. Quantitative and qualitative analysis of the sEVs showed 10× higher yields and enhanced anti‐inflammatory potency compared to current standards.

Summary/Conclusion : This scalable, 4D scaffold‐enabled platform provides a comprehensive solution for producing high‐quality sEVs for therapeutic applications, offering a seamLess transition from R&D to clinical and commercial‐scale production.

Stem cell‐derived extracellular vesicles in bioprinted corneal lenticules for regenerative healing

Parinita Agrawal 1 , Nisha Rajendran 1 , Ritu Raj 1 , Suvro K. Chowdhury 1 , Namit Dey 1 , Alka Bhat 1 , Sabyasachi Chattopadhyay 1 , Imrankhan Pathan 1 , Mujib Ullah 2 , Rita Mahapatra 1 , Abha Gour 3 , Anil Tiwari 3 , Virender S. Sangwan 3 , Arun Chandru 1 , Tuhin Bhowmick 1,2

1 Pandorum Technologies Pvt. Ltd., Bangalore, India; 2 Pandorum International Inc., Boston, Massachusetts, USA; 3 Dr. Shroff's Charity Eye Hospital, New Delhi, India

Introduction : Corneal blindness affects millions of people worldwide, and its treatment is impacted by the limited availability and sub‐par quality of donor cornea tissues. Mesenchymal stem cells (MSCs)‐derived extracellular vesicles (EVs), drawing attention for the treatment of various disorders, have been explored here as therapeutics for corneal wound healing. We developed bioprinted corneal lenticules as a means for targeted and sustained delivery of EVs while studying corneal regeneration.

Methods : EVs isolated from MSCs by ultracentrifugation and purified by density gradient separation were evaluated by nanoparticle tracking analysis and electron microscopy for size estimation and western blot for protein profile following MISEV2023‐guidelines. Establishing therapeutic function in cell lines, the study investigated their encapsulation in a cornea‐mimetic biopolymeric ink by bioprinting corneal lenticules using the digital light processing technique. Their therapeutic application and regenerative potential were explored by release profile and in vitro functional studies.

Results : EVs, with a size of 70–150 nm, appeared as cup‐shaped vesicles in transmission electron microscopy. Expressed characteristic markers CD9, CD63, CD81, TSG101, Flotillin, and Alix, with remarkable re‐epithelialization, anti‐inflammatory, anti‐fibrosis, and neurogenesis activities. The bioprinted lenticule exhibited cornea mimetic properties, with light transmittance > 85% and compressive modulus comparable to that of a native cornea. The average release rate of 22% over 120 days, showcased their potential as a sustained EV delivery platform. Demonstrating 98% compatibility with human corneal stromal cells, this system proved to conserve EVs activity and support corneal regeneration.

Summary/Conclusion : MSCs derived EVs emerged as promising bioactive agents for stimulating re‐epithelization and supporting corneal regeneration. Their delivery mediated by bioprinted corneal lenticules ensures regeneration, preserving structural and functional requirements of the tissue. The study describes a promising strategy for clinical translation of EVs towards the treatment of corneal keratopathy involving inflammation and removing the dependency on donor corneas for transplants.

Next‐generation hollow fibre bioreactors for scalable and monitorable microfluidic production of EVs

Celia Atalaya 1 , María Mantecón‐Oria 1 , Nazely Diban 1 , Ana Villar 2 , Ane Urtiaga 1

1 University of Cantabria, Santander, Spain; 2 Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), Santander, Spain

Introduction : The clinical application of extracellular vesicles (EVs) is limited by the scalability of traditional 2D cell culture methods. Hollow fibre bioreactors (HFBR), as 3D culture systems, support high cell density and increased EV yield, addressing this scalability challenge. However, current commercial HFBR systems lack transparency, limiting cell culture monitoring. This study introduces an advanced HFBR designed for high EV yields and improved visualization of EV‐producing cells. The system incorporates translucent fibres with optimal porosity for EV enrichment. EVs were generated from genetically modified HaCaT cells with enhanced adhesion to fibres composed of 100‐200 nm pore‐polymer.

Methods : We generated various polymer hollow fibres (HF) to build a hollow fibre bioreactor (HFBR), each offering different translucency levels and tailored material properties. These HFs were produced using a lab‐scale wet‐dry HF spinning system, and parameters were optimized to extrude fibres from synthetic polymers, including polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), polyethersulfone (PES), and an alginate hydrogel [1]. Porosity was carefully controlled between 100 and 200 nm. HaCaT cells were modified via viral infection with a CAR‐T‐like plasmid encoding a fibroblast activating protein (FAP) antibody variant, enhancing cell membrane adhesion for attachment to HFBR cartridges. Cell colonization on the fibres’ surface was tracked using epifluorescence visualization of the fluorophore‐tagged CAR‐T‐like FAP construct. Extracellular vesicles (EVs) were characterized for size and concentration by nanoparticle tracking analysis (NTA), morphology by transmission electron microscopy (TEM), and fluorescence by confocal microscopy.

Results : We observed differences in terms of areas colonized by HaCaT CAR‐T cells and EVs yield among conventional 2D methods, commercial HFBR and transparency custom Laboratory‐engineered microfluidic HFBR.

Summary/Conclusion : These findings provided strong evidence for the high potential of Laboratory‐engineered HFBRs and adherent engineered HaCaT cells as an efficient and monitorable system for EV production. This system showed potential to become a high‐efficiency platform for EV production with characteristics similar to biological production. [1] N. Diban, M. J. Rivero, A. Urtiaga, M. Mantecón‐Oria, 10 June, 2024, Spanish patent application P202400038 Fibras huecas poliméricas porosas de alta transmitancia.

Funding : MICIU/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR [grant PDC2022‐133704‐I00]. SUBVTC‐2023‐0006 T. Gobierno de Cantabria. C: Fomento de la transferencia de conocimiento en la Comunidad Autónoma de Cantabria

Colorectal Cancer Derived Extracellular Vesicles Modulate Antitumour Immune Response

Presenter: Subbaya Subramanian

University of Minnesota, Minneapolis, Minnesota, USA

Introduction : Colorectal cancer (CRC) is the third leading cause of cancer‐related deaths, and most patients are resistant to immune checkpoint inhibitors due to the immunosuppressive microenvironment. Understanding resistance mechanisms to ICIs could lead to novel combinatorial intervention strategies that make patients eligible for ICI treatment. Our previous studies have identified resistance mechanisms where extracellular vesicles (EVs) containing miR‐424 as cargo can lead to the downregulation of costimulatory CD28 on T cells and CD80/86 on dendritic cells (DCs). Furthermore, we have demonstrated that tumour EVs (TEVs) are captured and processed by DCs and are presented as antigens to T cells. The consequence of miR‐424 expression within CRC TEVs and the potential alterations in antigenic cargo within TEVs remain unexplored. In this study, we sought to understand how the expression of miR‐424 in CRC cells alters the antigenic cargo within CRC TEVs.

Methods : TEVs from CT26 cells with or without miR‐424 were isolated using ultracentrifugation methods. The EVs were characterized for size and frequency using nanoparticle tracking analysis. EV tetraspanins, CD63, CD81, and Alix, were characterized using western blotting. We employed a label‐free quantitative proteomic method using mass spectrometry to characterize TEVs isolated from CT26‐miR‐424i (antimiR inhibition), CT26‐miR‐424‐KO (CRISPR/Cas9), and CT26‐miR‐424‐control cells and compared proteomic cargo to TEVs derived from CT26‐WT cells. We used FragPipe followed by data‐independent analysis using neural networks (DIA‐NN) to determine signal intensities of proteins throughout TEV samples and MSstats for statistical comparisons.

Results : We observed significant differences between TEVs derived from CT26‐WT and CT26‐miR‐424KO cells. Proteins of interest enriched in TEVs derived from CT26 cells lacking miR‐424 include Tsta3, P4hb, Cdk2, and Etf1. Additionally, we saw the proteins P4hb, Ube3a, and Glb1 significantly enriched in CT26‐miR‐424KO TEVs compared to CT26‐miR‐424i TEVs. We intend to study further the antigenic potential and immune regulatory role of the protein cargos in the TEVs.

Summary/Conclusion : The proteins identified in TEVs have been implicated in modulating cellular signalling. Future studies will focus on quantitative assessments of these proteins as potential tumour antigens and understanding their possible role in immune regulation using immunopeptidomics.

Funding : This study was funded by the Mezin Koats Colorectal Cancer Research Funds and the Minnesota Colorectal Cancer Research Foundation.

A nano‐cocktail strategy regulating circadian clock and re‐establishing bone‐immune balance for the treatment of osteoporosis

Presenter: Ke Zhao

Wangjing Hospital, Beijing, People's Republic of China

Introduction : Circadian rhythm disorder impairs the efferocytosis function of macrophages; thus, non‐professional efferocytosis by bone mesenchymal stem cells (BMSCs) will function as compensation. However, the BMSC efferocytosis may decrease their osteoblastic differentiation and the consequent bone loss. Thus, circadian rhythm disorder and abnormal efferocytosis by BMSCs are considered to play a vital role in the pathogenesis of senile osteoporosis (SOP). At present, most pharmaceuticals for SOP treatment trying to re‐establish osteoblast/osteoclast balance overlook the significance of restoring circadian clock and efferocytosis function of macrophages.

Methods : Here, we developed a nano‐cocktail strategy utilizing melatonin‐loaded bone‐targeting nanovesicles (NVs) prepared from gene‐edited BMSC membrane. Owing to the receptor activator of nuclear factor‐𝜿B (RANK) and (C‐X‐C motif chemokine receptor 4) CXCR4 overexpression, the NVs can specifically clear the RANK ligand (RANKL) in the bone microenvironment, thus inhibiting osteoclastogenesis.

Results : After (AspSerSer)6 modification of partial NVs, they are redistributed onto the bone formation surfaces and activate RANKL reverse signalling in osteoblasts, thus promoting osteogenesis. More critically, most of these NVs are proved to be phagocytosed by bone marrow‐derived macrophages (BMDMs), which recover their expression of crucial circadian rhythm genes and efferocytosis function. Thus, BMSCs are liberated from the non‐professional efferocytosis, and their osteogenic/adipogenic shift is also rectified.

Summary/Conclusion : In conclusion, this study proposed a promising nano‐cocktail strategy for treating SOP via regulating the circadian clock and restoring normal efferocytosis function.

Exploring plant derived extracellular vesicles for cancer treatment

L. Simon 1 , Z. Zhao 2 , N. Sanchez‐Ballester 1 , I. Soulairol 1 , J. Lacombe 2 , F. Zenhausern 2 , S. Begu 1

1 ICGM, CNRS, ENSCM, Université de Montpellier, Montpellier, France; 2 Center for Applied NanoBioscience and Medicine, University of Arizona, Tucson, Arizona, USA

Introduction : Extracellular vesicles (EVs) are nanosized vesicles naturally produced by cells (animal or human) and are key players of intercellular communication. Given their natural cell internalization and intrinsic properties, they have a strong potential as a tunable bio‐inspired drug delivery system. However, their development is still limited by low production capacity at high quality, low drug loading and poor resistance to environmental factors. Thus, recent evidence of plant‐derived extracellular vesicles (pEVs), with similarities in morphology, size, properties and drug delivery potential, has opened up new treatment perspectives. In this context, our study intends to evaluate the potential of pEVs as nanovectors for doxorubicin (Dox) delivery in cancer treatment.

Methods : pEVs were isolated from black olives using differential centrifugation on blended olives, followed by sucrose density gradient ultracentrifugation on the remaining supernatant.

Results : The pEVs produced and purified from black olives displayed a size of 110 nm and a surface charge of ‐20 mV. Microscopy, lipidomic and ELISA showed the presence of a rich lipid bilayer with PEN1 and TET8 markers confirming the extracellular origin. To evaluate their resistance to physical stresses, pEVs were exposed to high temperature (70°C), pH (5–10), salt and repeated extrusion through a membrane of 50 nm. Interestingly, their size and recovery yield remained unchanged after these stresses, showing their robustness. Then, the potential of pEVs was evaluated for Dox intracellular delivery and tested on ovarian cancer cells (SKOV‐3) in 2D and 3D. Thus, after testing different loading methods from freeze‐thaw cycles to sonication, 1 µM of Dox was loaded by simple passive incubation. The Dox‐pEVs were able to enhance the effect of Dox on cell viability after 72 h with a decrease to 13% compared to 28% with free Dox. This improvement was also confirmed on SKOV‐3 spheroids after 8 days with a significant volume decrease with pEVs compared to free Dox.

Summary/Conclusion : Therefore, pEVs represent a promising candidate as a drug nanocarrier for cancer treatment, as demonstrated by their biocompatibility, high resistance to stress, good stability in minimal environment, and improvement of anticancer drug efficacy on cancer cells.

Funding : This study was supported by the NIAID CMCR 5U19AI067773 Desert Mountain CARE #40779.

Engineered small extracellular vesicles for targeted delivery of antifibrotic drugs in cardiac fibrosis

Helena Soto Pérez‐Cejuela, Ana Victoria Villar

Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Santander, Spain

Introduction : Cardiac fibrosis, characterized by the accumulation of extracellular matrix, particularly collagen, is a common protective process occurring in cardiovascular diseases which usually leads to tissue rigidity and potential fatality. Since there is a lack of antifibrotic therapies specifically targeting this process, this study proposes the use of engineered small extracellular vesicles (sEVs) as targeted nanocarriers of existing antifibrotic drugs towards activated fibroblasts, the key cells responsible for fibrosis, increasing their efficiency and reducing side effects. In pursuit of this objective, the HaCaT cell line was genetically modified to produce engineered EVs with a theragnostic purpose (CART HaCaT). These EVs were designed to target fibroblast activation protein (FAP), since it is a protein that has been demonstrated to be overexpressed in cardiac diseases and therefore in the active cardiac fibroblasts responsible for the fibrosis. The design is inspired by the CART cell technology. It consists in the construction of a chimeric protein that I will call FAP‐CAR that contains a FAP binding domain, a hinge and transmembrane region of CD63 for the insertion of the construction in the EVs membranes, a His tag and a fluorescent protein for tracking.

Methods : EVs from CART HaCaT were extracted from conditioned media and purified by ultracentrifugation (serial centrifugations of 10,000 × g and 100,000 × g ). Characterization of the sEVs was performed by nanoparticle tracking analysis (NTA), flow cytometry and transmission electron microscopy (TEM). The specificity of the sEVs was assessed by colocalization assays with FAP in different human cell lines and human cardiac organoids.

Results : The engineered sEVs derived from CART HaCaT cells were successfully characterized, demonstrating an average size of 107 nm as confirmed by nanoparticle tracking analysis (NTA). Colocalization assays revealed that the engineered sEVs exhibited a significantly higher affinity for the tested cell lines compared to unmodified sEVs as observed by confocal microscopy.

Summary/Conclusion : In conclusion, this study highlights the promising potential of engineered sEVs as targeted nanocarriers for antifibrotic therapies in cardiac fibrosis. The genetically modified CART HaCaT cells successfully produced sEVs that not only maintain the therapeutic efficacy of existing antifibrotic drugs but also enhance their specificity and reduce potential side effects.

Prolonged continuous intravenous infusion of bone marrow mesenchymal stromal/stem cell‐derived small extracellular vesicles greatly improves functional recovery after contusive spinal cord injury

Presenter: Masahito Nakazaki

Sapporo Medical University, Sapporo, Hokkaido, Japan

Introduction : In previous studies, we showed that three intravenous infusions of bone marrow mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEVs) over three days starting one week post‐spinal cord injury (SCI), improved motor recovery and growth in young adult rats, whereas a single equivalent dose did not. Tracing DiR‐labelled MSC‐sEVs indicated uptake by M2 macrophages at the lesion, with excess cleared by the kidneys within 24 h. These findings suggest that extending MSC‐sEV exposure through prolonged, continuous delivery may improve their therapeutic efficacy by sustaining interaction with target cells at the lesion site. In this study, we evaluated the therapeutic efficacy of continuous intravenous infusion of MSC‐sEVs in an SCI rat model.

Methods : Severe contusive SCI (T9) rats were randomized into eight treatment groups based on administration method (daily or continuous), duration of treatment (3 or 6 days) and treatment type (human MSC‐sEVs or PBS control) starting on Day 7 post‐SCI. Continuous infusion was administered via osmotic pumps connected to an intravenous catheter, while daily injections were delivered through the tail vein. Open field locomotor recovery was assessed weekly until day 70 post‐SCI using the Basso–Beattie–Bresnahan (BBB) scoring protocol, and body growth was evaluated by weekly body size measurements. For mechanistic analysis microRNA sequencing was performed, and data were analysed using Ingenuity Pathway Analysis (IPA) software.

Results : We found that continuous delivery of MSC‐sEVs over three days accelerated the onset of recovery compared to daily injections, and continuous infusion over six days significantly enhanced recovery compared to daily injections with the same total dosage. The timing of recovery in growth trajectories was comparable across all MSC‐sEV treatment conditions. Mechanistic analyses revealed that numerous miRNAs associated with fibrosis pathways were highly enriched in MSC‐sEVs compared to control sEVs.

Summary/Conclusion : These results suggest that prolonged continuous exposure to MSC‐sEVs is essential for maximizing specific therapeutic benefits, particularly in enhancing functional motor recovery in SCI. Furthermore, at least part of the therapeutic effect of MSC‐sEVs may be mediated by microRNA changes related to fibrosis pathways.

Cyclic protein‐charged extracellular vesicles as anti‐inflammatory oral nanocarriers in the gut

Presenter: Yeonju Lee

Hanyang University, Seoul, Republic of Korea

Introduction : Oral drug delivery offers a patient‐friendly and minimally invasive approach for treating inflammatory bowel disease (IBD). However, high immune tolerance and oxidative stress in the gastrointestinal (GI) tract pose significant challenges. While extracellular vesicles (EVs) are promising carriers for IBD therapeutics, engineered EVs loaded with multi‐regulatory proteins are essential for sustained anti‐inflammatory effects.

Methods : Herein, we report a smart therapeutic approach using cyclic protein‐charged EVs (cPC‐EVs) to rapidly attenuate oxidation and inflammation in the gut. Taking advantage of the enhanced permeability and stability of cyclic proteins, we designed cyclic versions of three immune‐regulatory proteins (human metallothionein 1A, hMT1A; cytosolic domain of cytotoxic T‐lymphocyte antigen 4, ctCTLA4; T cell protein tyrosine phosphatase, TC‐PTP) by knotting their N‐ and C‐termini via a split intein‐mediated process, which function as anti‐inflammatory agents within EVs. We examined natural EVs ( Lactobaccillus plantarum ‐derived EVs, LpEVs, or plant‐derived EVs, pEV) in combination with N‐C cyclization of these proteins.

Results : Through in silico and in vitro comprehensive analyses of enhanced green fluorescent protein (EGFP) with peptide tags, we identified optimal peptide tags for improved membrane permeability in cyclic proteins. Consequently, multifunctional cPC‐EVs containing three cyclic immune‐regulatory proteins demonstrated synergistic anti‐inflammatory effects in both IBD organoids and orally administered IBD mouse models, significantly outperforming naïve LpEV or pEVs.

Summary/Conclusion : These findings suggest that cPC‐EVs are very useful as a versatile oral drug platform for various inflammatory diseases.

The administration of MSC human primed‐IL‐1β‐derived conditioned medium enriched in extracellular vesicles combined with soluble factors is effective in a rodent model of traumatic haemorrhagic shock at the early stage

Guillaume Valade 1,2 , Clément Devautour 1,2 , Marion Grosbot 1,2 , Nathalie Oru 3 , Muriel Nivet 1,2 , Marine De Taddeo 1,2 , Sylvie Goulinet 2 , Kelly Aubertin 4 , Vincent Jung 5 , Gérard Pehau‐Arnudet 6 , Sébastien Banzet 1,2 , Marina Trouillas 1,2 , Juliette Peltzer 1,2

1 French Armed Forces Biomedical Research Institut (IRBA), Clamart, France; 2 INSERM U1197, Villejuif, France; 3 Université Paris Cité, UMRS942, Paris, France; 4 Plateforme IVETH UMR7057, Paris, France; 5 Plateforme Protéomique de Necker CNRS UAR 3633, Paris, France; 6 Plateforme de Bio‐Imagerie Ultra structurale—Institut Pasteur, UMR 3528, Paris, France

Introduction : Traumatic haemorrhagic shock (THS) can quickly induce death by bleeding or later by organ failure. The administration of IL‐1β‐primed human bone marrow mesenchymal stromal cells (MSCil) modulates the inflammatory response in this context and reduces organ dysfunction. Our previous work indicates that the EV + SF fraction has the most complete immunological effect. We explore the therapeutic potential in vivo, of concentrated fractions of MSC‐derived conditioned medium (CM) enriched in extracellular vesicles (EV) combined with soluble factors (SF). These acellular products are easier to store than MSC and immediately available for emergencies.

Methods : CM are obtained after 72 h of MSCil (CM‐MSCil) or MSC naive (CM‐MSCn) secretion from a pool of 5 human donors. The CM are purified/concentrated by tangential flow filtration (TFF) with a 10 kDa filters, allowing an enrichment in EV + SF. We diafiltrated our CM in Ringer's lactate. The characterization of CM is carried out in terms of quantity, size and functional monocytic anti‐inflammatory assay. We developed a Sprague Dawley rat THS model consisting in hindlimb rhabdomyolysis by administration of glycerol at 2 mL/Kg, and haemorrhagic shock with controlled blood pressure (35 mmHg ± 2), followed by fluid resuscitation and blood re‐transfusion. Two doses of CM from MSCil, one dose of MC from naive MSC, or a blank are administered at the end of the resuscitation phase. Animals are sacrificed 6 h after treatment administration. Whole blood, urine and organs (liver, kidney, intestine, lung, muscle and heart) are collected for histological, protein and mRNA analysis. Water content in organs is assessed by the difference between wet and dry weight.

Results : Preliminary results obtained on plasma and urine samples suggest a better efficacy of CM‐MSCil than CM‐MSCn on renal and hepatic dysfunction. A dose‐response effect also emerged for CM‐MSCil. Histological, protein and mRNA evaluations are currently ongoing.

Summary/Conclusion : This promising work is consistent with previous data indicating a beneficial effect of IL‐1β‐primed MSCs to prevent organ dysfunction after haemorrhagic shock and provides the first evidence supporting the efficacy of acellular products in a THS context.

Engineered exosomes target synovial fibroblasts to promote ferroptosis to ameliorate rheumatoid arthritis and its potentiation by photodynamic therapy

Presenter: Yanan Gao

Third Hospital of Shanxi Medical University, Taiyuan, People's Republic of China

Introduction : Rheumatoid arthritis (RA) is a chronic autoimmune disease where synovial fibroblasts (SFs) play a critical role in its pathogenesis. An imbalance in iron homeostasis leads to iron overload, promoting SF proliferation and inhibiting apoptosis, which worsens the disease. Regulating microRNA (miRNA) expression in SFs can induce ferroptosis. Photodynamic therapy (PDT) enhances ferroptosis through lipid peroxidation and laser irradiation, but its effectiveness is limited by challenges in drug delivery and unstable photosensitizers. Exosomes (Exos) are promising carriers for therapeutic drugs due to low immunogenicity and good permeability. This study investigates Exos for targeting SFs and promoting ferroptosis in RA.

Methods : We screened differential miRNAs from human umbilical cord mesenchymal stem cells (hUCMSCs) in RA SFs related to ferroptosis, selecting miR‐181 as the target gene. The relationship between miR‐181 and arachidonate 5‐lipoxygenase (ALOX5) was analysed using bioinformatics. SFs were extracted from RA patient synovial tissues to assess the effects of miR‐181 on SFs and iron metabolism. We reprogrammed hUCMSCs‐Exos to load them with the photosensitizer ICG and miR‐181, along with anti‐FAP‐α antibody 28H1 and fluorescent probe Cy5 to create engineered Exos. We measured particle size and encapsulation efficiency. SFs were co‐cultured with Exos and irradiated with an 808 nm laser to observe phagocytosis, targeting, ICG changes, and the cell viability and biological function of SFs.

Results : Both miR‐181 and ALOX5 were expressed at low levels in RA SFs. Overexpression of miR‐181 increased ALOX5 and raised reactive oxygen species (ROS) and lipid peroxides (LOPs) levels, including MDA and GSH ( p < 0.05). The loading rates of ICG and miR in Exos were 23% and 0.25%, with release efficiencies of 65% and 87%. Exos showed longer retention in the joint cavity compared to ICG alone. Fluorescence microscopy indicated aggregation of ICG and Cy5 in RA SFs ( p < 0.05). Laser treatment decreased ICG in Exos, increased ROS and LOPs, and reduced SF survival. In CIA rats, miR‐181 and ALOX5 were significantly upregulated in synovial tissues, improving joint inflammation.

Summary/Conclusion : MiR‐181 promotes ferroptosis by targeting ALOX5 in RA SFs. Engineered Exos effectively target SFs and synergize with PDT, presenting a promising therapeutic strategy for RA.

Burn wound repair potential of extracellular vesicles and soluble factors from IL‐1β‐primed mesenchymal stromal cells

De Taddeo M. 1,2 , Valade G. 1,2 , Grosbot M. 1,2 , Nivet M. 1,2 , Langle C. 1,2 , Goulinet S. 2 , Mauduit P. 2 , Jung V. 3 , Guerrera C. 3 , Banzet S. 1,2 , Peltzer J. 1,2 , Trouillas M . 1,2

1 French Armed‐Forces Biomedical Research Institute (IRBA), Clamart, France; 2 INSERM U1197, Villejuif, France; 3 Necker Proteomics, Paris, France

Introduction : Despite the great diversity of approaches in regenerative medicine, severe burns still lead to delayed healing and abnormal scarring. The secretome of mesenchymal stromal cells (MSC) has strong potential in tissue repair, with immunomodulatory and remodelling properties similar to MSC themselves. Composed of extracellular vesicles (EV) and soluble factors (SF), its composition is modified by environmental stimuli. Our team leveraged this property by priming MSC with IL‐1β to enhance burn wound healing. However, the specific secretome components with pro‐repair properties and their mechanism of action remain elusive. This study aims to identify key components of the IL‐1β‐primed MSC secretome that promote cell proliferation, migration, and remodelling in vitro.

Methods : MSC were primed with IL‐1β, and the conditioned medium was collected after 72 h. EV or EV + SF fractions were isolated and concentrated using tangential flow filtration with filter cutoffs at 500 or 10 kDa, respectively. Characterization was performed by cryo‐electron microscopy, NTA, flow cytometry phenotyping and mass spectrometry analysis. Various doses of these products, reflecting MSC numbers producing the secretome, were tested in proliferation, migration and extracellular matrix production assays involving keratinocytes and dermal fibroblasts under serum‐deprived conditions. PCR analyses were performed to assess myofibroblast activation.

Results : EV or EV + SF fractions were characterized according to MISEV guidelines. Our results indicated no difference in particle concentration and size. Both fractions expressed EV markers (CD9, CD63, CD81) and MSC markers (CD90). Mass spectrometry analysis revealed differences between EV and EV+SF but few differences among priming doses. In functional assays, the EV + SF fraction more effectively promoted proliferation and migration of keratinocytes and dermal fibroblasts under stressed conditions than the EV fraction, regardless of priming dose. Nucleic acid and protein compositions with related reactomes supported these results, revealing more growth factors, interleukins, chemokines linked to matrix remodelling.

Summary/Conclusion : Our research highlights the role of both soluble factors and extracellular vesicles in wound healing. We aim to validate these findings using an in vivo model. This study will help optimize scalable production methods for a product designed to improve severe burn healing.

Effect of extracellular vesicles derived from glycolytic mesenchymal stem/stromal cells on the phenotypic and metabolic reprogramming of synovial macrophages in patients with osteoarthritis

Presenter: César A. Merino

Universidad de Los Andes, Santiago, Chile

Introduction : Osteoarthritis (OA) is a chronic inflammatory disease for which no cure exists; only palliative treatments are available. OA is characterized by an exacerbated inflammatory response primarily driven by the activity of synovial macrophages (SM). Mesenchymal stem/stromal cells (MSCs) are well‐known for their therapeutic potential; however, alternative strategies are essential to enhance their immunomodulatory properties. We show that glycolytic MSCs (MSCglyco) and their small extracellular vesicles (EVs) hold promise for developing new therapies for OA.

Methods : MSCs were isolated from the umbilical cords of healthy donors, with informed consent obtained for the use of these samples. MSCs were treated with oligomycin for 24 h to induce glycolytic metabolism. EVs were isolated from the MSCglyco (MSCglyco‐EVs) using ultracentrifugation, quantified by nanoparticle tracking analysis (NTA), and characterized by transmission electron microscopy (TEM) and flow cytometry. Macrophages were isolated from the synovial membranes of OA patients, for which informed consent was also obtained. EVs from MSCs or MSCglyco were added to the culture media of the macrophages at various doses. After 24 h, macrophages were recovered to evaluate EV internalization using qPCR, surface marker expression through flow cytometry, cytokine secretion via ELISA, and glycolytic flux using SCENITH, a flow cytometry assay.

Results : The metabolic reprogramming of MSCs does not alter the phenotype or size of the released EVs, nor their capacity of internalization. Synovial macrophages from OA patients (OAM) treated with MSCglyco‐EVs internalize these EVs, leading to an increased expression of CD206, which indicates an anti‐inflammatory phenotype, while decreasing HLA‐DR and CD86 associated with a proinflammatory phenotype. Additionally, OAM treated with MSCglyco‐EVs exhibits a reduction in the secretion of inflammatory mediators.

Summary/Conclusion : EVs from metabolically reprogrammed MSCs have improved therapeutic properties. We demonstrate that MSCglyco‐EVs dose‐dependently reduce inflammatory profiles in OAM and increase anti‐inflammatory profiles, providing compelling evidence for their enhanced therapeutic effects. This offers a promising approach for the development of novel acellular therapies for OA, focusing on immunomodulation using EVs from metabolically reprogrammed MSCs.

Funding : This research was supported by the ANID‐Chile through Beca Doctorado Nacional folio 21220015; FONDECYT regular N#1211353; FONDECYT Iniciación N#11220549; FONDEF‐ID: 21I10194; IMPACT‐FB 210024; and Tecnologías Avanzadas TA24I10054.

Characterization of extracellular vesicles (EVs) derived from umbilical cord mesenchymal stromal cells (UC‐MSCs) loaded with miR‐365a‐5p on CD4+ memory T cells

Presenter: Yesenia Flores‐Elías

Universidad de Los Andes, Santiago, Chile

Introduction : Autoimmune and inflammatory diseases exhibit an imbalance between pro‐inflammatory and regulatory CD4+ T cells, making immune response regulation a persistent challenge. Mesenchymal stromal cells (MSCs) are a promising therapeutic option due to their immunosuppressive capacity, mainly attributed to their secretion of extracellular vesicles (EVs). However, clinical outcomes are divergent, leading to proposals for enhancing their therapeutic potential by reprogramming MSCs towards glycolysis. Additionally, studies suggest that miRNAs within EVs may influence immunomodulation. Our studies with glycolysis‐reprogrammed UC‐MSC‐derived EVs (EVs‐glyco) demonstrated enhanced immunosuppressive activity on memory CD4+ T cells compared to non‐reprogrammed EVs and a high expression of miR‐365a‐5p. Therefore, we characterized EVs internalization through C. elegans miR‐39 expression and evaluated the effect of miR‐365a‐5p on memory CD4+ T cells.

Methods : EVs were isolated from basal UC‐MSCs (EVs) and glycolysis‐reprogrammed (EVs‐glyco) by ultracentrifugation. They were subsequently loaded with miR‐39 via electroporation (EVs‐miR‐39) and characterized by nanoparticle tracking analysis and FACS. The internalization of miR‐39 in EVs and treated memory CD4+ T cells was analysed by qPCR. To assess the effect of miR‐365a‐5p, EVs were loaded with this miRNA, and its levels were detected by qPCR. Memory CD4+ T cells were treated with EVs, EVs‐glyco, and EVs‐miR‐365a‐5p, followed by FACS analysis to determine Th1, Th17, and Treg phenotypes.

Results : The characterization of EVs‐miR39 did not reveal significant differences in size or surface markers CD9/CD63/CD81 when compared to non‐electroporated EVs. qPCR confirmed the presence of miR‐39 in EVs‐miR‐39 and its internalization into CD4 T cells. Analyses showed elevated levels of miR‐365a‐5p in EVs‐miR‐365a‐5p compared to non‐electroporated EVs. In memory CD4+ T cells treated with EVs‐miR‐365a‐5p, a similar trend of immunosuppression was observed as that seen with EVs‐glyco on Th1, Th17, and Treg subpopulations.

Summary/Conclusion : It is suggested that the electroporation process does not alter the essential characteristics of EVs, and effective transfer of miR‐39 as well as miR‐365a‐5p was demonstrated. Our results point out that miR‐365a‐5p seems to be critical for the enhanced suppressive activity of glyco EVs. This opens possibilities for further investigation into the role of miR‐365a‐5p in the immunosuppressive function of EVs derived from UC‐MSC.

Funding : Impact FB 210024, Fondecyt Regular 1211353

Development and functional evaluation of anti‐KRAS extracellular vesicles for the treatment of pancreatic cancer

Presenter: Diego Baranda Martínez Abascal

Vall d'Hebron University Hospital, Barcelona, Spain

Introduction : We explored the loading of therapeutic affimers, small proteins mimicking antibody activity, into EVs in three distinct nanoformats: extravesicular, luminal membrane‐bound and luminal. Affimers were validated to inhibit the difficult‐to‐drug oncoprotein KRAS. Vehiculization on EVs was expected to guarantee affimer protection and to enhance delivery upon vesicle internalization into cancer cells. EVaffimer nanoparticles were produced following scalable methods and are being tested, aiming to develop an innovative nanomedical treatment for pancreatic ductal adenocarcinoma (PDAC).

Methods : DNA constructs were transfected into EV‐producing HEK293F cells. Recombinant proteins were expressed on vesicles from the outset. Through fusion to the CD63 C‐terminus, the affimer was located on the lumen of the vesicle, while fusion with the 2nd loop of CD63 granted external location. Luminal, no membrane‐bound, loading was reached by fusion to EV soluble protein N‐Syntenin. Constructs included mClover3 as a reporter. EVs were isolated by size exclusion chromatography. Cargo loading was quantified by flow cytometry as well as western blot (WB). Functional evaluation was performed by treating PDAC cell lines with EVs and checking the KRAS signalling cascade by WB along with cytotoxicity MTT and cell cycle inhibition assays.

Results : Single particle analysis by flow cytometry showed 88% of fluorescent vesicles from CD63 C‐terminus construct production, while the N‐syntenin construct generated 25% of fluorescent particles and CD63 2nd loop corresponded to 16%. WB showed affimer concentration in CD63 C‐terminal EVs was around 20 times higher than in N‐Syntenin EVs and 100 times higher than in the CD63 2nd loop EVs. Surprisingly, none of the EV conformations showed significant efficacy when functionally evaluated.

Summary/Conclusion : Affimer was found in EV cargo with all tested strategies, with CD63 C‐terminus loading being the most effective approach. Additional assays will be done to identify the reason for EVs inefficacy and their further optimization.

Funding : Project ‘PI22/1301,’ funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union, EMBO Scientific Exchange Grant, VIII Becas Carmen Delgado/Miguel Pérez Mateo a la investigación en cáncer de páncreas (ACANPANC), Horizon 2020 Mimic‐KeY Project ( GA964386 ).

Comparative analysis of EVs isolation methods for enzyme delivery: yield, purity, and functional activity

Verónica Villagrasa‐Araya 1,2,3 , Marc Moltó‐Abad 1,2 , José Luis Corchero 2,4,5 , María Fidel 1 , Diego Baranda Martínez Abascal 1,2 , Roser Ferrer 1,3 , Simó Schwartz Jr. 1,3 , Joaquin Seras‐Franzoso 1,2,4 , Ibane Abasolo 1,2,6,3

1 Clinical Biochemistry, Drug Delivery and Therapy Group (CB‐DDT), Vall d'Hebron Research Institute (VHIR), Vall d'Hebron University Hospital, Barcelona, Spain; 2 Centro de Investigación Biomédica en Red, Bioingeniería, Biomateriales y Nanomedicina (CIBER‐BBN), Barcelona, Spain; 3 Nanomedicine for Therapeutics Applications (NM4T), Instituto de Química Avanzada de Cataluña (IQAC), CSIC, Barcelona, Spain; 4 Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain; 5 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain; 6 Clinical Biochemistry Service, Vall d'Hebron University Hospital, Barcelona, Spain

Introduction : Extracellular vesicles (EVs) are under investigation for their potential as delivery vectors in enzyme replacement therapies (ERT) in lysosomal storage disorders (LSD). Our group aims to leverage this approach for Fabry disease, a specific LSD, where the affected enzyme is alpha‐galactosidase A (GLA).

Methods : This study evaluates three isolation and purification methods to compare their performance in terms of yield, purity and functional activity of EVs and GLA. EVs were obtained from HEK293, CHO and immortalized mesenchymal stem cells (MSC) following transient and stable transfection. The tested isolation methods include precipitation (PP), tangential flow filtration (TFF) combined with size exclusion chromatography (SEC), and benchtop filtration combined with SEC (BTF‐SEC). EVs were quantified using nanoparticle tracking analysis (NTA), purity was determined by western blot, and GLA's potency was assessed through enzyme activity assays and in vitro efficacy assays.

Results : Results indicate that although the PP method renders fewer EVs, it offers a higher quantity of GLA, probably due to the co‐precipitation of free GLA. The EVs BTF‐SEC and TFF‐SEC showed similar EV and GLA yields, with a slight advantage in GLA for the BTF‐SEC method. In terms of activity and efficacy, enzymatic activity was the highest in the EVs obtained with PP, followed by BTF‐SEC. When EV extracts were incubated in cell cultures derived from Fabry KO mice, the efficacy was higher in those extracts with higher enzymatic activity. Among different cell lines, HEK293 cells grown in suspensions produced the highest EV yields but exhibited the lowest enzymatic activity. This finding suggests post‐transcriptional modifications of the protein, such us glycosylation, are important to keep the enzyme's functionality also in EVs.

Summary/Conclusion : In summary, our results suggest that the BTF‐SEC method ensures a high activity of the GLA enzyme in EV fractions in different cell cultures, making this a more economical and straightforward method. However, for studies prioritizing EV purity, TFF‐SEC may be a preferred method.

Funding : This study was supported by the Instituto de Salud Carlos III, co‐funded by the European Union and the Catalan Government's Strategic Plan for Research and Innovation in Health: NEX‐TRY (PI21/0936), DipEV (PI22/1301); European Commission: Mimic‐KEY ( GA964386 ); PERIS (SLT01720000181)

Human dental pulp stem cell derived extracellular vesicles as a therapeutic strategy in modulating inflammation and alleviating periodontitis

Presenter: Hyekyoung You

Chonnam National University, Gwangju, Republic of Korea

Introduction : Periodontitis is one of the most prevalent infectious diseases worldwide, causing damage to the soft tissue surrounding the teeth. It results in the inflammatory loss of alveolar bone, eventually leading to tooth loss. Human dental pulp stem cells derived extracellular vesicles (hDPSC‐EV), nano‐sized vesicles containing genetic material, have emerged as a promising cell source for regenerative medicine and tissue engineering due to their accessibility, versatility, low immunogenicity, and ethical advantages. This study examines the therapeutic potential of hDPSC‐EV for periodontitis using both in vitro and in vivo periodontitis models.

Methods : hDPSCs were isolated from healthy supernumerary teeth, and exosomes were extracted using Exoquick‐TC reagent according to the manufacturer's protocol. An in vitro periodontitis model was established by exposing human periodontal ligament cells (hPDLs) to lipopolysaccharide (LPS) derived from oral pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis . The effects and mechanisms of hDPSC‐EV on inflammation in LPS‐induced hPDLs were examined using RT‐qPCR and western blot analysis. An in vivo mouse model of periodontitis was generated by placing a 5‐0 silk ligature and subsequently treated with either hDPSC‐EV or PBS. After 4 weeks, maxillae were isolated from the mice and examined using micro‐computed tomography (micro‐CT) and histological analysis.

Results : hDPSC‐EV treatment significantly downregulated pro‐inflammatory cytokines, including IL‐1β, TNF‐α, IL‐6, and IL‐8, in LPS‐induced hPDLs by modulating the NF‐κB pathway. In vivo, mice treated with hDPSC‐EV showed reduced alveolar bone loss and overall periodontitis severity compared to the control group.

Summary/Conclusion : hDPSC‐EVs offer a promising therapeutic potential for treating periodontitis by reducing inflammation and preserving alveolar bone. These findings underscore the potential of hDPSC‐EV in addressing the challenges in periodontitis treatment.

Activation of OT‐1 T cells by tumour‐targeting small extracellular vesicles loaded with SIINFEKL peptide leads to proliferation of T cells and redirection of the cytotoxic activity of T cells to targeted tumours in OT‐1 mice

Presenter: Yang Lu

The University of Texas, Austin, Texas, USA

Introduction : Cancer immunotherapy has been successful in treating some cancer patients, but it still fails in most cases. Despite novel potential tumour neoantigens being discovered, cancer is in general an internal disorder lacking potent antigens to elicit strong anti‐tumour immune responses compared with foreign antigens. In this study, we tested a new strategy using tumour‐targeting small extracellular vesicles (sEVs) to redirect host preexisting T cell‐mediated non‐cancer immunity toward cancer cells through targeted delivery of relevant antigens to tumours.

Methods : To develop a tumour‐targeting sEV platform, we designed a cell membrane‐anchoring approach to display a single chain variable fragment (scFv) antibody (derived from anti‐HER2 trastuzumab) on sEV surface. For proof of concept, we used HER2‐targeting sEVs for delivering ovalbumin (OVA)‐derived peptide SIINFEKL to targeted tumours in OT‐1 transgenic mice. We developed a method to load SIINFEKL peptides on the sEVs in an MHC‐I‐dependent manner.

Results : We confirmed a targeting role of the scFv on the sEVs in facilitating receptor‐mediated endocytosis of the sEVs through binding to the HER2 overexpressed on targeted tumour cells. Treatment of OT‐1 T cells with SIINFEKL‐loaded sEVs activated OT‐1 T cells, shown by a marked increase in the percentage of CD69‐positive T cells, and stimulated proliferation of OT‐1 T cells, shown by a proliferation assay of T cells labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Co‐culture of OT‐1 T cells with HER2‐overexpressing tumour cells induced massive apoptosis of the tumour cells only after treatment with the cells with SIINFEKL‐loaded HER2‐targeting sEVs. In our studies carried out in mice, we found that intraperitoneal administration of SIINFEKL‐loaded HER2‐targeting sEVs also activated CD8+ T cells in OT‐1 mice. Compared with the findings from the OT‐1 mice treated with the HER2‐targeting sEVs without loading with SIINFEKL or treated with SIINFEKL‐loaded untargeted sEVs, treatment of the mice with SIINFEKL‐loaded, HER2‐targeting sEV strongly inhibited growth of HER2‐overexpressing tumours implanted in OT‐1 mice.

Summary/Conclusion : Our findings support our ongoing and future work that pre‐existing T cell‐mediated immunity, such as anti‐viral immunity acquired through natural infection or vaccination, can be recalled and redirected to targeted tumours by tumour‐targeting sEVs loaded with MHC‐I‐compatible peptides.

Funding : This study was supported by the CPRIT (RP200271) and BCRF (23‐051).

Therapeutic targeting of triple negative breast cancer using engineered small extracellular vesicles for delivery of miRNA‐204‐5p

Presenter: Kavitha Unnikrishnan

Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, India

Introduction : Breast cancer is the leading cause of cancer‐related mortality among women worldwide, with triple‐negative breast cancer (TNBC) accounting for 10%–20% of cases. Despite recent advancements in cancer treatment, TNBC patients often confront poor prognosis and limited targeted therapeutic options compared to other subtypes, mainly because of the lack of oestrogen, progesterone, and HER2 receptors. Strategies to deliver small RNA molecules such as miRNAs are an exciting and potential therapeutic option. However, a robust gene delivery system is generally recognized as a bottleneck in translating the technology. Utilizing small extracellular vesicles (sEVs) to deliver therapeutic miRNAs to the target tumour site will provide a proof of concept for the functional sEVs to act as natural nanovesicles.

Methods : sEVs were isolated from TNBC cells and were characterized by western blotting, DLS, and TEM analysis. The isolated sEVs were then conjugated to GE11 peptide by the EDC/sulpho NHS reaction. Further, the uptake efficiency of sEVs‐GE11 was determined in non‐TNBC and TNBC cell lines by FACS analysis, and the therapeutic carrier ability of sEVs was also analysed.

Results : In this study, we surface engineer sEVs by conjugating with cancer specific peptides to enhance the targeting potential of these vesicles towards TNBC. We show that the conjugation of EGFR targeting peptide, GE11, enhances the specificity and selectivity of sEVs towards TNBC. Our big data analysis reveals that miR‐204‐5p is significantly downregulated in TNBC patients and possesses therapeutic value. We encapsulate miR‐204‐5p in the engineered sEVs and show that the miRNA affects several hallmarks of cancer, including proliferation and migration.

Summary/Conclusion : Our study shows that GE11 conjugated sEVs specifically deliver miR‐204‐5p to TNBC cells, and miR‐204‐5p inhibits the proliferation and migration of TNBC cells. This approach offers a rapid and efficient method for the targeted delivery of nucleic acids into cancer cells, thereby expanding the potential for cancer treatment.

Funding : This study was supported by the Intramural funding of the Rajiv Gandhi Centre for Biotechnology.

Targeted LOXL1‐AS1‐siRNA delivery using peptide‐modified extracellular vesicles and focused ultrasound to suppress medulloblastoma metastasis

Anh Duy Do 1 , Shing‐Shung Chu 1 , Tai‐Tong Wong 1 , Shian‐Ying Sung, 1,2,3 , Chia‐Ling Hsieh 1,3

1 Taipei Medical University, Taipei, Taiwan; 2 Chung Shan Medical University, Taichung, Taiwan; 3 Chung Shan Medical University Hospital, Taichung, Taiwan

Introduction : Medulloblastoma (MB) metastasis is a leading cause of death in paediatric patients with brain tumours. Current treatments, primarily relying on radiotherapy, aim to control metastatic recurrence but often lead to long‐term neurocognitive deficits. Thus, there is a critical need for targeted therapies against metastatic MB.

Methods : This study explores a gene therapy approach using extracellular vesicles (EVs) to deliver small‐interfering RNAs (siRNAs) targeting MB. We generated MB‐targeted EVs (MB‐tEVs) by modifying EV‐producing cells to express the E1‐3 peptide, specific to MB cells. These MB‐tEVs were loaded with siRNAs targeting LOXL1‐AS1, a pro‐metastatic long non‐coding RNA in sonic‐hedgehog MB (SHH‐MB). To enhance brain delivery, we applied microbubble‐enhanced focused ultrasound (FUS) to increase EV accumulation in the mouse brain.

Results : The expression of the E1‐3 peptide significantly increased MB‐tEV uptake by MB cells, facilitating effective silencing of LOXL1‐AS1. This silencing led to suppression of LOXL1‐AS1‐mediated metastatic characteristics in SHH‐MB cells in vitro. In vivo, systemic administration of siRNA‐loaded MB‐tEVs, coupled with FUS, reduced LOXL1‐AS1 expression in the mouse brain, decreased metastatic burden, and extended survival in an SHH‐MB orthotopic model.

Summary/Conclusion : This study presents promising evidence supporting the combined use of tumour‐targeted EVs and microbubble‐enhanced FUS for therapeutic siRNA delivery in metastatic MB. Our findings suggest that this approach may enhance conventional therapies and improve clinical outcomes for children with this aggressive paediatric brain tumour.

Funding : NSTC 113‐2314‐B‐040 ‐031

Development of a PSMA‐targeted therapy based on extracellular vesicles for the treatment of castration‐resistant prostate cancer (CRPC)

Shian‐Ying Sung 1,2,3 , Mafewu Olga Raboshakga 1 , Anh Duy Do 1,4 , Chia‐Ling Hsieh 1,3

1 Taipei Medical University, Taipei, Taiwan; 2 Chung Shan Medical University, Taichung, Taiwan; 3 Chung Shan Medical University Hospital, Taichung, Taiwan; 4 Pham Ngoc Thach University of Medicine, Ho Chi Minh City, Vietnam

Introduction : Prostate cancer (PCa) is one of the most common cancers in men, particularly in older populations. While often slow‐growing, aggressive forms of PCa are resistant to conventional treatments, highlighting the need for innovative therapeutic strategies. Current therapies suffer from non‐specificity and toxicity. Mesenchymal stem cells (MSCs) have shown potential due to their anti‐inflammatory, immunomodulatory, and tumour‐targeting capabilities, but tumourigenic risks and immune rejection limit their use. Exosomes, nano‐sized vesicles, offer a promising cell‐free alternative due to their biocompatibility, stability, customizable cargo, and targeted delivery potential.

Methods : This study aimed to develop a targeted exosome‐based drug delivery system for PCa therapy. MSC‐derived exosomes were engineered to express a prostate‐specific membrane antigen (PSMA)‐targeting single‐chain variable fragment (ScFv) on their surface to enhance specificity. These exosomes were loaded with siRNA against ADAM9 (siADAM9), a gene associated with PCa progression, to improve therapeutic efficacy further.

Results : Engineered exosomes exhibited high purity and intact structure, ensuring effective drug delivery. Protein analysis confirmed successful PSMA‐ScFv expression on the exosomal membrane via LAMP2 anchoring. This modification enabled targeted binding to PSMA‐expressing PCa cells (LNCaP and CWR22V1), resulting in significantly higher uptake than PSMA‐negative cell lines (DU145 and PC3), thereby reducing off‐target effects. siADAM9 encapsulation within these exosomes further enhanced therapeutic efficacy. In vitro results showed a significant reduction in tumour cell invasiveness, as evidenced by decreased migration and invasion.

Summary/Conclusion : Combining PSMA‐ScFv‐modified exosomes for targeted delivery and siADAM9 for gene silencing offers a promising therapeutic strategy for PCa. This dual‐action approach effectively targets multiple pathways involved in disease progression and represents a potential strategy to improve treatment efficacy and specificity in castration‐resistant PCa.

Funding : This study was supported by the NSTC 113‐2331‐B‐038‐010

ExoVia, a bioactive umbilical cord MSC‐derived exosome product, enhances hair follicle revitalization and protection

Presenter: Martin Sieber

BIONET Corp, Taipei, Taiwan (Republic of China)

Introduction : Hair loss is a significant concern for many individuals, prompting the exploration of innovative approaches for hair follicle revival. One promising strategy involves the use of exosomes derived from umbilical cord mesenchymal stem cells (UCMSCs), which are known to carry various bioactive factors that can influence cellular behaviour. This study investigates the potential of UCMSC‐derived exosomes to enhance the proliferation of hair cells and improve the overall health and function of hair follicles. We employ bioinformatics tools to analyse the biological pathways and molecular networks associated with these exosomes. Our findings not only enhance the understanding of the role of exosomes in hair regeneration but also provide valuable insights for the development of new therapeutic strategies.

Methods : Cell proliferation assays were conducted using the MTS assay to assess cell viability. The mRNA gene expression in the cells was measured using real‐time PCR. Cell migration was evaluated using a scratch assay. The secretion levels of inflammatory factors were determined through ELISA. Additionally, ingenuity pathway analysis (IPA) (Qiagen) was employed to identify canonical pathways and upstream regulators.

Results : UCMSC‐derived exosomes demonstrate a range of beneficial effects on hair follicle stem cells, hair follicle dermal papilla cells, and keratinocytes. They promote significant proliferation of hair follicle cells in a dose‐dependent manner and markedly enhance keratinocyte migration within 24 h. Additionally, these exosomes upregulate hair growth‐related genes, including VCAN, ALP, WNT5A, VEGFA, and SOD1, while downregulating the hair loss gene DKK1. Under oxidative stress conditions, UCMSC exosomes facilitate the restoration of cell numbers and reduce IFN‐γ secretion by 54% in stimulated PBMCs, indicating their anti‐inflammatory properties. Pathway analysis suggests that UCMSC exosomes are involved in key signalling pathways associated with hair growth, with IPA analysis revealing significant engagement in the PI3K/Akt signalling pathway.

Summary/Conclusion : UCMSC‐derived exosomes demonstrate significant potential for revitalizing hair follicles by promoting the proliferation of various types of hair cells, enhancing their migratory capacity, and combating inflammation. Importantly, we have identified key molecular mechanisms that enhance our understanding of how UCMSC‐derived exosomes support hair regeneration. By harnessing these benefits, UCMSC‐derived exosomes emerge as a promising option for clinical applications in hair regeneration therapies.

Subtopic Biologic activity and therapeutic potency of EVs 2

Dual‐mode action of scalable, high‐quality engineered stem cell‐derived SIRPα‐extracellular vesicles for treating acute liver failure

Presenter: Seonghyun Kim

SHIFTBIO Inc., Seoul, Republic of Korea

Introduction : Acute liver failure (ALF) is a critical inflammatory condition characterized by rapid hepatocyte death, impaired liver regeneration, and high mortality rates. This study introduces a novel dual‐mode action therapeutic approach using extracellular vesicles expressing Signal Regulatory Protein Alpha (SIRP‐EVs) derived from genetically engineered mesenchymal stem cells (MSCs). These SIRP‐EVs are designed to concurrently resolve necroptosis and promote liver regeneration.

Methods : We evaluated CD47 expression across diverse ALF models using histological analysis, western blot, confocal microscopy, and spatial transcriptomics. EVs were harvested through a 3D bioreactor‐based process and subsequently purified. These purified EVs underwent established characterization assays. After a single systemic injection, CD47‐dependent accumulation of SIRP‐EVs in liver tissues was evaluated using the in vivo imaging system. The therapeutic efficacy of SIRP‐EVs was determined by measuring liver enzyme levels, examining histological changes, tracking survival rates, assessing principal component analysis of RNA sequencing data, and analysing inflammatory cytokine profiles.

Results : Our studies identified CD47 and SIRPα as promising therapeutic targets for ALF. We developed a scalable 3D bioreactor process for producing high‐purity SIRP‐EVs with significant yields of SIRP‐EVs, while preserving MSC properties. SIRP‐EVs effectively eliminate necroptotic hepatocytes by targeting CD47 and simultaneously enhance the regenerative capabilities of myeloid cells through delivery of MSC‐derived cargo. Comprehensive in vivo studies demonstrate that SIRP‐EVs exert significant therapeutic effects in ALF.

Summary/Conclusion : These findings highlight SIRP‐EVs' potential as a dual‐mode action therapeutic for ALF, offering prospects for application in other inflammatory diseases. Moreover, these results pave the way for advancing EV‐based therapies toward clinical implementation.

Funding : This research was supported by SHIFTBIO INC. and funded by Grant Numbers: 23C0111L1, H123C0883, HI23C154100, and S3252275.

Mesenchymal stem cell derived small extracellular vesicles promote mitochondrial repair of dopaminergic neuronees via Homer 3 in a mouse model of Parkinson's disease

Presenter: Chan‐Lin Lian

Department of Neurology, China (People's Republic)

Introduction : Parkinson's disease (PD) is a common neurodegenerative disease. Mitochondrial dysfunction has been increasingly recognized as a critical factor in the pathogenesis of various neurological diseases, especially PD. Emerging studies reveal that mesenchymal stem cell‐derived small extracellular vesicle (MSC‐sEV) treatment presents a promising cell‐free approach to alleviate neurodegenerative disorders.

Methods : Here, a mouse model of PD was induced using 1‐methyl‐4‐phenyl‐1,2,4,5‐tetrahydropyridine (MPTP). MSC‐sEV was intranasally administered and the behavioural tests were performed, including total stere, rearing times and rotarod speed. The dopaminergic (DA) neuronees and mitochondrial function were evaluated, and RNA‐seq and Homer3‐knockout mice were used to investigate the potential signalling pathways.

Results : We found that the intranasal administration of MSC‐sEV significantly increased the behavioural parameters of total stere, rearing times and rotarod speed, and protected the injured dopaminergic (DA) neuronees in the striatum nigra. Furthermore, the treatment of MSC‐sEV suppressed mitochondrial dysfunction and increased the number of restored mitochondria in the substantia nigra. RNA‐seq showed that the administration of MSC‐sEV reversed the levels of Homer3 in the PD mouse model. We further found that Homer3 knockout mice presented behavioural disorders and showed dopaminergic neuronal deficits. However, the treatment of MSC‐sEV did not present the neuroprotection in Homer3 knockout mice.

Summary/Conclusion : Thus, our findings indicate that mesenchymal stem cell‐derived small extracellular vesicles promote mitochondrial repair of dopaminergic neuronees via Homers 3 in a mouse model of Parkinson's disease. It provides novel insights into the treatment of PD.

Developing extracellular vesicles as next‐generation gene therapy vectors for the treatment of glioblastoma

Presenter: Munzur Lacin

Erasmus Medical Center, Rotterdam, The Netherlands

Introduction : The treatment of glioblastoma (GB), the most aggressive brain tumour, remains a significant clinical challenge. Despite extensive treatment, tumour recurrence is inevitable, leading to a median survival of only 10 to 13 months. The tumour's ability to evade therapy is driven by inherent mechanisms like genetic instability and immune evasion. By engineering extracellular vesicles (EVs) as a new delivery vehicle, with the potential to load multiple therapeutic cargos at once, we aim to advance targeted therapy for GB cells, addressing limitations of conventional drug delivery.

Methods : To engineer therapeutic EVs, we employed a method to encapsulate full‐length therapeutic DNA derived from adenoviral vectors within EVs, resulting in hybrid particles termed ‘Adenosomes.’ To target GB cells, we developed novel Adenosome variants carrying DNA encoding kinase‐inhibiting peptides (KIPs). We hypothesized that this approach will enable highly efficient delivery of gene therapy to GB cells, leading to targeted disruption of aberrant kinase signalling pathways and subsequent cell death.

Results : We initially pre‐screened several KIP peptides (Myr‐KIPs) on GB cell cultures to identify the most effective variants. This screening included Myr‐KIPs targeting DNA‐PK and CDK1, with the CDK1‐targeting KIP demonstrating a significant anti‐tumour effect on U87‐MG and GS.607 GB cells. Building on these results, we next created adenosomes loaded with DNA encoding GFP‐KIPs. The adenosomes were characterized using q‐PCR for DNA payload quantification and nanoparticle tracking analysis for particle concentration. Mass spectrometry proteomics confirmed an overall protein profile consistent with exosome‐like characteristics. Cell viability assessments (Cell‐Titer Glo assay) and microscopic morphological analyses demonstrated growth‐inhibitory effects of adenosome‐KIPs on primary GB cultures, with a reduction in cell count compared to controls. Importantly, astrocytes (non‐cancerous cells) remained relatively unaffected.

Summary/Conclusion : We have developed adenosomes, we have employed a novel technology (adenosomes) for encapsulating therapeutic DNA within EVs. In our GB model, adenosomes expressing KIPs exhibited selective cytotoxicity toward GB cells, underscoring their potential as a targeted gene therapy tool for cancer treatment applications.

Functional recovery induced by extracellular vesicles from adipose tissue stem cells 24 h post‐stroke in hypertensive rats

Francieli Rohden 1 , Luis Pedro Bernardi 1 , Thomas Hugentobler Schlickmann 1 , Felippo Bifi 1 , Debora Branchi 1 , Diogo de Souza 1

1 Federal University of Rio Grande do Sul, Porto Alegre, Brazil

Introduction : Approximately 17 million people worldwide experience a stroke each year, making it one of the leading causes of adverse outcomes and mortality. Systemic arterial hypertension (SAH) is the main risk factor for developing stroke. This study aims to propose a therapeutic approach for post‐stroke neuroregeneration using extracellular vesicles (EVs) derived from human mesenchymal stem cells isolated from adipose tissue (hAT‐MSC), administered intranasally.

Methods : Spontaneously hypertensive Wistar Kyoto rats (SHR) and normotensive control Wistar Kyoto (WKY) rats, both male and female (90–120 days), were subjected to focal permanent ischaemic stroke and treated with hAT‐MSC‐derived EVs (200 µg/kg) 24 h after the stroke. The symmetry of the forepaws (cylinder task) and short‐ and long‐term memory (Open Field) were evaluated.

Results : In this study we observed that in WKY rats, spontaneous recovery in naive animals began on day 21 post‐stroke, while treated animals started to show recovery as early as Day 7. In contrast, the response to ischaemia in SHR rats was significantly different, demonstrating a chronic effect with no spontaneous recovery of forepaw symmetry. After treatment with EVs, female SHR rats exhibited improvements in asymmetry; however, the males only showed partial recovery of forepaw symmetry. Although memory tests are still ongoing, it has already been noted that untreated (naive) SHR animals display memory deficits.

Summary/Conclusion : These findings underscore the potential of hAT‐MSC‐derived EVs as a promising therapeutic strategy for stroke treatment. The observed partial recovery of forepaw symmetry in SHR males treated with EVs suggests that this approach may mitigate some of the chronic motor deficits associated with hypertensive ischaemic events. Additionally, the ongoing analysis of memory outcomes is expected to provide further insights into the cognitive benefits of EV treatment. Future studies should explore the underlying mechanisms by which EVs promote neuroregeneration and angiogenesis, as well as optimize dosing strategies and administration timing to enhance therapeutic efficacy. The use of EVs offers a non‐invasive and innovative method that could potentially be translated into clinical applications, improving recovery and quality of life for stroke patients, especially those with underlying conditions such as systemic arterial hypertension.

Funding : This study was supported by the INCT/CNPQ and UFRGS.

Engineered plant‐derived nanovesicles‐based Janus aerogel inhibits energy metabolism in multidrug‐resistant bacteria to accelerate infected wound healing

Presenter: Tong Liao

Southern Medical University, Guangzhou, People's Republic of China

Introduction : The development of an effective wound dressing for combating bacterial infections and simultaneously addressing complications such as delayed healing, chronic inflammation, and reinfection is highly desirable in clinical practice.

Methods : In this study, we proposed an engineered grapefruit‐derived nanovesicle (GDN) combined with a Janus aerogel for multidrug‐resistant‐bacteria infected wound. The GDN are modified with silver nanodots (GDN@Ag, GAg) via a mild in situ deposition method and integrated into a Janus aerogel reconstructed from grapefruit peel.

Results : The resulting Janus aerogel dressing demonstrated strong capabilities in wound exudate absorption on one side and anti‐external contamination on the other side. The gradual release of GAg provides effective bacterial clearance and biofilm inhibition against multidrug‐resistant Staphylococcus aureus (MRSA). Transcriptomic and metabolomic analysis revealed that the aerogel dressing disrupts bacterial organelle membrane potential, impairs the tricarboxylic acid cycle, downregulates enzyme activities, and increases the production of reactive oxygen species (ROS), thereby facilitating bacterial elimination. The dressing could also modulate antioxidant and energy pathways in damaged skin tissues, re‐polarize inflammatory macrophages into anti‐inflammatory phenotypes, reduce mast cell infiltration, and enhance angiogenesis, thus accelerating tissue regeneration. In animal models, omics analysis reveals the dressing exhibited exceptional sustained antimicrobial activity alongside significant wound healing promotion by modulating the functional balance among bacteria, immune cells, and skin tissues.

Summary/Conclusion : This study tackles the challenges of difficult‐to‐heal infected wounds by presenting a multifunctional Janus antimicrobial dressing, offering a gentle yet efficient strategy for the engineering of plant‐derived nanovesicles with metallic nanoparticles. Furthermore, this research provides a framework for the cross‐regulatory use of plant nanovesicles in tissue repair and introduces potential strategies for the development of advanced wound dressings.

Funding : This study was supported by the National Natural Science Foundation of China (82302639). National Natural Science Foundation of China (8217081536). National Science Fund for Distinguished Young Scholars (82025024).

MSC‐EVs treatment changes microglial activation parameters in ischaemic stroke models: a systematic review with meta‐analysis

Presenter: Luis Pedro Bernardi

Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Introduction : Mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) have shown promising results for treating ischaemic stroke. Their immunomodulatory properties may influence microglial responses and local inflammation following ischaemia. However, a comprehensive investigation of MSC‐EVs' effects on microglia is lacking. Therefore, this systematic review and meta‐analysis aimed to assess whether MSC‐EVs treatment alters microglial responses in in vivo or in vitro ischaemic stroke models.

Methods : In accordance with the PRISMA 2020 Statement, we searched PubMed, Web of Science, and EMBASE up to October 2023 for studies assessing microglial activation, morphology, autophagy, phagocytosis, or viability responses to MSC‐EVs treatment in in vivo or in vitro ischaemic stroke models. We calculated standardized mean differences and confidence intervals using an inverse‐variance weighted random‐effects model. Heterogeneity was estimated using the I 2 statistic, and the risk of bias was assessed with an adapted version of the SYRCLE's questionnaire.

Results : The search identified 297 studies, and 27 met the inclusion criteria. In in vivo ischaemic stroke models, MSC‐EVs reduced the number (SMD = −1.45 [95% CI = −2.19, −0.71]; adjusted (adj.) p value < 0.001), surface area (SMD = −1.10 [95% CI = −2.10, −0.11]; adj. p value = 0.03), and fluorescence intensity (SMD = −1.25 [95% CI = −2.09, −0.4]; adj. p value = 0.005) of Iba1+ cells, as well as the number of Iba1+ cells co‐expressing pro‐inflammatory markers (SMD = −2.13 [95% CI = −2.96, −1.30]; adj. p value < 0.001). Conversely, MSC‐EVs increased the number of Iba1+ cells co‐expressing anti‐inflammatory markers (SMD = 1.79 [95% CI = 1.06, 2.52]; adj. p value < 0.001). In in vitro models, our results revealed decreased TNF‐α (SMD = −6.16 [95% CI = −8.95, −3.37]; adj. p value < 0.001), IL‐1β (SMD = −3.45 [95% CI = −5.57, −1.32]; adj. p value = 0.002), and IL‐6 (SMD = −2.95 [95% CI = −4.50, −1.40]; adj. p value < 0.001) concentrations in the culture medium.

Summary/Conclusion : Our meta‐analysis showed that MSC‐EVs modulate microglial responses to ischaemia, decreasing pro‐inflammatory and increasing anti‐inflammatory responses. These findings highlight the potential of microglia as therapeutic targets in MSC‐EVs‐based treatments for ischaemic stroke.

Funding : This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

A comparative analysis of anti‐inflammatory properties in nano‐ and microvesicles from Allium cepa : does size influence efficacy?

Presenter: Razieh Dalirfardouei

Hamadan University of Medical Sciences, Hamadan, Iran

Introduction : Nano‐ and micro‐vesicles naturally derived from plants and medicinal herbs are attracting considerable interest in the medical and pharmaceutical fields due to straightforward and efficient scaling‐up methods for isolation. The conventional extract of Allium cepa, with a long history in traditional medicine, has a striking therapeutic function, especially anti‐inflammatory and antioxidant activity. Despite this interest, the biological activity of A. cepa ‐derived nanovesicles (AcDNV) remains unclear. Our study aims to investigate and compare the reparative effects of large and small EVs derived from A. cepa in a full‐thickness animal wound healing model.

Methods : After isolation and characterization of AcDNVs by TEM, DLS, and BCA, the anti‐oxidant activity of AcDNVs was examined using the DPPH assay. A full‐thickness excisional wound was created on the dorsal skin of each C57BL/6 male mouse. Twenty mice were divided into four groups: the PBS group, the ethanolic extract group, the lEV group, and the sEV group. The wound tissues were excised at days four and seven to evaluate inflammation through M1/M2 polarization and RelA gene expression.

Results : The cup‐shaped vesicles isolated from A. cepa showed strong free radical scavenging activity. Moreover, gene expression analysis revealed that lEV and sEV significantly downregulated iNOS/ARG1 compared with the control group. The NF‐κB signalling pathway, demonstrated as a pro‐inflammatory pathway, was analysed by RelA expression. The results showed that the expression of RelA was remarkably decreased in both the lEV and sEV groups ( p value < 0.01).

Summary/Conclusion : These results demonstrated that the lEV and sEV derived from A. cepa have comparable anti‐inflammatory activity. Both lEV and sEV preferentially lead to M1/M2 polarization and decrease NF‐ K B expression. Therefore, microvesicles from A. cepa can be a promising anti‐oxidant candidate for oxidative stress and inflammation conditions without specific equipment requirements.

Funding : The study was supported by the Hamadan University of Medical Sciences [grant number: IR.UMSHA.AEC.1402.024].

Effect of red blood cell‐derived Klotho‐engineered extracellular vesicles on renal cell pro‐fibrotic changes via TGF‐β inhibition

Tunahan Ergunay 1 , Michela Arena 1,2 , Alessia Brossa 1 , Alessandro Gori 3 , Benedetta Bussolati 1

1 University of Turin, Turin, Italy, 2 University of Naples Federico II, Naples, Italy, 3 Consiglio Nazionale delle Ricerche, Rome, Italy.

Introduction : Klotho is an anti‐ageing protein whose expression significantly declines with ageing and kidney injury. Studies have also shown that increasing Klotho expression or supplementing it externally provides therapeutic benefits in kidney disease models, particularly by reducing kidney fibrosis. Klotho has been reported to bind to the TGF receptor, thereby blocking TGF binding and inhibiting TGF‐β signalling. We here engineered red blood cell (RBC)‐derived EVs with Klotho peptide (RBC‐EVKP) to investigate its effects on epithelial mesenchymal transition (EMT) and kidney fibrosis.

Methods : EVs were isolated from human donors' RBCs via ultracentrifugation. Klotho peptide was synthesized and linked to the EV membrane using click chemistry. To confirm engineering of vesicles and delivery to cells, a fluorophore was added to the peptide. To evaluate the effects of engineered EVs, human kidney cells (HK2 and ciPTEC) were pre‐treated with RBC‐EVKP and subsequently exposed to TGF‐β. Changes in fibrosis and mesenchymal markers were evaluated by real‐time PCR and flow cytometry analyses. To assess the functional outcome of engineered EVs, wound‐healing, migration, and SMAD binding element (SBE) reporter assays were performed.

Results : The engineering and delivery of RBC‐EVKP to target cells were confirmed by flow cytometry. Fluorophore labelled Klotho peptide showed membrane and cytoplasmic localization on target cells. Treatment of kidney cells with RBC‐EVKP and TGF‐β showed decreased RNA levels of Fibronectin, Collagen 1 and N‐cadherin compared to cells treated with TGF‐β. Functional assays showed reduced wound closure and migration levels on RBC‐EVKP‐treated cells compared to TGF‐β‐treated cells. Cell treatment with TGF‐β and RBC‐EVKP decreased luciferase activity by SBE assay compared to cells treated with control EVs.

Summary/Conclusion : We successfully engineered RBC‐EVs, delivering them to kidney cell models. These engineered EVs effectively prevented fibrosis and EMT induction. Further investigations will prioritize elucidating the mechanisms underlying Klotho peptide‐driven prevention of EMT and fibrosis, including an exploration of alterations in the TGF‐β pathway and the fate of engineered EVs post‐uptake by target cells.

Funding : This project is funded by PNRR; the National Center for Gene Therapy and Drugs Based on RNA Technology, CN3_Spoke8.

Targeted therapy for colorectal cancer stem cells using engineered extracellular vesicles

Farzaneh Malekian, Alireza Shamsian, Van Manh Hung Le, Biljana Gigic, Christoph Kahlert

Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany

Introduction : Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer‐related deaths worldwide. Its tumours contain heterogeneous cell populations, complicating treatment. Among these, cancer stem‐like cells (CSLCs) contribute significantly to tumour progression, chemoresistance, and metastasis. CSLCs, characterized by markers such as CD133, are challenging therapeutic targets. Developing strategies to selectively target and eliminate CSLCs is essential for improved treatment outcomes. Extracellular vesicles (EVs), natural vehicles for intercellular communication, have emerged as promising carriers for targeted therapy due to their ability to deliver therapeutic agents and their potential for modification for targeted delivery.

Methods : In this study, EVs have been isolated from HEK293T cells that were modified to present CD133 antibodies on their surface. EV isolation and characterization followed MISEV 2023 guidelines to ensure purity and quality. EVs were decorated with CD133 antibodies using a chemistry‐based technique and loaded with the cytotoxic agent 5‐fluorouracil (5‐Flourouracil (5‐FU) and Doxorubicin) via electroporation, achieving an efficient cargo‐loading process. The success of EV decoration was assessed using a MACSQuant Analyzer 10. To evaluate targeting specificity, CRC cell lines were genetically modified using CRISPR/Cas9 to create two distinct target cell populations: CD133‐overexpressing cells (DLD1 cell line) and CD133 knockout cells (SW620 cell line). The targeting ability of the modified EVs was assessed by comparing their binding and uptake in these cell populations with their wild‐type counterparts.

Results : The strategies for extracellular vesicle (EV) decoration and cargo loading achieved approximately 80% efficiency, with target specificity enhanced twofold compared to unmodified EVs. Uptake assays were conducted on wild‐type DLD1 and SW620 cells, with further analysis planned for engineered cell lines. The cytotoxic effects of modified EVs will be evaluated across various colorectal cancer (CRC) cell lines, alongside an investigation of their mechanisms of action.

Summary/Conclusion : Primary results from our study suggest that CD133‐modified EVs can target CSLCs, delivering therapeutic cargo effectively in vitro and potentially in vivo. However, further in vitro and in vivo studies are needed to validate and expand these findings. This approach holds promise for targeting chemoresistant populations within CRC tumours, advancing the potential for more precise and effective therapies.

Funding : This study was supported by the DAAD Scholarship.

Neutralizing microparticles inhibits the propagation of Japanese Encephalitis Virus into macrophages

Anjali Singh, Alok Kumar

Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.

Introduction : Microparticles (MPs), annexin‐positive vesicles ranging from 100 to 1000 nm, are critical players in cellular communication, transferring active biomolecules that influence cellular functions. Recent studies highlight that disease states, including viral infections, can alter MPs composition, messageing, and function. This study examined the characteristics of MPs shed by Japanese Encephalitis Virus (JEV)‐infected murine macrophages (RAW 264.7) and their role in promoting infection and inflammation. Furthermore, we demonstrated that neutralizing MPs can control JEV replication, subsequent propagation of JEV copy numbers into macrophages, and inflammation.

Methods : MPs were isolated from cell‐conditioned media using differential ultracentrifugation. MPs were characterized through scanning electron microscopy, flow cytometry, and nanoparticle tracking analysis (NTA). Viral copy numbers and cytokine levels were assessed using RT‐PCR, flow cytometry, and Western blotting. Furthermore, enriched MPs were neutralized, co‐cultured with macrophages, and used for viral propagation assays.

Results : Macrophage activation following JEV infection was evidenced by cytopathic changes, cell death, increased viral copy numbers, and M1‐associated inflammation. NTA and flow cytometry analysis revealed a significant increase in the concentration of Annexin V+ MPs post‐infection. We further noted that JEV‐infected MPs exhibited unique protein profiles, with elevated levels of flotillin‐1 and Annexin V compared to control MPs, indicating altered biogenesis of the MPs. Additionally, we detected enriched JE viral RNA and elevated cytokine levels in the MPs after infection. Furthermore, we found that MPs from JEV‐infected cells carry infectious viruses and facilitate their spread to uninfected cells. Notably, MPs‐mediated infection was inhibited by neutralizing MPs with Annexin V antibody, heat inactivation, and PEG‐TB surface neutralization.

Summary/Conclusion : Together, these results provide insight into the mechanisms of disseminating JEV infections. MPs containing JEV viral particles facilitate the seeding and spread of the infection, representing a potential therapeutic target for controlling JEV propagation.

Funding : This study was supported by the Anusandhan National Research Foundation (ANRF) Science & Engineering Research Board (SERB) Core Research Grant (FILE NO. CRG/2023/006849) and SRF Fellowship (DBT/2020/SGPGIMS/1373) from DBT, Govt. of India to Anjali Singh.

Aloe vera‐derived exosomes as a skin dressing for enhancing wound healing in diabetic patients

Presenter: Lei Zhang

Nankai University, Tianjin, People's Republic of China

Introduction : This study aimed to investigate whether exosomes derived from Aloe vera (Aloe‐Exo) could accelerate the healing of skin wounds in diabetic patients. Specifically, we evaluated the therapeutic potential of Aloe‐Exo when used as a bioactive dressing to treat skin injuries associated with diabetes.

Methods : Exosomes were isolated from Aloe vera extracts and characterized for their size, morphology, and protein markers using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting. Aloe‐Exo was incorporated into a hydrogel‐based dressing and applied to diabetic mouse models with skin wounds. Wound healing progression, inflammation reduction, and tissue repair were evaluated through histological analysis, immunohistochemistry, and biochemical assays. In vitro, human dermal fibroblasts and keratinocytes were treated with Aloe‐Exo to assess cellular proliferation, migration, and anti‐inflammatory responses using cell viability assays, scratch tests, and ELISA.

Results : Aloe‐Exo significantly promoted faster wound closure and enhanced collagen deposition in diabetic mice compared to control groups. Inflammatory cytokine levels were reduced, while the proliferation and migration of skin cells were markedly increased. Aloe‐Exo demonstrated strong antioxidative and anti‐inflammatory properties, aiding in the reduction of oxidative stress and inflammation at the wound site. In vitro studies further revealed that Aloe‐Exo stimulated fibroblast proliferation and keratinocyte migration, both critical for effective wound healing.

Summary/Conclusion : Aloe vera‐derived exosomes provide a promising therapeutic approach for treating skin injuries in diabetic patients. By enhancing wound healing through anti‐inflammatory and antioxidative mechanisms, Aloe‐Exo offers a potential alternative as bioactive dressings for diabetic wound care.

Funding : This work was supported by the Natural Science Foundation of Tianjin Science and Technology Bureau, China [grant number 21JCZDJC01050]; Tianjin Key Medical Discipline (Speciality) Construction Project, China [grant number TJYXZDXK‐047A]; and Tianjin Municipal Health Science and Technology Project, China [grant number TJWJ2021ZD003].

Natural killer cell‐derived extracellular vesicles show therapeutic potential against triple‐negative breast cancer and cancer stem cell populations

Frederic St‐Denis‐Bissonnette 1,2 , Melanie Kirkby 1,2 , Marena Diab 2 , Sarah Cummings 1 , Gauri Muradia 1 , Andrew Stalker 1 , Michele Ardolino 2,4 , Seung‐Hwan Lee 2 , Dylan Burger 2,4 , Lisheng Wang 2,4 , Jessie R. Lavoie 1,2

1 Health Canada, Ottawa, Canada; 2 University of Ottawa, Ottawa, Canada; 3 Carleton University, Ottawa, Canada; 4 Ottawa Hospital Research Institute, Ottawa, Canada

Introduction : Triple‐negative breast cancer (TNBC) is an aggressive breast cancer subtype, accounting for 10%–15% of all breast cancers, with a 5‐year survival rate of 77%. It lacks targeted therapies and posts a high risk of recurrence within the first few years, resulting in poor prognosis and disproportionately impacting younger women. Natural killer cell‐derived extracellular vesicles (NK‐EVs) are promising cancer immunotherapeutics for hard‐to‐treat cancers like TNBC, owing to their cytotoxic, immunomodulatory and biocompatibility properties, enabling them to target local and distant tumours. This study evaluates NK‐EVs' efficacy against various TNBC models (in vitro, ex vivo, and in vivo) and their effects on cancer stem cells (CSC), a subpopulation with stem cell‐like properties linked to TNBC recurrence.

Methods : Clinical‐grade NK‐EVs were produced from the NK92‐MI cell line using a scalable biomanufacturing process, following MISEV guidelines (published in JEV in 2023). Efficacy was evaluated against TNBC cell lines, mammospheres, and patient‐derived xenografts (PDX). Cell viability was measured using a resazurin‐based assay in time‐ and dose‐dependent studies to assess NK‐EVs' cytotoxicity. CSC populations were evaluated using flow cytometry. In vivo studies utilized an immunocompromised TNBC‐PDX mouse model to assess NK‐EVs’ efficacy and safety. Testing includes tumour growth, mouse body weight, CSC populations, and tumour‐infiltrating macrophages. Ethical approval was obtained by the relevant animal use committees.

Results : NK‐EVs induced rapid, dose‐dependent TNBC cell line death, with effects persisting up to 48 h, indicating strong and sustained cytotoxicity. Ex vivo TNBC‐PDX slices further confirmed a robust decrease of TNBC cell viability after 24 h of NK‐EV treatment, with sustained effects over five days. Ongoing in vivo studies are focused on tumour growth following intratumoural and intravenous NK‐EV injections. Preliminary toxicology data suggest high‐dose intravenous injections of NK‐EVs are well‐tolerated (10 13 EVs/kg).

Summary/Conclusion : This is the first study evaluating NK‐EVs' efficacy and safety in a clinically relevant TNBC‐PDX model. Preliminary results indicate NK‐EVs effectively target tumours, underscoring their potential as a novel TNBC therapy. Future studies will investigate NK‐EV biodistribution, pharmacokinetics, and their ability to target metastatic sites.

Novel approach for lung cancer brain metastases: iRGD‐Decorated extracellular vesicles loaded with pemetrexed

Presenter: Linhao Yuan

Beijing Tiantan Hospital, Beijing, People's Republic of China

Introduction : Non‐small cell lung cancer (NSCLC) brain metastases have a poor clinical prognosis due to the existence of the blood‐brain barrier and the limited efficacy of commonly used clinical treatments such as pemetrexed combined with platinum drugs. There is an urgent need for more effective chemotherapeutic approaches to treat NSCLC brain metastases.

Methods : Exosomes were extracted from HEK‐293F cells and loaded with pemetrexed via electroporation. The tumour‐targeting peptide iRGD was conjugated to these extracellular vesicles. The resulting Peme‐EVs‐iRGD was characterized using electron microscopy and nanoflow cytometry. Its efficacy in binding and inhibiting tumour growth was tested in vitro and in vivo using the H2030 lung cancer cell line. Biosafety assessments included blood biochemistry and histopathological analysis of organs and bone marrow.

Results : NSCLC brain metastases exhibit high αVβ3 integrin expression. Compared to free pemetrexed, Peme‐EVs‐iRGD demonstrated superior tumour penetration and inhibition in a non‐small cell lung cancer brain metastasis model by targeting the binding and increasing the local drug concentration of pemetrexed. Additionally, Peme‐EVs‐iRGD showed no significant hepatorenal toxicity or bone marrow suppression.

Summary/Conclusion : The engineered Peme‐EVs‐iRGD effectively targets NSCLC brain metastases, offering enhanced therapeutic efficacy while maintaining high biosafety. This approach presents a promising new strategy for treating brain metastases in NSCLC.

MSC‐derived sEVs combined with a low dose of BMP‐2 synergistically enhance osteoporotic bone repair but fail to improve implant osseointegration

Presenter: Christian Deininger

Paracelsus Medical University, Salzburg, Austria

Introduction : With the increasing incidence of osteoporosis, there is a critical need for innovative therapies to improve osteoporotic fracture repair. Effective fracture healing relies on enhanced bone regeneration alongside robust fracture fixation and implant osseointegration. Extracellular vesicles derived from mesenchymal stem cells have shown potential in promoting bone repair. Building on this, our study aimed to evaluate the effects of GMP‐grade human umbilical cord MSC‐sEVs in enhancing BMP‐2‐mediated bone repair and implant integration. This approach may not only improve therapeutic outcomes but also reduce safety concerns associated with high BMP‐2 doses applied in clinical settings.

Methods : After inducing osteoporosis via bilateral ventral ovariectomy and a specialized diet, 64 rats underwent distal femoral metaphyseal osteotomy. Defects were stabilized with custom Y‐shaped mini‐locking plates and treated with one of four interventions: alginate alone, alginate with hUC‐MSC‐sEVs (2 × 10 9 particles), rBMP‐2 (1.5 µg), or a combination of sEVs and rBMP‐2. In a second study, drillhole defects were created in 42 rats and treated with alginate containing sEVs, rBMP‐2, or both to evaluate screw implant integration. Animals were euthanized 6 weeks post‐surgery, and femora were analysed by micro‐CT, histology, and biomechanical testing to assess bone healing and osseointegration.

Results : Radiographs and µCT showed improved bone union in metaphyseal defects treated with hUC‐MSC‐sEVs and a low dose of BMP‐2 at 6 weeks, which was confirmed by histological analysis. Torsional testing indicated increased stiffness with rBMP‐2 alone or combined with sEVs, while torque was significantly enhanced only in the combination group. Interestingly, sEVs alone did not improve osseointegration, and only the localized application of 1.5 µg rBMP‐2 improved biomechanical outcomes. Further, no additive effect was observed when sEVs were combined with rBMP‐2.

Summary/Conclusion : Combined rBMP‐2 and sEVs improved osteoporotic defect healing compared to either treatment alone but did not enhance implant osseointegration. sEVs alone slightly reduced bone formation around implants, and rBMP‐2 alone was the most effective for implant integration, with no added benefit from combined sEV treatment.

Funding : This work was supported by the Lorenz Böhler Fonds (#1/20), Land Salzburg/IWB/EFRE 2014‐2020 (P1812596; “EV‐TT”), and a PMU‐RIF‐PRE fellowship (2023‐ PRE‐007).

Use of autologous conditioned serum, an extracellular vesicle (EV)‐saturated cell free whole blood secretome triggers pain relief, resolution of inflammation and regeneration

Julio Reinecke

Practice Wehling & Partner, Düsseldorf, Düsseldorf, Germany

Introduction : Customary medications for pain and inflammation obstruct physiologic healing responses and increase the risk of chronification. ACS contains a complex composition of mediators released upon whole blood incubation. Animal and human studies show that this autologous secretome improves acute pain and inflammation, kicks off tissue regeneration and counteracts the notorious chronifying effects of glucocorticoid and NSAID treatment. In addition to, or subsequent to, ACS, numerous techniques have surfaced that uniformLy claim to exploit physiological self‐healing mechanisms. Such include, but are not limited to, fat or bone marrow derived cell or cell fragments and platelet‐rich plasma. The multitude of preparations is as heterogeneous as are the clinical results. Allogenic mesenchymal stem cells as pharmaceutical products have also been developed. The unifying principle of such approaches is the fact that not the cells, but their releasates or secretomes are now believed to be the main active principle. As a therapeutic autologous secretome, ACS has a high standard of reproducibility, reflecting in consistently good clinical efficacy.

Methods : ACS results from in vitro coagulation and incubation of whole patient blood at physiologic temperature for > 3 h employing a CE‐marked medical device. It can be routinely filtered by 0.22 µm, is routinely aliquoted and can be kept frozen for up to 12 months at −20°C or colder. ACS strategy does not aim for a purified single component but employs a complex mixture released by coagulating whole blood.

Results : NTA (Zeta View) determined ACS particle counts regularly are between 10 9 and 10 13 per mL and sizes range from 30 to 200 nm. Composition of ACS secretome (proteins, EV, lipid mediators) differs significantly from baseline blood plasma. We summarize treatment options and results for ACS and illustrate feasible application routes. We present a graphic comparison of processing techniques for the autologous and allogenous preparations described above, demonstrating the simplicity and safety of the method.

Summary/Conclusion : Compared to some other technologies ACS is simple and safe to generate. Treatment efficacy has been demonstrated in numerous studies. One strength is that the therapeutic mechanism is based on multiple mediators, including EV.

Spleen‐derived extracellular vesicles loaded in protein hydrogel for tertiary lymphoid structure formation

Presenter: Jihoon Han

Korea University, Seoul, Republic of Korea

Introduction : Cancer immunotherapy, particularly immune checkpoint blockade (ICB), has transformed cancer treatment but often struggles in tumours lacking pre‐existing immune responses. Our novel hierarchical hydrogel platform encapsulating spleen‐derived extracellular vesicles (EVs) addresses this by fostering tertiary lymphoid structure (TLS) formation, thereby enhancing ICB efficacy. Combined with ICB therapy, this approach shows potential to significantly boost anti‐tumour immunity.

Methods : We followed MISEV2023 guidelines for EV separation and characterization. All animal experiments were approved by the animal department at KIST.

Results : We engineered EV‐encapsulating hydrogels using an EV‐binding CP05 peptide sequence and verified its ability to capture spleen‐derived EVs via binding assay. We then implanted spleen EV‐loaded hydrogels into mice and observed that after 10 days, TLS‐like cell aggregates were formed inside the hydrogel. Finally, we assessed the antitumour effect of this TLS hydrogel on a murine CT26 colon cancer model along with anti‐PD‐1 antibody therapy and demonstrated that the combination group showed dramatic tumour regression with enhanced immune cell infiltration.

Summary/Conclusion : Our study highlights the immune‐enhancing effects of spleen EV‐loaded hydrogels in promoting tertiary lymphoid structure (TLS) formation within tumour environments. By using spleen‐derived EVs, we successfully triggered high immune cell infiltration, aggregation, vessel formation, and germinal centre‐like structures. This immune‐rich environment significantly boosts the efficacy of immune checkpoint blockade (ICB) therapy, converting the tumour microenvironment (TME) into an “immune hot” phenotype. This approach addresses limitations in current immunotherapies, offering broad applicability across diverse cancer types.

Large‐scale isolation of natural killer (NK) cell‐derived extracellular vesicles with anti‐tumour activity to treat patients with relapsed/refractory cancer

Presenter: Emily C. Matchett

Saint Louis University, St. Louis, Missouri, USA

Introduction : New treatments for relapsed, refractory and rare cancers are needed. Glioblastoma (GBM) is an aggressive brain tumour that develops rapidly and lacks effective treatment. Patients have a median survival of 15 months and a 5‐year survival rate of 5.8%. They experience progressive and severe neurological impairment from the disease and from the treatments. Equally devastating is cognitive decline, personality changes and behavioural disorders that significantly diminish quality of life. Another cancer with a poor prognosis is acute myeloid leukaemia (AML), which is characterized by rapid clonal growth of myeloid lineage stem cells in the bone marrow. The 5‐year survival rate is ∼30% as patients become resistant to chemotherapy. We are investigating using EVs derived from natural killer (NK) cells as a new treatment for these diseases. To address the issue of efficient and large‐scale production, we immortalized the human NK cell line NK3.3 and developed an NK EV isolation protocol to maximize yield and activity.

Methods : NK3.3 EVs were isolated from culture supernatants by differential high‐speed centrifugation, sterile filtration, followed by tangential flow filtration (TFF). EVs were characterized by protein concentration, nanoparticle tracking analysis, and enzyme linked immunosorbent assays. Cell counts, cytotoxicity assays, and flow cytometry were used to measure viability of GBM and AML cells after NK EV treatment. Tumour growth in vivo was monitored by bioluminescence imaging.

Results : EVs isolated by TFF range in size from 100 to 200 nm, contain NK receptors and cytolytic proteins important for tumour recognition and killing and decrease tumour growth and viability both in vitro and in vivo. They kill drug‐resistant tumour cells but are nontoxic to healthy cells. Levels of the caspase activator granzyme B in NK EVs correlates with their anti‐tumour activity, making granzyme B a reliable and quantitative biomarker.

Summary/Conclusion : EVs isolated from immortalized NK3.3 by TFF display strong anti‐tumour activity, with no toxicity to normal cells, making them a safe and effective treatment option for patients when current therapeutics fail, such as with AML, or for difficult to treat cancers, such as GBM.

Anti HER2 chimeric antigen receptor (CAR)‐T extracellular vesicles (EVs), an innovative and safe cancer therapy

Presenter: Anat Aharon

Tel Aviv University, Tel Aviv, Israel

Introduction : Chimeric antigen receptor (CAR)‐T cells are genetically engineered T cells, directed against tumour‐associated antigens. Extracellular vesicles (EVs), derived from CAR‐T cells (CAR‐T EVs), preserve CAR‐T function and may overcome CAR‐T cell therapy challenges in patients with solid tumours by better infiltration and less cytokine storm. This study explored whether CAR‐T EVs can be used for effective immunotherapy for solid tumours. For that, EVs obtained from CAR‐T cells, stimulated by HER2 expressing cells or by Her2 recombinant‐coated beads, were compared.

Methods : Anti‐HER2 CAR‐T cells were produced and stimulated by SKOV‐HER2+ cells or by recombinant HER2+coated beads. EVs were isolated from the cell media at multiple time points and compared with EVs of un‐transduced (UT) cells. All EVs types were characterized for their size, concentration, proteins content and functions, using Nano tracking analysis, protein array, and western blot (WB). EVs cytotoxic effects were assessed on breast cancer cells (SKBR‐HER2+), ovarian cells (SKOV‐HER2+ and OVCAR‐HER2‐), glioblastoma cells (U251‐HER2+), and on primary healthy cell culture (kidney, lung, skin, bone marrow mesenchymal stem cells). Cytotoxic effects were measured by IFNγ secretion level (ELISA), methylene blue killing assay and caspase 3/7 activity assay, using live cell imaging (Incucyte).

Results : CAR‐T EVs contained a mixture of small and large EVs. Higher CAR‐T EVs concentrations were found in SKOV‐stimulated cells, compared to CAR‐T EVs of Her2+ coated beads. This is related to the fact that EVs of SKOV‐stimulated cells include a mixture of EVs derived from the cancer cells (∼60%) and CAR–T EVs (∼40%), while EVs of Her2+ coated beads include only CAR‐T EVs. EVs obtained after CAR‐T cells stimulation with Her2+ coated beads contained higher levels of cytokines (e.g., GM‐CSF, I‐309, IL‐13, IL‐17, RANTES) and induced significantly higher death of Her2+cancer cells, compared to UT EVs. That is similar to the cytotoxic effects that were induced by SKOV stimulated CAR T EVs. Moreover, EVs from CAR‐T cells stimulated by Her2+ coated bead did not affect the viability of healthy cells.

Summary/Conclusion : CAR T EVs produced by Her2+coated beads may provide a novel, pure and potent immunotherapy approach, with an effective cytotoxicity and without effect on healthy cells.

Funding : This study was supported by the KAMIN‐ Israel innovation Authority‐68014.

Genetically engineered extracellular vesicles with chimeric antigen receptor of human Stem Cell‐Derived Neutrophils for Treating Glioblastoma

Colin Esmonde 1 , Li Sun 1,2 , Yun Chang 3 , Changchun Zeng 4 , Alfredo Quiñones‐Hinojosa 5 , Xiaoping Bao 3 , Yan Li 1

1 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering, Tallahassee, Florida, USA; 2 Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA; 3 Purdue University, West Lafayette, Indiana; 4 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering, Tallahassee, Florida, USA; 5 Mayo Clinic, Jacksonville, Florida, USA

Introduction : Glioblastoma multiforme (GBM) is the most commonly occurring tumour in the central nervous system, accounting for 80% of brain tumours. Work in gene editing and immunology has led to the development of chimeric antigen receptors (CARs). CARs employing a chlorotoxin receptor domain have recently been incorporated into neutrophils for GBM treatment. However, such cells are liable for inducing body wide off‐ target effects that can be deadly. Extracellular vesicles (EVs) secreted by the cells have been shown to share properties with their parent cells including the expression of CARs and tumour‐lytic effects. However, this has never been investigated in CAR neutrophils.

Methods : The DNA for the CAR‐CLTX was inserted into the AAV1 safe locus of induced pluripotent stem cells, which were differentiated into neutrophils. Conditioned media were collected along the differentiation and went through the extraPEG EV isolation. EVs were quantified by nanoparticle tracking analysis, electron microscopy, and western blot. Proteomics analysis of protein cargo and microRNA (miRNA) sequencing of the CAR EVs was performed. The cytotoxicity of the EVs was performed with U87MG and LN229 glioblastoma cells in 2D culture and 3D organoids, as well as in vivo.

Results : EVs are secreted in high quantities in the size range of 100–200 nm. The EVs expressed exosomal markers and displayed exosomal morphology. Proteomics analysis indicate that neutrophil markers and proteins related to regulation of neutrophil migration, chemotaxis, and degranulation were identified in EVs. miRNA profiling reveals high levels of miRNAs (e.g., miR‐182) active in apoptotic and tumour suppressive pathways, including T cell receptor signalling pathway, cytokine‐cytokine receptor interactions, Jak‐STAT, Hedgehog signalling pathways, and natural killer cell‐mediated cytotoxicity. EV uptake in an in vitro glioblastoma model was demonstrated. CAR Neutrophil EVs are cytotoxic to multiple glioblastoma cell lines and organoids, and in vivo.

Summary/Conclusion : EVs of CAR Neutrophils contain anti‐tumour cargo. The CAR EVs have shown uptake in glioma cells and induced cytotoxicity of the glioblastoma cells. These results demonstrate the feasibility to establish correlation between protein and miRNA cargo of CAR Neutrophil EVs and the function of EVs in GBM treatment.

Funding : This project is supported by NSF 1917618 and NIH of USA.

Natural killer cell‐derived extracellular vesicles enhance the cytotoxicity of immune cells against triple‐negative breast cancer

Frederic St‐Denis‐Bissonnette 1,2 , Karan Mediratta 2 , Andrew Stalker 1 , Gauri Muradia 1 , Michele Ardolino 2,4 , Seung‐Hwan Lee 2 , Dylan Burger 2,3 , Lisheng Wang 2,3 , Jessie R. Lavoie 1, 2

1 Health Canada, Ottawa, Canada; 2 University of Ottawa, Ottawa, Canada; 3 Ottawa Hospital Research Institute, Ottawa, Canada

Introduction : Natural Killer cell‐derived extracellular vesicles (NK‐EVs) are promising cancer immunotherapeutics due to their immunomodulatory and cytotoxic effects. However, the mechanisms by which administered NK‐EVs interact with host immune cells in immunosuppressed environments like triple‐negative breast cancer (TNBC) remain unclear. This study investigates NK‐EVs' immunomodulatory effects on immune cells from TNBC patients and healthy controls using a multi‐OMIC approach (i.e., flow cytometry and single‐cell RNA sequencing—scRNA‐seq).

Methods : Clinical‐grade NK‐EVs were produced from the NK92‐MI cell line using a scalable biomanufacturing process, following MISEV guidelines (published in JEV in 2023). Human peripheral blood mononuclear cells (PBMCs) obtained from TNBC patients (3 donors) and healthy controls (3 donors) were exposed to NK‐EVs in a time and dose‐dependent manner. The effects of NK‐EV treatment on immune cells were assessed using flow cytometry and scRNA‐seq. NK‐EV‐stimulated immune cells were co‐cultured with TNBC cells to monitor cytotoxicity using a live‐cell monitoring assay. NK‐EV uptake was evaluated using CFSE‐labelling (dye associated with EV membrane) and flow cytometry. Ethical approval and informed consent were obtained.

Results : Preliminary assessment of healthy PBMCs by flow cytometric analysis, performed using an unsupervised data reduction algorithm (i.e., fitSNE) and a state‐of‐the‐art clustering algorithm (i.e., FlowSOM), revealed significant activation of T, NK, and myeloid cells (e.g., macrophage, monocyte, and dendritic cells) following a 4‐h NK‐EV treatment. EV uptake experiments confirmed that these immune cell subtypes were selectively associated with NK‐EV treatment, with myeloid cells showing a disproportionately higher association with NK‐EVs than other immune subtypes after a 48‐h treatment. Interestingly, NK‐EV‐stimulated immune cells exhibited increased tumour‐killing potential (72‐h treatment) over the non‐stimulated counterparts. scRNA‐seq analysis of NK‐EV‐stimulated PBMCs is ongoing to decipher activation pathways.

Summary/Conclusion : This is the first multi‐OMIC study combining flow cytometry and scRNA‐seq to investigate NK‐EV‐induced immunomodulation in TNBC‐derived immune cells. Our findings align with previous research showing that NK‐EVs can educate the immune system (i.e., T cell activation and macrophage polarization), further reinforcing the therapeutic potential of NK‐EVs against cancer.

Pharmacokinetics, biodistribution, and immunogenicity of syncytin‐1‐bearing EVs in primates

Zach Troyer, Miranda Soumakis, Erin Shirk, Olesia Gololobova, Sarah Marquez, Martina Fabiano, Natalie Castell, Isabella Baumann, Suzanne Queen, Kenneth W. Witwer

Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Introduction : It is increasingly recognized that extracellular vesicles (EVs) fusion do not fuse promiscuously with cellular membranes. Fusion proteins of several exogenous viruses have been used to enhance EV‐cell fusion; however, immunogenicity may limit clinical applications. Endogenous retroviruses (ERVs) are found in various primate genomes, and their fusogenic envelope proteins may be less immunogenic due to expression in early development. Here, we engineered the human endogenous retrovirus protein (HERV) Syncytin‐1 onto EVs and assessed pharmacokinetics, biodistribution, and immunogenicity after intravenous injection into to pig‐tailed macaques ( Macaca nemestrina ).

Methods : Expi293F cells were used to produce EVs loaded with PalmGRET, an EGFP‐Nanoluciferase (NLuc) fusion and displaying Syncytin‐1. EVs were separated by ultrafiltration and size exclusion chromatography and characterized per MISEV by electron microscopy, Western blot, and nanoflow cytometry. EVs were administered to macaques intravenously every two weeks for four doses total. Pharmacokinetics and biodistribution in the primate model were assessed by NLuc assay with blood plasma and organ/tissue homogenates. Immunogenicity was assessed by detecting cytokines and EV‐specific IgG in plasma.

Results : Syncytin‐1 EVs displayed a significantly different distribution of association with blood cell subtypes compared with those of unmodified EVs from a previously published study. In particular, T cell and monocyte associations were diminished. In addition to negligible cytokine responses at early administrations, we now also report on adaptive immune responses and tissue distribution of these EVs.

Summary/Conclusion : Syncytin‐1+ EVs display different cell association characteristics compared with unmodified EVs, providing proof‐of‐principle that not all exogenous EVs are processed identically in the primate bloodstream and beyond.

Anti‐tumoural effect of osteoblasts‐derived extracellular vesicles loaded with methotrexate, doxorubicin, and cisplatin for osteosarcoma treatment

Luca Giacchi 1 , Elisa Pucci 1 , Argia Ucci 1 , Chiara Puri 1 , Letizia Clementi 1 , Maria Maggi 2 , Antonio Angelo D'Archivio 1 , Adriano Angelucci 1 , Nadia Rucci 1

1 University of L'Aquila, L'Aquila, Italy; 2 Hortus Novus Srl, Rome, Italy

Introduction : Gold standard chemotherapy for osteosarcoma is the combined administration of high dose methotrexate (MTX), doxorubicin (DXR) and cisplatin (CPT). Currently, synthetics liposomes and nanoparticles are investigated as potential vehicle for drug administration, to reduce related side effects and increase specificity.

Methods : We employed extracellular vesicles (EVs) isolated from the human foetal osteoblast (hFOB) cell line to deliver MTX, DXR, and CPT to MNNG/HOS human osteosarcoma cells. FOB‐EVs were loaded with MTX by incubation for 30 min with 20 mg/mL MTX in 0.1 M NaOH at 37°C (hFOB‐EVs + MTX).

Results : MNNG/HOS treatment with hFOB‐EVs + MTX significantly reduced cell metabolic activity, number, and proliferation, while increasing apoptosis, compared to tumour cells treated with empty hFOB‐EVs or vehicle. However, passive incubation and electroporation failed to load DXR or CPT into hFOB EVs, so we adopted an endogenous loading strategy: hFOBcells were treated with 4.6 µg/mL DXR or 30.0 µg/mL CPT, and EVs were harvested from conditioned medium. hFOB EVs + DXR showed a similar cytotoxic effect on MNNG/HOS as exerted by hFOB‐EVs + MTX—significantly reducing viability, proliferation, and increasing apoptosis compared to tumour cells treated with empty hFOB‐EVs or vehicle—whereas hFOB‐EVs + CPT produced a marked trend toward reduced metabolic activity ( p = 0.0633) while significantly reducing cell number, proliferation and increasing apoptosis, when compared to tumour cells treated with empty hFOB‐EVs or vehicle. Intriguingly, hFOB cells treated with drug‐loaded EVs maintained normal metabolic function, underscoring the safety of this delivery platform.

Summary/Conclusion : These findings demonstrate that hFOB derived EVs can efficiently deliver MTX, DXR, and CPT to osteosarcoma cells with no observable toxicity on normal cells, offering a promising strategy to enhance chemotherapeutic specificity and minimize off target toxicity.

Funding : This research was funded by an AIRC investigator grant to NR, grant number 24823.

Cancer‐derived extracellular vesicles for targeted delivery of EGFRvIII siRNA to glioblastoma

Presenter: Fereshteh Shojaeighahrizjani

McGill University, Montreal, Quebec, Canada

Introduction : EVs are a heterogeneous group of lipid‐bound cellular particles with the size of 50–150 nm in diameter and they act as a natural delivery system. Our aim is to evaluate the tumour tropism of glioma cell‐derived EVs towards tumour and to harness this tropism to deliver siRNA against epidermal growth factor receptor variant III (EGFRvIII) in glioma.

Methods : Glioma cell line (U373P) was cultured and EVs were extracted from the conditioned media. The first EV preparation included filtration of conditioned media with syringe filters and concentration with 100 KDs filters followed by ultracentrifugation while the second preparation, the filtration steps were avoided. Then, EVs were labelled with fluorescent dye indocyanine green (ICG). After modelling a mouse model with U373vIII tumour, the in vivo biodistribution was evaluated through IVIS imaging. For the second part of our project, we evaluated different methods for loading therapeutic siRNA into EVs such as endogenous loading and exogenous loading (passive incubation, sonication, electroporation and transfection) with the aim of delivering this siRNA directly to the tumour. The efficiency of each method was evaluated by nano flowcytometry followed by uptake assays and immunoblot analysis.

Results : ICG‐labelled U373P EVs showed a preferential accumulation at the tumour site compared to control groups while filtration could interfere with the tropism by removing the EV protein corona. Regarding the cargo loading part, endogenous loading, passive loading and sonication showed the least efficiency and electroporation showed false positive results as it caused aggregation of siRNA. Transfection of U373P Evs with siRNA by cell penetrating peptide showed better functionality in terms of knocking down the EGFRvIII expression in the cells.

Summary/Conclusion : The tropism of cancer‐derived EVs needs further validation, and if the challenges regarding external loading are addressed, they could be considered a promising tool for the direct delivery of therapeutic cargoes to tumours.

Funding : University of Milan and McGill University

CTHRC1+CXCL12+ fibroblasts‐epithelia communication via exosome foster MDMs accumulation and aggravate lung injury

Presenter: Chen Hongbo

Sun Yat‐sen University, Guangzhou, People's Republic of China

Introduction : Alveolar fibroblast subsets exhibit considerable heterogeneity and functional diversity after lung injury. However, the role of CTHRC1+ fibroblasts in responding to and transmitting damage signals caused by SARS‐CoV‐2 remains unclear. Here, utilizing publicly available data, we reveal that CTHRC1+ fibroblasts associated with epithelial cells and monocyte‐derived macrophages (MDMs). Further study demonstrates that these CTHRC1+ fibroblasts exhibit a pro‐inflammatory phenotype, primarily receiving damage signals from type 2 alveolar epithelial cells via exosomes uptake and subsequently secreting CXCL12 to recruit MDMs—a process closely related to ARDS progression. Based on these findings, we develop and preclinical test an inhalable, room‐temperature‐stable recombinant antibody‐exosome conjugated drug that, after ultrasonic nebulization, reduces inflammatory infiltrates and attenuate lung injury in mice.

Methods : (1) scRNA‐seq and CellChat analysis identified fibroblast subsets involved in communication with MDMs. (2) TFA analysis and co‐culture assays were conducted to explore the mechanisms driving these fibroblast subsets to secrete elevated CXCL12 levels following SARS‐CoV‐2 infection. (3) miRNA‐seq identified regulatory miRNAs, with their effects confirmed via transfection and qRT‐PCR. (4) ALGGEN PROMO and TransmiR v2.0 predicted transcription factors potentially regulating the miRNAs. 5.Reverse chimeric‐Bebtelovimab was engineered and conjugated to MSCs‐Exo using a DSPE‐PEG linker.

Results : (1) CTHRC1+ fibroblasts secrete elevated CXCL12 levels after infection, thereby recruiting excessive MDMs. (2) Exosome mediated communication between epithelia and fibroblasts. (3) EDEVs‐delivered miR‐126 downregulates CXCL12 mRNA levels in fibroblasts. (4) S protein inhibits the expression of miR‐126 by suppressing TFAP2A expression. (5) Fabrication and characterization of mBebtelovimab@MSCs‐Exo and its superior stability. (6) mBebtelovimab@MSCs‐Exo reduce inflammatory infiltrates and attenuate lung injury in vivo after nebulization.

Summary/Conclusion : In conclusion, we demonstrated that the SARS‐CoV‐2 spike (S) protein, but not influenza virus, downregulated miR‐126 levels in epithelial cells by decreasing TFAP2A expression. This TFAP2A suppression reduced miR‐126 levels in exosomes derived from epithelial cells (EDEVs). Upon uptake of these EDEVs by CTHRC1+CXCL12+ fibroblasts, their capacity to downregulate CXCL12 expression was impaired, resulting in elevated CXCL12 secretion and increased MDMs recruitment. Based on these findings, we developed an aerosolized, S protein‐specific recombinant antibody‐conjugated MSC exosome therapy, which effectively reduced inflammatory cell infiltration and mitigated lung injury in a murine model.

Subtopic Disease/therapy relevant mechanism/s of action 2

Physiologically cultured adipose‐derived stem cell‐derived extracellular vesicles for wound healing

Presenter: Julia K. Monola

University of Helsinki, Helsinki, Finland

Introduction : Conventional 2D cell culture in 5% CO 2 with uncontrolled oxygen does not necessarily represent physiological conditions, leading to production of less in vivo‐like extracellular vesicles (EVs). Stem cell‐derived EVs can enhance the regenerative capacity of damaged tissues, but reproducible production of therapeutic in vivo‐like EVs remains challenging. This study aims to find an optimised protocol to produce therapeutic EVs from mesenchymal stromal cells (MSCs) for wound‐healing applications.

Methods : Human mesenchymal adipose stem cells are cultured under normoxic (∼21% O 2 ) and physioxic (5% O 2 ) conditions in two 3D culture systems: ultra‐low attachment (ULA) conditions and NFC hydrogel (NFCh). The EVs were isolated by ultracentrifugation and size‐exclusion chromatography. The protein phenotype will be evaluated by western blotting and immunocytochemistry. The EV yield was determined by NTA and other EV characteristics will be evaluated by proteomics and qPCR. The effects of EVs with NFC hydrogel on wound healing were studied using an in vitro scratch wound model to model the rates of cell migration and proliferation.

Results : Cells produced EVs for 7 days after which, the experiment was terminated. The metabolic activity of the cells was higher in ULA compared to NFCh and in physioxia compared to normoxia. The EV yield was greater in ULA plates than NFCh and in physioxia compared to normoxia. Cells cultured under all conditions exhibited typical mesenchymal stem cell markers (CD73, CD90, CD105) but differences in the amount of expression were observed that were favouring ULA over NFCh and 2D and physioxia over normoxia. The physioxic EVs accelerated relative wound closure compared to normoxic EVs and control (no EV treatment).

Summary/Conclusion : Oxygen levels and culture conformation influences EV yield and phenotype. Additionally, EVs produced under lower oxygen levels seem to accelerate wound healing. Next, the impact of 3D culture and controlled release of EVs from NFC hydrogel on wound‐healing will be studied, along with a detailed analysis of EV characteristics and effects on skin cells during scratch wound healing.

Hydrogel‐assisted transdermal delivery of extracellular vesicles: a novel therapeutic strategy for psoriasis treatment

Yu‐Chen Fa 1 , Bor‐Luen Chiang 2,3,4 , Li‐Chen Wu 5 , Ja‐an Annie Ho 1,6,7,8

1 BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan; 2 College of Life Science, National Taiwan University, Taipei, Taiwan; 3 Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan; 4 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 5 Department of Applied Chemistry, National Chi Nan University, Nantou, Taiwan, 6 Department of Chemistry, National Taiwan University, Taipei, Taiwan; 7 Center for Emerging Materials and Advance Devices, National Taiwan University, Taipei, Taiwan; 8 Center for Biotechnology, National Taiwan University, Taipei, Taiwan Bor‐Luen Chiang, Li‐Chen Wu, and Ja‐an Annie Ho are co‐senior authors .

Introduction : Psoriasis is a chronic inflammatory skin condition characterized by red, scaly lesions due to an overactive immune response that accelerates keratinocyte proliferation. While topical corticosteroids are commonly used, prolonged use leads to significant side effects, affecting 70%–90% of patients globally. This study aims to develop a novel transdermal drug delivery system utilizing extracellular vesicles‐containing hydrogel (hydrogel@EVs) to reduce inflammation in psoriasis. hydrogel@EVs are designed to suppress keratinocyte activity, potentially providing a safer alternative treatment.

Methods : We developed a biocompatible hydrogel loaded with EVs and evaluated it for moisture retention, stability, and transdermal delivery efficiency. In an Imiquimod (IMQ)‐induced psoriasis mouse model, we compared the therapeutic effects of hydrogel@EVs with the hydrogel alone.

Results : The hydrogel@EVs exhibited significant anti‐inflammatory effects in an inflamed keratinocyte model, effectively targeting both the epidermal and dermal layers and showing excellent moisture retention. In the IMQ‐induced psoriasis mouse model, all treatments reduced symptoms, but hydrogel@EVs achieved the greatest improvements in the psoriasis area severity index (PASI), histopathological assessments, and immunohistochemical analysis, with results nearing those of healthy controls.

Summary/Conclusion : The hydrogel@EVs offers a promising topical treatment for psoriasis, combining moisture retention, EV protection, and strong anti‐inflammatory effects. This approach could serve as a safer, long‐term alternative to corticosteroids, meeting the need for more effective psoriasis therapies.

The use of hyaluronidase and centella asiatica exosomes in the treatment of facial complications: a case series report”

Presenter: Paula A. Arroyo

Universidad de Chile, Santiago, Chile

Introduction : This case series examines the combined use of hyaluronidase and Centella Asiatica derived exosomes to treat complications arising from the use of hyaluronic acid fillers, specifically focusing on vascular occlusions. By leverageing the dissolving properties of hyaluronidase and the regenerative potential of exosomes.

Methods : The study aimed to evaluate the efficacy and safety of a combined treatment using hyaluronidase and Centella Asiatica derived exosomes. Patients included in the study had a confirmed diagnosis of vascular occlusion or filler‐related complications within 12 h of symptom onset. The dosage of hyaluronidase was determined based on the severity and extent of the occlusion per affected area. Following successful dissolution of the filler material and confirmation of restored vascularization, these exosomes were applied via localized microinjections directly into the affected area. Patients were monitored closely for any immediate adverse reactions to hyaluronidase or exosome therapy. Follow‐up assessments were conducted a reduction inflammation, and overall skin quality.

Results : in all six patients treated for facial complications following hyaluronic acid filler injections. The primary objective—restoring vascular function and promoting tissue regeneration the administration of hyaluronidase successfully dissolved the obstructing. By the fourth week, all patients demonstrated a substantial decrease in erythema and swelling, with no signs of persistent inflammatory responses. The exosome therapy contributed to the accelerated reduction of oedema compared to typical hyaluronidase‐only the application of Centella asiatica derived exosomes was associated with enhanced tissue regeneration and improved skin quality. All patients reported high levels of satisfaction, and no serious adverse effects were observed.

Summary/Conclusion : This combination therapy offers significant advantages over hyaluronidase alone, not only resolving the immediate issue of vascular compromise but also enhancing long‐term tissue healing and reducing the risk of scarring.

Funding : This study was partially supported by Skymedic, which provided the clinical supplies, including hyaluronidase and exosomes. The research team contributed additional resources to carry out the clinical study, patient care, and follow‐up assessments.

Subtopic Immunotherapy 2

Enhancing natural killer (NK) cell‐derived extracellular vesicles (EVs) for targeted cancer immunotherapy via chimeric antigen receptor (CAR) engineering

Ofir Bar, Tomer Cooks, Angel Porgador, Ishai Luz, Olga Radinsky

Ben‐Gurion University of the Negev, Be'er Sheva, Israel

Introduction : Extracellular vesicles (EVs) derived from immune cells hold promise for cancer immunotherapy. This study investigates the potential of enhancing the anti‐tumour capabilities of natural killer (NK) cell‐derived EVs by engineering their parental NK cells with chimeric antigen receptors (CARs) targeting HER2+ cancers. This strategy aims to combine the inherent cytotoxicity of NK cells with the specificity of CARs to enhance the targeted delivery of anti‐cancer agents via EVs.

Methods : NK‐92 cell line was modified to express two generations of CARs: a first‐generation (Gen‐0) with an anti‐HER2 scFv linked to mouse CD3ζ and a second‐generation (Gen‐2) with an anti‐HER2 scFv linked to human CD28 and CD3ζ. These constructs were chosen to investigate the impact of co‐stimulatory domains on CAR‐NK cell activity and EV production. EVs were isolated from these CAR‐NK cells using differential centrifugation and ultracentrifugation techniques. EVs were then characterized using nanoparticle tracking analysis (NTA). The anti‐tumour activity of CAR‐NK‐EVs was evaluated in vitro against the HER2+ (JIMT1) breast cancer cell line using flow cytometry‐based cytotoxicity assays.

Results : CAR‐NK cells expressing Gen‐0 and Gen‐2 anti‐HER2 CARs were generated, as confirmed by flow cytometry analysis. The EVs exhibited typical characteristics of exosomes, with most EVs falling within the size range of 100‐200 nm. NTA analysis revealed a slight increase in size heterogeneity in CAR‐NK‐EVs, particularly in Gen‐2, which may reflect alterations in EV biogenesis or cargo sorting. Cytotoxicity assays showed that CAR‐NK cells and CAR‐NK‐EVs enhanced activity against HER2+ cancer cells, with Gen‐2 exhibiting the highest efficacy, followed by Gen‐0, compared to untransduced NK cells and their EVs.

Summary/Conclusion : Engineering NK cells with CARs can improve their cytotoxic abilities and enhance the effectiveness of their EVs. Cytotoxicity assays indicated that the Gen‐2 CAR construct had the highest antitumour efficacy against HER2+ cancer cells. Further investigations will validate these findings with additional experiments and evaluate CAR‐NK‐EVs' in vivo efficacy and safety in pre‐clinical animal models to assess their therapeutic potential. This approach holds promise for developing targeted cancer immunotherapies, particularly against HER2+ tumours.

Checkpoint molecules on antigen‐loaded extracellular vesicles modulate their anti‐tumoural effects in a mouse model of melanoma

L. Steiner 1,2,4 , L. Teeuwen 1,2,4 , A. Offens 1,2 , G. Güçluler Akpinar 1,2 , J. Kuipers 1,2 , D. Martinez‐Martinez 1,2 , J. Mazouin 1,2 , B. Chambers 3 , S. Gabrielsson 1,2

1 Division of Immunology and Allergy, Department of Medicine, Karolinska Institutet, Solna, Stockholm, Sweden; 2 Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden; 3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 4 These authors contributed equally to the work .

Introduction : Extracellular vesicles (EVs) from dendritic cells have been tested in humans as cancer immunotherapy, but the treatment needs improvement to induce strong T cell immunity. In mice, EVs secreted by bone marrow derived dendritic cells (BMDCs) induce an antigen‐specific immune response and significantly delay tumour growth. Our previous work showed that BMDC EVs combined with checkpoint blockade therapy (anti‐PD1 or anti‐PD‐L1) synerize. As the BMDC EVs contain both PD1 and PD‐L1, we speculated that EVs from PD1 or PD‐L1 knock out (KO) mice would induce stronger anti‐tumour responses than WT EVs.

Methods : EVs from were prepared from BMDCs by Tangential flow filtration followed by differential ultracentrifugation and characterized by nanoparticle tracking analysis, bead‐based flow cytometry and EM. Mice were immunized intravenously with WT, PD1 KO and PD‐L1 KO BMDC EVs twice, and immune responses were detected on day 21 by ELISA, ELISPOT and flow cytometry.

Results : All EV‐injected groups showed an increased and similar number of antigen‐specific CD8 T cells in the spleen. However, following ex vivo restimulation of splenocytes, OVA specific CD8 T cells from both PD1 and PDL‐1 KO EV injected mice produced more IFN‐g than the WT. In contrast, when EVs were injected as tumour therapy in a mouse melanoma model, WT EVs performed better than the two KO in delaying tumour growth. All the EV treatments induced a potent anti‐tumour response by recruiting several immune cell types at the tumour sites, but the absence of checkpoint molecules on the EVs dampened the antigen specific response. This contrasts with earlier findings in tumour‐derived EVs, where tumour‐derived PD‐L1 on EVs inhibit immune responses to the tumour.

Summary/Conclusion : We confirmed that EVs loaded with antigen are potent immune stimulators and promising tools for immunotherapy. Additionally, the checkpoint molecules present on theses EVs may play a functional role in immune activation and are important in eliciting a strong anti‐tumour response. Our results also highlight the need to further investigate the PD1/PD‐L1 axis in cancer therapy.

Funding : The study was funded by the Swedish Medical Research Council, the Swedish Cancer Society, the Cancer Research Foundations of Radiumhemmet, and the Karolinska Institutet's KID grant.

Generation of designer vesicles for the activation of primary NK cells

Xiaoling Wu, Susanne Rößner, Katja Blume, Jan Van Deun, Miriam Zentgraf, Andreas S. Baur

Department of Dermatology, Unikilinikum Erlangen, Friedrich‐Alexander‐Universität (FAU) Erlangen‐Nürnberg, Deutsches Zentrum Immuntherapie (DZI); Erlangen Germany

Introduction : Ex‐vivo amplified autologous Natural killer (NK) cells, similar to CAR T cells, have attracted great interest as an anti‐tumour therapy. The generation of these cells, however, is complex, time consuming and expensive. Injection of designer extracellular vesicles (DesEV) that activate NK cells in vivo may overcome a number of these issues including the most relevant HLA‐mismatch problem. However, still many technical and biological issues need to be addressed before GMP‐grade Designer Vesicles can be generated for in vivo application. This includes the EV‐producer cell, its optimal genetic manipulation, the identification of the subcellular targeting structure and most notably, the potency of DesEV to activate NK cells.

Methods : We used different primary cells and cell lines that were genetically transduced with different methods to introduce different GFP‐labelled cytokines, reported to activate primary NK cells. EV were purified from culture supernatants using Ultracentrifugation and incubated with primary NK cells. Confocal microscopy, FACS, proteomics and real‐time PCR analysis were used to analyse and measure the effects on NK cells.

Results : While differently generated EV were readily taken up by NK cells, only certain primary cells, stimulated by a defined procedure, resulted in EV able to correctly stimulate NK cells. The cells proliferated and expressed high levels of perforin and granzyme B. One critical factor was the targeting of the internalized vesicle to the perinuclear region.

Summary/Conclusion : A number of factors need to be considered when generating DesEV to stimulate immune cells. Such vesicles are able to efficiently generate functional NK cells.

Enhancing adenovirus‐based cancer vaccines: the role of extracellular vesicles in targeted delivery and anti‐tumour response in humanized melanoma models

Mariangela Garofalo 1 , Sara Mathlouthi 1 , Maria Giovanna Lupo 2 , Beate Rinner 3 , Nicola Ferri 2,4 , Stefano Salmaso 1 , Paolo Caliceti 1 , Lukasz Kuryk 5

1 Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy; 2 Department of Medicine, University of Padova, Padua, Italy; 3 Division of Biomedical Research, Medical University of Graz, Graz, Austria; 4 Veneto Institute of Molecular Medicine, Padua, Italy; 5 Department of Virology, National Institute of Public Health NIH—National Research Institute, Warsaw, Poland

Introduction : Malignant melanoma, a rapidly spreading form of skin cancer, is increasingly prevalent worldwide. While surgery can effectively manage early‐stage melanoma, patients with advanced disease such as those with NRAS gene mutations, face a grim prognosis and limited survival chances. Consequently, there is a pressing need for novel treatment strategies for this group of patients. One promising approach involves leverageing cancer vaccines to enhance the body's immune response against tumours. However, their efficacy remains suboptimal, necessitating exploration of alternative delivery mechanisms. Recent advances in nanotechnology highlight the potential of extracellular vesicles (EVs) as tumour‐selective targeting agents in cancer therapy. These nano‐sized lipid vesicles, generated by nearly all cell types, play a significant role in intercellular communication and show promise in drug delivery. Our research focuses on harnessing cancer cell‐derived EVs, which demonstrate a unique ability to selectively target tumour tissue. The study presents a novel adenovirus‐based cancer vaccine, using a newly cloned oncolytic adenovirus and clinically relevant melanoma antigens, specifically targeting NRAS mutations to enhance anticancer efficacy.

Methods : We initially investigated the antineoplastic effects using two‐dimensional (2D) and three‐dimensional (3D) co‐culture models that included NRAS‐mutated melanoma cells and peripheral blood mononuclear cells. Subsequently, we established a humanized mouse model implanted with NRAS‐mutated melanoma to assess the systemic delivery of the vaccine via EV‐formulations.

Results : In vivo and ex vivo imaging in the humanized mice revealed that EVs could selectively deliver the vaccine to both primary and metastatic tumours. The EV‐tumour targeting was also confirmed by confocal microscopy analysis perfomed on section of explanted tumour. The anticancer outcomes observed included a reduction in tumour volume and an increase in tumour‐infiltrating lymphocytes, particularly activated cytotoxic T‐cells (GrB+CD8+). These results support the notion of a synergistic anti‐tumour effect, with a clear relationship identified between tumour volume and the presence of activated CD8+ tumour‐infiltrating lymphocytes.

Summary/Conclusion : This research overall highlights the potential of EVs as a safe and effective means for personalized cancer therapy, offering new opportunities for the systemic delivery of immunogenic vaccines in difficult melanoma cases.

Funding : STARS Starting Grant (GARO_STARS_MUR22_01), EU funding within the MUR PNRR (CN00000041, CN3 − Spoke #8), PRID project (GARO_BIRD23_01)

Tumour‐Derived Extracellular Vesicles‐Based Cancer Vaccine Using Tunicamycin

Chan Ho Kim 1 , Kyung Hee Han 1 , So Hee Kim 1 , Jae Hyung Park 1,2,3*

1 School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu‐ro, Jangan‐gu, Suwon, 16419, Republic of Korea 2 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, Republic of Korea 3 Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, 2066 Seobu‐ro, Jangan‐gu, Suwon, 16419, Republic of Korea

Introduction : Tumour‐derived extracellular vesicles (T‐EVs) have been implicated in promoting tumour growth and metastasis and modulating the tumour microenvironment by influencing surrounding stromal cells. T‐EVs have been shown to suppress immune cell function and recruit immunosuppressive cell populations, including regulatory T cells and tumour‐associated macrophages, thereby facilitating immune evasion. Paradoxically, accumulating evidence suggests that T‐EVs carry tumour‐associated antigens, which can elicit anti‐tumour immune responses, underscoring their potential utility as components in cancer vaccine strategies.

Methods : In this study, we aimed to develop a T‐EV‐based cancer vaccine by enhancing immune‐stimulatory molecules, including damage‐associated molecular patterns (DAMPs), while reducing immunosuppressive proteins. To achieve this, B16F10, a murine melanoma cell line, was treated with tunicamycin, known as an endoplasmic reticulum (ER) stress inducer, and the resulting T‐EVs (tT‐EVs) were subsequently isolated from the supernatants.

Results : Western blot analysis revealed that representative DAMPs, such as HMGB1 and HSP70, were upregulated in tT‐EVs, whereas immunosuppressive proteins, including CD47 and S100A9, were reduced. Treatment of DC2.4, a murine dendritic cell line, with tT‐EVs resulted in enhanced maturation, evidenced by increased expression of CD80, CD86, and CCR7 compared to DCs treated with bare T‐EVs (bT‐EVs). In a therapeutic efficacy study using a B16F10 tumour‐bearing mouse model, tT‐EVs significantly suppressed tumour growth and triggered an anti‐tumour immune response, as shown by flow cytometry analysis revealing an increased population of cytotoxic T cells within the tumour. These outcomes were corroborated in both post‐surgery and bilateral tumour models, underscoring the ability of tT‐EVs to mediate systemic anti‐tumour immunity.

Summary/Conclusion : One major advantage of cancer vaccines over other treatments is their ability to robustly induce a systemic anti‐tumour immune response, which can prevent tumour recurrence and inhibit metastatic growth following surgery. The tT‐EVs developed in this study showed their feasibility as a cancer vaccine, which showed enhanced DAMPs and reduced immunosuppressants. Also, as tT‐EVs can be isolated from resected tumours as a personalized cancer vaccine, tT‐EVs demonstrated the potential to treat tumours regardless of their heterogeneity.

Subtopic EV Therapeutics Development 2

Quality by Design (QbD) Analysis for GMP Manufacturing of Human Mesenchymal Stem/Stromal Cell‐derived Extracellular Vesicles

Presenter: Chadaporn Attakitbancha

Chulalongkorn University, Thailand

Introduction : Quality by Design (QbD) represents a systematic approach to pharmaceutical development that begins with predefined objectives and emphasizes product and process understanding and strategic control. For the emerging field of therapeutic extracellular vesicles (EVs), implementing QbD principles is crucial to ensure product quality, patient safety, and optimal therapeutic outcomes. This study applies QbD methodology to develop a robust GMP manufacturing process for human mesenchymal stem/stromal cell (MSC)‐derived EVs, based on ICH guidelines (Q8‐Q11), PDA recommendations, and ISPE baseline guides.

Methods : We implemented a comprehensive QbD framework incorporating iterative risk assessments throughout the product lifecycle. The process began with defining the Quality Target Product Profile (QTPP) for MSC‐derived EVs, followed by systematic identification of Critical Quality Attributes (CQAs), Critical Process Parameters (CPPs), and Critical Material Attributes (CMAs). Process Development Risk Assessment utilized Failure Mode and Effects Analysis (FMEA) to establish links between raw materials, starting cell quality, process parameters, and final product attributes. The Control Strategy was developed through risk‐based approaches and multivariate analysis, culminating in Process Performance Qualification (PPQ).

Results : Our QbD implementation established a knowledge‐based manufacturing process with reduced product risk through enhanced process understanding. The analysis yielded: 1) A comprehensive QTPP defining target specifications for therapeutic EVs; 2) Risk‐prioritized CQAs, CPPs, and CMAs with established acceptance criteria, including critical attributes for starting materials and raw materials; 3) A documented Control Strategy incorporating in‐process controls and release specifications; and 4) A Continued Process Verification (CPV) program enabling continuous improvement through systematic monitoring and feedback loops. The process demonstrated consistent production of EVs meeting predefined quality attributes across multiple validation batches.

Summary/Conclusion : Applying QbD principles to hMSC‐derived EV manufacturing resulted in a well‐characterized process where quality is proactively designed for each step of production, ensuring consistent product quality critical for patient safety and therapeutic efficacy. This systematic approach enhances process robustness, facilitates regulatory compliance, and establishes a framework for continuous improvement in therapeutic EV manufacturing for clinical uses.

Funding : This study is supported by the National Research Council of Thailand (NRCT) and Chulalongkorn University, Thailand

EV Sources and Basic Biology

Subtopic EV Biogenesis 2

The role of RBBP7 in sEV secretion in hepatocellular carcinoma

Presenter: Wing Lam Tam

The University of Hong Kong, Hong Kong

Introduction : Hepatocellular carcinoma (HCC) poses a significant global health challenge, with Asia bearing a disproportionate burden of its cancer‐related mortality. Recent studies emphasize the critical role of small extracellular vesicles (sEV) in coordinating communication within the HCC microenvironment. These nanosized vesicles facilitate the exchange of molecular cargo among cancer cells and various cell types, thereby promoting tumour growth and metastasis.

Methods : This study explores the interplay between sEV secretion and HCC progression. Through screening a chemical library, a promising compound was identified that attenuated sEV release in HCC cells.

Results : Subsequent analyses identified RBBP7 as a key mediator in regulating sEV biogenesis and secretion. Silencing RBBP7 significantly decreased sEV production, highlighting its role in modulating tumour‐derived sEV dynamics. Furthermore, the decreased multivesicular body (MVB) count and enhanced endolysosomal fusion upon RBBP7 knockdown reveal a link between intracellular trafficking pathways and sEV secretion. Clinical data show the overexpression of RBBP7 in HCC and its positive correlation with proteins associated with sEV biogenesis.

Summary/Conclusion : These findings not only highlight RBBP7 as a potential therapeutic target in HCC management but also illustrate the intricate interplay between cellular trafficking mechanisms and intercellular communication in cancer progression. By elucidating the molecular underpinnings of sEV‐mediated communication in HCC, this study contributes to a deeper understanding of tumour biology and unveils new avenues for therapeutic intervention strategies targeting sEV biogenesis and secretion pathways.

ESCRT‐0/HRS Modulates Lipid Profiles in Tumour‐Derived Small Extracellular

Presenter: Beike Wang

Wuhan University, China

Introduction : The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery plays a pivotal role in the biogenesis and cargo sorting of small extracellular vesicles (sEVs). While it is established that ESCRT machinery is crucial for the selective inclusion of proteins in sEVs, its impact on the lipid composition of sEVs remains unexplored. This study investigates the role of ESCRT‐0/HRS in regulating the lipid content of sEVs derived from tumours.

Methods : We generated HRS knockdown (KD) and knockout (KO) cell lines using human head and neck squamous cell carcinoma Cal27. Tumour‐derived sEVs were characterized using Nanoparticle Tracking Analysis, transmission electron microscopy (TEM), and western blotting. Lipidomics profiling of both tumour cells and their sEVs was performed using Liquid Chromatography‐Mass Spectrometry (LC‐MS).

Results : Significant differences in lipid composition were observed between tumour cells and their derived sEVs. Cal27 cells predominantly contained phosphatidylethanolamine (PE) and triacylglycerols (TAGs), whereas Cal27‐derived sEVs were rich in phosphatidylcholine (PC) and PE. In HRS KD Cal27 cells‐derived sEVs, there was a notable increase in ceramide (CE), lysophosphatidylcholine (LPC), and phosphatidic acid (PA). Enrichment analysis indicated activation of glycerophospholipid and glycerolipid metabolic pathways. Lipid droplet accumulation in HRS KD cells, as evidenced by Oil Red and BODIPY staining, suggested impaired lipid metabolism. The lipolysis rate‐limiting enzyme, ATGL, was significantly downregulated in HRS KD cells, potentially contributing to the observed lipidomic alterations in sEVs.

Summary/Conclusion : Significant differences in lipid composition were observed between tumour cells and their derived sEVs. Cal27 cells predominantly contained phosphatidylethanolamine (PE) and triacylglycerols (TAGs), whereas Cal27‐derived sEVs were rich in phosphatidylcholine (PC) and PE. In HRS KD Cal27 cells‐derived sEVs, there was a notable increase in ceramide (CE), lysophosphatidylcholine (LPC), and phosphatidic acid (PA). Enrichment analysis indicated activation of glycerophospholipid and glycerolipid metabolic pathways. Lipid droplet accumulation in HRS KD cells, as evidenced by Oil Red and BODIPY staining, suggested impaired lipid metabolism. The lipolysis rate‐limiting enzyme, ATGL, was significantly downregulated in HRS KD cells, potentially contributing to the observed lipidomic alterations in sEVs.

Investigating exosome biogenesis using vesicular trafficking inhibitors

Presenter: Deborah Polignano

Istituto Superiore di Sanità, Italy

Introduction : Exosomes are small extracellular vesicles (sEV) formed within late endocytic compartments/multivesicular bodies (MVB). Various machineries have been described to regulate their biogenesis, however several aspects of these processes have not been fully elucidated. We developed a methodology to obtain fluorescent exosomes (Bodipy exo) by using Bodipy FL C16. To gain insight into exosome biogenesis we combined this approach with the use of a panel of inhibitors of vesicular trafficking.

Methods : Bodipy exo secreted by melanoma cells pulsed with BODIPY FL C16 and treated with inhibitors of cellular pathways were isolated by differential ultracentrifugation and quantified by Flow Cytometry (FC) and Nanoparticle Tracking Analysis (NTA). Colocalization between Bodipy exo and anti‐tetraspanins fluorescent antibodies was determined by FC. Characterization of sEVs protein content was performed by Western Blot. Transmission Electron Microscopy (TEM) analysis of cells treated with inhibitors was also performed.

Results : Significant differences were observed in the secretion of Bodipy exo as evaluated by FC upon treatment of melanoma cells with selected inhibitors of cellular pathways (U18666A, Trimeprazine, GW4869, Desipramine, Bafilomycin‐A1, Cytochalasin D). Notably, GW4869 specifically reduced the secretion of Bodipy exo, while the total amount of sEVs released remained comparable to that of control cells. Moreover, treatment with Thioperamide, which increases lysobisphosphatidic acid (LBPA) levels, and Monensin, which interferes with acidification by a different mechanism than Bafilomycin‐A1, had no significant effect on sEV secretion. Interestingly, with the exception of Bafilomycin‐A1 and Brefeldin A treated cells, the percentage of tetraspanins (CD63, CD81, and CD9) positive Bodipy exo, remained similar. However, striking differences were observed in Western blot analysis of the total sEV population even in cases where inhibitors treatment did not affect exosomes secretion. TEM analysis further revealed variations in the intracellular number of MVBs and of intraluminal vesicles within MVBs.

Summary/Conclusion : The use of selected inhibitors of intracellular pathways affects not only the secretion of sEV, but also their protein composition. This suggests that alterations in cellular pathways can induce modifications in protein trafficking, consequently impacting the exosome biogenetic pathway. In conclusion, this approach has the potential to provide further insights into the process of exosome biogenesis.

Funding : Italian Ministry of Health (grant RF‐2019‐12369719)

Rab GTPases cooperate with the exocyst complex to release skeletal muscle extracellular vesicles upon contraction signalling

Presenter: Luke T. Nelson

University of Hawaii, USA

Introduction : In response to exercise, skeletal muscle (SkM) cells are known to enhance the secretion of small (∼50‐150 nm) extracellular vesicles (EVs). These EVs facilitate inter‐organ communication by carrying specialized molecular cargo that likely mediate the metabolic benefits of physical activity. However, the mechanisms underlying contraction‐induced EV secretion from SkM remain poorly understood. We investigated the role of Rab27a, a Rab GTPase, in regulating EV release using exercise‐mimetic treatments on cultured SkM cells in vitro.

Methods : Immunofluorescence and biochemical assays were employed to study the localization and molecular associations of EV‐containing intracellular vesicles (known as multivesicular bodies (MVBs)) during this process. We analysed intracellular protein‐protein associations using proximity ligation assays (PLA) in fixed SkM cells. For characterization of EVs produced by SkM, we utilized Tunable Resistive Pulse Sensing (TRPS), Western blotting, and Transmission Electron Microscopy (TEM).

Results : Our findings reveal that Rab27a plays a role in the translocation of MVBs to the plasma membrane in response to contraction‐signalling. Additionally, we identified the octameric exocyst protein complex, which is responsible for vesicle tethering prior to membrane fusion, as part of the intracellular trafficking mechanism that facilitates critical steps for EV secretion.

Summary/Conclusion : This study highlights Rab27a and the exocyst complex as key regulators of contraction‐induced EV release in SkM. Our findings provide insights into the molecular basis of SkM‐derived EV secretion and its potential role in mediating interorgan signalling during exercise. These insights could pave the way for further exploration of SkM EVs as therapeutic targets.

Funding : NIH OLA Hawaii #5U54MD007601

Extracellular vesicle‐carried DNA during adipocyte differentiation

Presenter: Paraskevi Apalaki

Democritus University of Thrace, Greece

Introduction : Adipose tissue functions as a crucial regulator of endocrine and immune homeostasis. The intercellular communication is mainly mediated by extracellular vesicles (EVs) and adipocytes are one of their major sources. Extensive research has been done on EV‐carried protein and RNA molecules. However, there is limited understanding of their DNA cargo. Moreover, EV‐carried DNA may contribute to the pool of total cell free DNA (cfDNA), a liquid biopsy biomarker of increasing interest for clinical applications. Here we are set to unravel the basic qualitative and quantitative characteristics of EV‐carried DNA during adipocyte differentiation. This information is valuable to the understanding of its subcellular source and contribution to intercellular communication.

Methods : 3T3‐L1 cells were differentiated to adipocytes in vitro. EVs were isolated by ultracentrifugation of supernatants collected during differentiation and their presence was verified by Western blot analysis. Total DNA extracted from EVs and DNase treated EVs along with total cfDNA from culture supernatants were quantified with Qubit fluorimeter. Their length was analysed by capillary electrophoresis while Western blot analysis and quantitative real time PCR were applied for the identification of their nuclear or mitochondrial origin.

Results : EV‐carried DNA and total cfDNA are increased during adipocyte differentiation, while arrested adipogenesis is related to lower levels. EVs carry long DNA fragments, the concentration of which decreases after DNase treatment, indicating the internal and external topology of DNA on the vesicle. Furthermore, histones and lamins as well as mitochondrial related genes are detected in the protein and DNA cargo of the isolated EVs respectively, showing both genomic and mitochondrial origin. Correlation of these results with the observed decreasing number of micronuclei and curved nuclear membranes in differentiated adipocytes are being investigated as important events related to the subcellular origin and loading mechanism of DNA into the vesicles.

Summary/Conclusion : Extracellular vesicles carry large fragments of genomic and mitochondrial DNA either encapsulated or decorating the periphery of the vesicle. Further study will unfold the origin and loading mechanism of DNA in exosomes. Moreover, these results will help us understand better the functional role of these DNA fragments mediating interactions and effects with recipient cells.

Funding : HFRI

MHC‐II‐positive small extracellular vesicles are tethered to lymphoblastoid cell lines by the antiviral protein tetherin

Adam M Bourke, James R Edgar

Department of Pathology, University of Cambridge

Introduction : Major Histocompatibility Complex class II (MHC‐II) molecules traffic through the endosomal system to the multivesicular body (MVB), where peptide loading and editing occurs. Here, MHC‐II molecules are specifically enriched on intraluminal vesicles (ILVs). To ensure MHC‐II presentation at the cell surface, ILV retrofusion has been proposed—allowing a route back from ILVs to the plasma membrane. Recently, it has been shown that small extracellular vesicles can be retained and tethered to producer cells, a property dependent on the expression of the antiviral protein, Tetherin. My research investigates the prominence of this phenotype in Lymphoblastoid Cell Lines (LCLs) and the role that tethered, endosomal derived exosomes play in MHC‐II presentation.

Methods : Tetherin knockout 721.221 LCLs were generated using CRISPR/Cas9, where the presence of tethered small EVs was scrutinised using conventional and cryo‐immunogold transmission electron microscopy. Immunofluorescence was used to further compare MHC‐II distribution, particularly in relation to endosomal markers. Small EVs were isolated from wild type and knockout cells using differential centrifugation, and protein composition and abundance determined by western blot.

Results : We found small extracellular vesicles tethered to the cell membranes of tetherin‐expressing 721.221s which contain CD63 and MHC class II molecules. Immunofluorescence showed MHC‐II and CD63 colocalising in puncta at the cell surface, a phenotype lost in tetherin KO 721.221s. Western blotting confirmed that tetherin KO 721.221s secrete increased levels of MHC‐II, CD63, and syntenin positive EVs.

Summary/Conclusion : Our findings demonstrate, on the surface of LCLs, tethered sEVs enriched in MHC‐II specifically. Tetherin knockout cells exhibit decreased tethering, and increased release of sEVs into the culture media. The finding of MHC‐II positive tethered sEVs on the 721.221 cell surface highlights that ILV retrofusion may not occur in LCLs, and highlights a new route for MHC‐II presentation at the cell surface.

Funding : This work is funded by a PhD studentship from the Department of Pathology, University of Cambridge. JRE's lab is funded by a Sir Henry Dale Fellowship funded by the Wellcome Trust and Royal Society (216370/Z/19/Z). I have received research grants, such as through the British Society of Cell Biology and Pembroke College, Cambridge, to aid my learning of cryoimmuno‐gold electron microscopy.

The structural and catalytic role of PHOSPHO1 in Matrix Vesicle biogenesis: from cell to membrane mimetic model

Luiz Henrique da Silva Andrilli 1,2 , Flavia Amadeu de Oliveira 2 , Colin Farquharson 3 Pietro Ciancaglini 1 , José Luís Millán 2

1 University of Sao Paulo—Faculty of Philosophy, Sciences and Letters at Ribeirao Preto, Chemistry Department, Ribeirao Preto, SP, Brazil. 2 Sanford Burnham Prebys Medical Discovery Institute—Sanford Children's Health Research Center, La Jolla, CA, United States. 3 Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, United Kingdom

Introduction : A special class of extracellular vesicles, called Matrix Vesicles (MV), play a key role during endochondral ossification. Orphan Phosphatase 1 (PHOSPHO1) is responsible for generating Pi in the MV lumen. Here, were evaluated the role of PHOSPHO1 in MV biogenesis and mineral propagation through: i) pharmacological inhibition of native PHOSPHO1, in cell cultures; ii) studies of MV isolated from Phospho1‐/‐ mice and iii) studies of the interaction of catalytically inactive PHOSPHO1 (D32N‐PHOSPHO1, with the membrane, using monolayer Langmuir‐Blodgett isotherms as biomimetic model.

Methods : MV isolated from primary chondrocytes from Phospho1‐/‐ and Wild Type (WT) mice, with the addition or not of MLS‐0263839 (PHOSPHO1 inhibitor), were characterized using biochemistry and biophysics techniques. Native PHOSPHO1 and the catalytically inactive D32N‐PHOSPHO1 mutant were applied to monolayers composed of 1,2‐dipalmitoylphosphatidylcholine (DPPC), 1,2‐dipalmitoylphosphatidylserine DPPS, Cholesterol (Chol) or Sphingomyelin (SM) and compressed at constant temperature. The isothermal compressibility (Cs‐1) was performed showing the monolayer condensation state.

Results : Decreased Alpl, anxa5, and Runx2 gene expression was observed in Phospho1‐/‐ and WT‐chondrocytes treated with the PHOSPHO1 inhibitor. Chondrocyte‐derived MV from WT and Phospho1‐/‐ mice showed a diameter of ∼160 and 200 nm, respectively, with Zeta potential ∼ 28.0 mV. Alkaline phosphatase activity per number of particles was decreased in MV isolated from Phospho1‐/‐, reflected in a decrease in calcium deposition. WT‐chondrocytes containing PHOSPHO1 inhibitor also showed a decrease in mineralization capacity. DPPC‐based monolayers containing Chol, DPPS (both 9:1 and 8:2), and SM (8:2) showed a Cs‐1 decrease of 2 and 17.1% for native and inactive PHOSPHO1, resulting in monolayer lipid‐phase changes, while the presence of 10% SM showed a Cs‐1 increase of 8.3 and 25.36% for PHOSPHO1 and D32N‐PHOSPHO1, respectively.

Summary/Conclusion : Our work shows that PHOSPHO1 plays structural and catalytical role during endochondral ossification since its absence and inhibition result in decreased alkaline phosphatase gene expression and decreased mineral deposition using cell models. Using biomimetic models, inactive PHOSPHO1 showed the same interaction with the lipid interface compared to native PHOSPHO1. Using a more compact monolayer containing 10% SM, revealed a greater interaction of the PHOSPHO1 molecules at the interface, and this phenomenon is thermodynamically favourable.

Funding : FAPESP 2019/08568‐2, 2021/13140‐1, 2023/14655‐0; CNPq 305426/2021‐4.

New class of highly reactive large extracellular vesicles are shed by circulating tumour cells under fluid shear stress at microvascular bifurcations

Angelos Vrynas 1 , Salime Bazban‐Shotorbani 1 , Sara Arfan 2 , Karishma Satia 3,4,5 , Brian Cunningham 1 , Gauhar Sagindykova 1 , Mymuna Ashna 1 , Aoyu Zhang 1 , Diana Visan 1 , Aisher Chen 1 , Teige Matthews‐Palmer 6 , Mathew Carter 3,4,7 , Fiona Blackhall 4,7 , Kathryn L. Simpson 3,4,5 , Caroline Dive 3,4,5 , Paul Huang 2, , Sam H. Au 1

1 Imperial College London, United Kingdom. 2 The Institute of Cancer Research,, United Kingdom. 3 University of Manchester, United Kingdom. 4 Cancer Research UK Lung Cancer Centre of Excellence, United Kingdom. 5 SCLC Biology Group, Cancer Research UK Manchester Institute, United Kingdom. 6 Division of Structural Biology The Institute of Cancer Research, United Kingdom. 7 Christie Hospital National Health Service (NHS) Foundation Trust, United Kingdom.

Introduction : It is well‐established that primary tumours release extracellular vesicles (EVs) modifying the microenvironment of distant organs to facilitate later colonization by circulating tumour cells (CTCs). However, it remains poorly understood if entrapped CTCs can directly alter their local microenvironment. In this work, we have identified and characterized a new class of EV released by patient small cell lung cancer CTCs in response to shear stress in capillary‐sized bifurcations that influences critical cellular regulators of metastasis.

Methods : We emulated in vivo geometries and physiological pressures in engineered microfluidic capillary‐sized bifurcated microchannels. Transiting primary CTCs, CTC‐derived xenografts and six cancer cell lines were observed to shed large nuclei‐free fragments by live imaging. Purified fragments were characterized via immunofluorescence staining and proteomic analyses via mass‐spectrometry. Fragments were co‐cultured with human umbilical vein endothelial cells (HUVECs) or THP‐1 monocytes (1:3 ratio). Phenotypic changes were validated via immunofluorescence and PCR after 24hr exposure. Endothelium permeability by tumour cells and microparticles on transwell and microfluidic 3D endothelial networks was fluorescently quantified.

Results : All tested cells shed large extracellular vesicles (LEVs) (1.17‐11.32 µm, mean:4.9±1.9 µm) with up to 90% frequencies. LEV biogenesis depended on fluid shear stress and constricted transit through bifurcations, distinguishing them from other EVs like large oncosomes (LOs). LEVs matched the MISEV2018 requirements for EVs validation including transmembrane and cytosolic proteins, nuclei absence and visual characterization by two complementary techniques. Our proteomics approach identified the presence of 3382 distinct proteins within LEVs samples, about 8‐fold higher compared to distinct proteins in LOs. LEVs were ingested by monocytes and HUVECs and elicited diverse signalling relevant to pre‐metastatic niches. LEVs polarized monocytes towards CD206+ M2 tumour‐associated macrophage lineages. LEVs also disrupted vascular endothelial junctions and increased permeability of tumour cells and microparticles. Finally, LEVs activated HUVECs promoting co‐interactions with monocytes.

Summary/Conclusion : Concluding, we identified and characterized a new class of LEVs whose biogenesis is driven by tumour cells traversing capillary bifurcations under shear stress, which we named ‘shearosomes’. Their local generation in microvasculature at CTCs entrapment regions, their vast proteome and co‐interactions with immune and endothelial cells may serve to explain how CTCs form pre‐metastatic niches that enable their successful metastasis.

Small Extracellular Vesicle Tethering Promotes Extracellular Matrix Degradation

Presenter: James R. Edgar

University of Cambridge, UK

Introduction : In addition to diffusing away from cells, small extracellular vesicles (sEVs) can remain physically attached to the surface of the cell of origin. This tethering is dependent on the expression of tetherin (Bst2), a protein that plays asimilar role in anchoring enveloped viruses and retaining midbody remnants. Tetherin is overexpressed in several highly metastastic cancers; however, the mechanisms linking tetherin expression to cancer invasiveness remain unclear.

Methods : Endogenous MT1‐MMP was investigated using MDA‐MB‐231 cells, and tetherin knockout cells were generated using CRISPR/Cas9. sEVs were isolated from cell culture supernatants via size exclusion chromatography, and characterised using western blotting, electron microscopy and nanoFCM. Immunogold electron microscopy methods were employed to precisely localise MT1‐MMP within and outside of cells. ECM degradation was assessed using various in vitro functional assays.

Results : Endogenous MT1‐MMP was localised to tethered exosomes, and to the intraluminal vesicles of multivesicular bodies. MT1‐MMP was found to be enriched in sEVs, and the loss of tetherin increased MT1‐MMP secretion by promoting sEV release. Electron microscopy, western blotting and nanoFCM analyses confirmed that tetherin depletion enhanced sEV release from cells, and reduced the ability of cells to degrade the ECM. Reintroducing ectopic tetherin into tetherin‐knockout cells restored sEV tethering and ECM degradation.

Summary/Conclusion : Our findings demonstrate that tetherin regulates the balance between sEV retention and release. In breast cancer cells, tetherin overexpression promotes sEV tethering and impairs their release. Our data demonstrate MT1‐MMP as a cargo of sEVs from breast cancer cells, and we find that MT1‐MMP‐positive tethered exosomes promote ECM degradation. As a highly responsive interferon‐stimulated gene, tetherin's regulation by external stimuli may have significant implications for sEV function. Collectively, our results identify tetherin as a key regulator of sEV fate, with potential implications for cancer progression and metastasis.

Funding : J.R.E. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (216370/Z/19/Z).

The MicroRNA composition in cultured cells is virtually indistinguishable from that in extracellular vesicles and non‐vesicular extracellular particles

Presenter: Michiel M. Pegtel

Institution Amsterdam UMC, Netherlands

Introduction : Extracellular vesicles (EVs) carry a diverse repertoire of biomolecules, including microRNAs that despite a low stoichiometry 1 copy per 100‐1000 or more EVs may have functional relevance for cell‐cell communication. Studies that made use of qRT‐PCR identified EV‐enriched and EV‐depleted miRNAs when compared to cellular expression levels, suggesting murine cells sustain an active miRNA sorting mechanism. These EV‐enriched miRNAs are proposed to contain special sequence motifs (EXOmotifs) for RNA‐binding proteins (RBPs) that drive sorting into EVs although. Here we aimed to test the EV‐miRNA sorting hypothesis with small RNA sequencing in human cell‐lines.

Methods : We determined the EV‐miRNA composition and stoichiometry by using unbiased small RNA sequencing IsoSeek method (that minimizes amplification and ligation bias), digital droplet PCR and Tunable Resistive Pulse Sensing. We sequenced over 100 samples from 14 human cell lines followed by comprehensive bioinformatic analysis.

Results : We determined that in human cell‐lines very few, if any, miRNAs are enriched in either cells or EVs from any of the 14 cell types we analysed. Repression of both miRNA and EV biogenesis pathways with CRISPR‐CAS mediated gene depletion, affects miRNA abundance but not miRNA composition in cells nor EVs. Notably AGO2 depletion raised EV‐miRNA levels more than 100‐fold. Nevertheless, the miRNA composition in EV subtypes, the EV lumen, ‐surface or NVEP fractions are identical although amphisome‐derived exosomes sustain a more pronounced miRNA‐corona compared to small EVs budding from the PM. Consistent with non‐selective association, in none of the conditions statically significant enrichment of RBP‐binding motifs in EV‐associated miRNAs over cellular miRNA expression levels could be determined. However, changing the cellular transcriptome by interferon (pre)treatment strongly depleted a defined set of miRNAs from the EVs.

Summary/Conclusion : With extensive miRNA sequencing analysis we find no evidence for EXOmotifs driving miRNA sorting into EVs from human cells. This is consistent with proteomics data suggesting that RBPs with EXOmotifs are barely detectable in EVs. In fact, depletion of AGO2 lead to a massive increase in non‐selective miRNA sorting into EVs. We propose that the dynamic cellular transcriptome is a major determinant for passive miRNA sorting into EVs.

Funding : TKI AQrate

Investigating quorum sensing through extracellular vesicles in Leishmania major

Liudmyla Hrebenyk, Vyacheslav Yurchenko

The University of Ostrava, Czech Republic

Introduction : Quorum sensing (QS) is a key mechanism that allows unicellular organisms, including Leishmania spp. parasites, to coordinate behaviour depending on population density and external factors, ensuring differentiation synchronization, adaptation to stress, and interaction with the host. Recent studies suggest that trypanosomatid communication occurs through extracellular vesicles (EVs) carrying microRNA, heat shock proteins (Hsp70, Hsp90), virulence factors (GP63, LPG), and viral endosymbionts (Leishmania RNA Virus, LRV). Despite growing interest, QS molecular mechanisms in Leishmania remain understudied. This project aims to characterize EVs from L. major (LRV+ and LRV‐) and investigate their role in QS.

Methods : EVs were isolated from L. major Tg 44 strains with (LRV2+) and without (LRV2‐) virus by ultracentrifugation and PEG precipitation. Characterization followed MISEV2023, including TEM, NTA, and dot blot analysis (DBA) with anti‐LPG antibodies. The impact of environmental factors on EVs production was assessed at 26–37°C using DBA. To establish an algorithm for future QS studies, we analysed QS‐related signalling molecules through database searches (PubMed, GEO, ExoCarta) and designed an experimental approach for EV content investigation using mass spectrometry and RNA sequencing.

Results : EVs sizes ranged from 150–170 nm, varying with LRV presence and temperature. LRV+ strains produced larger EVs, suggesting viral influence and dynamic corona formation. DBA confirmed LPG in EVs, justifying its further investigation in quorum sensing. Temperature influenced EV yield and composition, highlighting environmental effects on communication.

Summary/Conclusion : EVs size and composition depend on viral presence and temperature, supporting their role in QS. Future studies integrating proteomic and transcriptomic approaches will identify specific molecules involved in QS, improving our understanding of Leishmania communication and adaptation mechanisms.

Funding : This research was made possible through funding from the MSCA4Ukraine project, supported by the European Union.

Cell‐specific effects of extracellular vesicles from white and brown adipocyte‐like cells

Zeinab Ghesmati 1 , Mohsen Rashid 1 , Shabnam Fayezi 2 , Effat Alizadeh 1 , Masoud Darabi 3, 4

1 Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Department of Gynecologic Endocrinology and Fertility Disorders, Women's Hospital, Heidelberg University, 69120, Heidelberg, Germany. 3 Department of Hematology and Oncology, Division of Experimental Oncology, University Hospital Schleswig‐Holstein, Lübeck, Germany. 4 University Cancer Center Schleswig‐Holstein (UCCSH), Lübeck, Germany

Introduction : Adipose tissue functions as a secretory organ, influencing processes beyond energy storage. White and brown adipocytes, which play opposing roles in energy metabolism, exhibit unique paracrine and endocrine roles. Extracellular vesicles (EVs) from these adipocytes show therapeutic potential, yet their cell‐specific effects remain poorly understood. This study aimed to assess the impact of EVs from white and brown adipocyte‐like cells (WALCs and BALCs) on various human cell types.

Methods : This study received local ethics committee approval, and all cell samples were collected under informed consent. Human adipose‐derived stem cells (hADSCs) were differentiated into WALCs and BALCs using induction media. Differentiation was verified by lipid droplet accumulation and quantification of adipocyte markers. EVs were isolated from conditioned media (CM) using differential ultracentrifugation and characterized by light scattering, electron microscopy, and marker immunoblotting. Four human cell types—representing stem, somatic, and cancer cells—were treated with purified EVs or crude CM. Cellular responses were evaluated using flow cytometry, qPCR, electrochemiluminescence, and lipidomic profiling via liquid chromatography‐tandem mass spectrometry.

Results : In hADSCs, all EVs and CM promoted osteogenic differentiation and reduced stemness markers Oct4 and Sox‐2, with BALC‐derived EVs showing the greatest potential to enhance differentiation and WALC‐derived EVs most effectively suppressing stemness. When treated with BALC‐derived EVs, hADSCs displayed the highest number of differentially expressed lipids, primarily upregulated hits, while umbilical cord stem cells exhibited minimal responses to the treatment. In MCF‐7 cancer cells, CM from both adipocyte types had a greater pro‐apoptotic effect than EVs. Additionally, BALC‐derived EVs induced higher estradiol secretion in ovarian granulosa cells compared to WALC‐derived EVs. Pathway analysis indicated that BALC‐derived EVs promoted lipid storage and membrane remodelling in hADSCs, decreased lipid storage reliance in umbilical stem cells, reduced fatty acid oxidation in MCF‐7 cells, and enhanced energy metabolism in granulosa cells.

Summary/Conclusion : EVs from WALCs and BALCs cells exert distinct, cell‐specific effects compared to soluble factors, underscoring their role in mediating cell‐cell communication. These findings provide insights into the potential applications of adipocyte‐derived EVs in non–cell‐based therapies.

Funding : This study was supported by the Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 66664) and the University Medical Center Schleswig‐Holstein, Leubeck, Germany. The presentation was supported by the COST Action CA21113 SENESCENCE2030, awarded to the last author.

A novel drug BCN512 induces exosomal wnt release

Elizabeth M. Singer, Ph.D. 1 , William H. McBride, Ph.D, DSc. 2 , Subhrajit Saha Ph.D. 3 , Julian Whitelegge Ph.D. 4 , Andrew J. Norris, Ph.D. 1,4 Payel Bhanja Ph.D 3

1 BCN Biosciences Pasadena, CA. 2 Department of Radiation Oncology, University of California Los Angeles, Los Angeles, CA. 3 University of Kansas Medical Center, Kansas City, Kansas., 4 Semel Institute for Neuroscience and Human Behaviour, University of California Los Angeles, Los Angeles, CA

Introduction : SRFP1 (Secreted Frizzled‐Related Protein 1) has been shown to be present in exosomes, suggesting a potential role in modulating their function. SRFP1 is known to inhibit Wnt signalling, a crucial pathway involved in cell growth, differentiation, and migration. By being present in exosomes, SRFP1 could potentially influence Wnt signalling in recipient cells. While the specific mechanisms are still under investigation SRFP1 has been detected in exosomes derived from various cell types, including fibroblasts and cancer cells. We present here a drug which binds to SRFP‐1 resulting in exosomal Wnt release affecting radiation induced lung fibrosis.

Methods : Characterization of exosomes and identification of exosomal Wnt. exosomes were purified from organoid condition medium by ultracentrifugation. To determine the role of BCN512 in pulmonary epithelium derived WNT release in the present study we first purified the exosome from organoid conditioned medium. Isolated exosomes were identified by NTA and transmission electron microscopy. Purified exosomes were tested for WNT activity by TOPFLASH assay. Testing for Wnt activity was performed using TOPFLASH assays.

Results : Our prior studies demonstrated Wnt ligands were secreted and transported through exosomes. The results here revealed that exosomal Wnt activity was several‐fold higher in samples prepared from irradiated organoids treated with BCN512 compared with irradiated control. BCN512 treatment increased lung organoid‐derived exosomal WNT levels compared with irradiated control at 6 and 24 h post‐irradiation (4 Gy, p < 0.0004 and p < 0.0003; 6 Gy, p < 0.0005, and p < 0.0004, respectively). Further, using an avidin biotin Wnt 5a eliza based binding study, we show direct disruption of SRFP‐1 and wnt5a by BCN512 at the nM level. These results suggest that BCN512 also induces endogenous WNT release via disruption of SRFP‐1s sequestration of the ligand. Further, we demonstrated when the Wnt receptor fzd5 was deleted, the efficacy of BCN512 was negated, however, this was not the case when fzd7 was deleted, underscoring the mechanism of action of the drug.

Summary/Conclusion : This work gives a framework to possibly impact exosomal Wnt release which can modulate the development of radiation induced lung fibrosis as well as a possible future use in immune polarization (M1/M2).

Funding : BAA‐NIAID‐DAIT‐NIHAI2014001

Isolation and Characterization of Extracellular Vesicles in Malaria: Insights into Pathogenesis and Potential for Biomarker Discover

Presenter: Muna Abubaker

University of Salford, UK

Introduction : Malaria, a widespread disease caused by Plasmodium parasites transmitted through mosquito bites, continues to pose significant global health challenges. Despite advances in treatment and prevention, key obstacles remain in fully understanding malaria pathogenesis and discovering dependable diagnostic biomarkers. Extracellular vesicles (EVs), which facilitate intercellular communication and contribute to disease progression, have emerged as promising candidates for diagnostic and prognostic applications.

Methods : This study aimed to isolate and characterize small extracellular vesicles (sEVs) from Plasmodium falciparum (K1 strain)–infected red blood cells (iRBCs) and uninfected red blood cells (RBCs) in vitro. EV isolation was achieved using differential centrifugation and size exclusion chromatography (SEC), and sEVs were subsequently characterized via nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), BCA protein assays, and western blotting.

Results : Our results indicate a direct correlation between parasitaemia levels and the quantity of sEVs released by iRBCs, with higher parasitaemia associated with increased vesicle secretion. In contrast, uninfected RBCs released significantly fewer sEVs (p = 3.8E‐05), producing over 70% fewer vesicles than iRBCs. Across all parasite stages (ring, trophozoite, or schizont), more than 75% of the isolated sEVs were under 200 nm in diameter. Preliminary western blot analysis identified the presence of Plasmodium falciparum aldolase and Flotillin‐1 in iRBC‐derived sEVs, though further confirmation is necessary.

Summary/Conclusion : These findings underscore the potential of sEVs as accessible biomarkers for early malaria detection, disease severity assessment, treatment monitoring, and the development of innovative disease management approaches.

Funding : Technician Commitment at the University of Salford/UK

PTOV1 enhances the epithelial‐mesenchymal transition (EMT) process in breast cancer (BrCa) cells by modulating the exosomal landscape

Piyush Kumar, Indranil Banerjee, Yuba Raj Pokharel

Faculty of Life Sciences & Biotechnology, South Asian University, SAARC Nations, New Delhi, 110068, India

Introduction : Breast Cancer (BrCa) is a complex, heterogeneous disease based on receptor status. Subtype TNBC is highly metastatic and shows recurrent characteristics. Exosomes are 150 nm cargo‐loaded extracellular vesicles that have been reported to promote cancer progression and metastasis. The translational significance of these vesicles can address the lack of early diagnostic and limited treatment options for TNBC patients. PTOV1, a conserved adaptor protein is associated with high‐grade breast tumours. It affects Wnt signalling by suppressing DKK‐1 activity and interacts with genes involved in modulating exosome biology. This could be particularly relevant if PTOV1 is directly or indirectly involved in exosome‐mediated BrCa pathogenesis.

Methods : Stable PTOV1 knockout and overexpression in MDA‐MB‐231 and MCF7 cells were created using pLJM‐1 and LentiCRISPRv2 strategy. For preparing exosomes, the conditioned medium was filtered through a 0.22‐µm and ultracentrifuged at 110,000 g for 70 min at 4 degrees C and pellets were resuspended in PBS. Exosomes were characterized by DLS (size < 150 nm) and exosome‐associated markers (CD9, CD63, and FLOT‐1, Negative marker BCL‐2) by western blot. The protein profile of cell lysates and exosome proteins was visualized by the Coomassie brilliant blue staining. Changes in exosomal cargo were studied through western blot and miRNA Real‐time PCR. The transformation potential of the exosomal fractionate on the mammary non‐tumourigenic epithelial cells (MCF10A) was studied after treatment with 80ug /experiment exosomal fractionate.

Results : Findings suggest that PTOV1 influences the exosome cargo selection and release. Moreover, Micro BCA Assay data indicate that PTOV1 expression influences the release of exosomal protein fractionates with no change in exosome size. This change in the exosomal landscape affects the activity of Akt1/2 and proliferation, cell cycle, 3 D spheroids formation, migration, and invasion properties in mammary non‐tumourigenic epithelial cells. The same trend was observed in studies with the serum samples from TNBC patients.

Summary/Conclusion : Conclusively, findings demonstrate that PTOV1 overexpression induces changes in the EMT process in MCF10A cells via changes in the exosomes‐mediated horizontal transfer of bioactive molecules.

Funding : Indian Council of Medical Research (ICMR).

Leverageing antitumour effects of non‐thermal biocompatible plasma‐primed ucMSC‐derived extracellular vesicles

Presenter: Seung Ah Choi

Seoul National University Hospital, Republic of Korea

ABSTRACT UNAVAILABLE

Kinesin‐3 family members cooperate on secretory MVB transport for EV secretion

Henrike Tietz 1,2 , Leonie Witte 2 , Davina Störmer 1 , Christof Lenz 3 , Mona Honemann‐Capito 2 , Wilhelm Walter 1 and Julia Christina Gross 1,2,

1 Institute of Molecular Medicine, HMU Health and Medical University Potsdam, Potsdam 2 University Medical Center Goettingen, Goettingen 3 Department of Clinical Chemistry, University Medical Center Goettingen, Goettingen

Introduction : Extracellular vesicles (EVs) are critical for intercellular communication, reflecting their cell of origin's molecular profile. In the last years, the machenery of small EV (sEV) biogenesis and secretion has been studied in great detail, yet a remaining question is, how secretory multivesicular bodies (sMVBs) are transported towards the plasma membrane for release. Intracellular anterograde transport along microtubules is mediated by the kinesin motor protein family. Specifically the kinesin‐3 family acts in the directed movement of endosomal compartments. Understanding which of these kinesins mediate sMVB transport is a key step towards unterstanding the balance between secretory versus degradative MVB trafficking, the release of EV subpopulations and the underlying regulation of sEV biogenesis.

Methods : The involvement of the kinesin‐3 family in endosomal trafficking and sEV release was analysed by siRNA mediated knockdowns (KD) of different kinesin candidates in combination with immunofluorescent staining (IF), western blotting (WB) and nanoparticle tracing analysis (NTA) of purified sEVs. In addition, using motorless kinesin constructs linked to a minus end‐directed dynein motor, we generated systematic kinesin‐endosome interaction profiles of 7 kinesins candidates with 13 different endosomal/sEV‐markers. Indeed, individual kinesin‐3 members were distinctly recruited to specific sEV marker‐positive endosomal compartments and we found other kinesins interacting with early Rab11‐endosomes then CD63‐bearing late endosomes. As both of these compartments participate in the release of sEVs, we consequently probed for the involvement in secretory MVB trafficking and found their knockdown associated with partially reduced sEV secretion levels.

Results : The linkage assay revealed cooperative effects between the kinesin members themselves or between multiple kinesins and members of the EV secretion machinery, such as nsmase‐2 and SYTL4.

Summary/Conclusion : We will present our findings showing the kinesin‐3 family cooperatively involved in the secretion of sEVs and thus integrating the intracellular transport of sMVB into the biogenesis pathway of sEVs.

Milk Exosomes Loaded with Natural Neuroprotective Compounds Improve Outcomes in a Mouse Model of Brain Injury

Presenter: Tehila Ben Ziv

Tel Aviv University, Israel

ABSTRACT UNAVAILABLE

Mechanistic Insight into Human Milk Extracellular Vesicle‐Intestinal Barrier Interactions

Presenter: Xiang Luo

King's College London, UK

Introduction : Human milk extracellular vesicles (EVs) are crucial mother‐to‐baby messengers that transfer biological signals. These EVs are reported to survive digestion and transport across the intestine. The mechanisms of interaction between human milk EVs and the intestinal mucosa, including epithelial uptake remain unclear. Here, we studied the interaction of human milk EVs with the gut barrier components, including intestinal biofluids, enzymes, mucus and epithelium.

Methods : EVs were isolated from human milk by a pH adjustment and differential ultracentrifugation. The characterization of EVs were determined by BCA, NTA, DLS, and TEM. The stability study in the intestinal environment was conducted in FaSSIF, FeSSIF and enzyme fluids. Cellular uptake of EVs was measured by flow cytometry and confocal microscopy. The transport of EVs across intestinal monlayers was conducted with Caco‐2 transwell model. The pharmacological inhibitors of endocytic pathways was used to determine the relative contribution of specific pathways (chlorpromazine, genistein, nocodazole, 5‐(N‐ethyl‐N‐isopropyi)‐amiloride, methyl‐β‐cyclodextrin and dynasore). The proteomic analysis was done by LC‐MS/MS. The nucleic acids were loaded into EVs with electroporation and the cellular study and transport were done.

Results : We show that human milk EVs are largely stable in the biochemical gut barriers and demonstrate high mucus diffusivity. EVs show a high level of epithelial cell uptake (∼70%), and efficient transport across Caco‐2 monolayers. Whilst cell uptake of EVs was mediated by multiple routes, none of the pathway‐specific inhibitors inhibited their epithelial translocation. Proteomic analysis of EVs transported across Caco‐2 monolayers identified 14 enriched EV proteins that may facilitate intestinal transport. These findings significantly expand our understanding of the interactions between human milk EVs and the gut barriers, including their intestinal uptake. The siRNA was loaded into EVs with electroporation and the permeability of EVs‐associated siRNA through the intestinal barrier was significantly higher than free siRNA.

Summary/Conclusion: The work strengthens the existing evidence that EVs in human milk may be important signalling mediators based on their ability to carry biological cargo into and across intestinal epithelial cells within the hostile biochemical environment of the gut.

Study of the mechanisms of extracellular vesicles‐mediated intercellular communication

Stefano Vecchione 1 , Ana Talamillo 1 , Amaia Ercilla 2,5,6 , Arkaitz Carracedo 2,5,6,7,8 , Juan Manuel Falcón‐Peréz 1,2,3,4,5 .

1 Exosomes Laboratory, Center for Cooperative Research in Biosciences, Basque Research and Technology Alliance,Derio, Spain. 2 Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Spain. 3 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Madrid, Spain. 4 Metabolomics Platform, Center for Cooperative Research in Biosciences, Basque Research and Technology Alliance, Derio, Spain. 5 IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. 6 Centro de investigación Biomédica En Red de Cáncer (CIBERONC),28029 Madrid, Spain. 7 Traslational Prostate Cancer Research Lab, CIC bioGUNE‐Basurto, Biobizkaia Health Research Institute, Spain. 8 Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU),P.O. Box 644,E‐48080 Bilbao, Spain.

Introduction : Extracellular vesicles (EVs) are cell‐derived particles delimited by a lipid bilayer that cannot replicate on their own. Exosomes are nanovesicles (30‐150 nm) whose biogenesis involves the endocytic pathway and subsequent formation of multivesicular bodies (MVBs). Early endosomes mature into late endosomes, during which they accumulate intraluminal vesicles. Following this process, MVBs can fuse with lysosomes to degrade their contents or with the plasma membrane (PM) to release their contents into the extracellular environment. The enormous heterogeneity of EVs and their biogenesis mechanisms, along with their involvement in multiple physiological and pathological processes, has sparked interest in identifying compounds capable of inhibiting these mechanisms to translate this knowledge from the bench to the bedside.

Methods : To study the mechanism of interchange material via vesicles, we co‐cultured human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs), labelled with the lipophilic dye DiO (5 µL/mL), and Mouse Liver Progenitor‐29 (MLP‐29) labelled with DiD (5 µL/mL) for 24 and 48 h. After this time point, we analysed the conditioned media using flow cytometry (Cytoflex), and the cells were fixed with formaldehyde 2% for image analysis with the Axiovert microscope. To assess whether drug treatment affects vesicle‐mediated intercellular communication we added GW4869 (5 µM), Manumycin A (5 µM), nocodazole (10 µM), and sodium butyrate (10 µM) to the co‐culture.

Results : Using our model, we observed material interchange between hUC‐MSCs and MLP‐29 after both time points under control conditions. After 24 h of treatment with GW4869, we observed a statistically significant decrease in EVs secreted by hUC‐MSCs in the co‐culture, with a trend toward decreased EV secretion observed with all tested drugs. The ratio of APC to FITC events indicated that the drugs do not affect a specific cell line.

Summary/Conclusion : These results demonstrate the feasibility of using these protocols for high‐throughput screening of various compounds to identify and characterize molecules that influence EV biogenesis and secretion, thereby enhancing their therapeutic applications and utility as research tools.

Funding : This work was funded by HORIZON‐HLTH‐2022‐STAYHLTH‐02‐01 halt‐RONIN project (Ref. 101095679). Proyecto PID2021‐125104OB‐I00 financiado por MCIN/ AEI / 10.13039 / 501100011033/ y por FEDER Una manera de hacer Europa.

Exploring the proteomic and lipidomic disparities between Bifidobacterium adolescentis CCDM 368 and its EVs

Dominikia Kozakiewicz 1 , Angelika Sysak 1 , Agnieszka Razim 1 , Zbyněk Zdráhal 2 , Sabina Górska 1

1 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 2 Mendel Centre of Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Introduction : Bifidobacterium adolescentis plays a significant role in gut health, with its benefits often attributed to its interactions with host tissues. EVs derived from bacteria serve as key mediators in these interactions, carrying bioactive molecules such as proteins and lipids that may differ substantially from those of the parent bacterial cells. Studying the distinctions between bacteria and its EVs is essential to understanding their unique roles in modulating host physiology. This study aims to provide a comparative analysis of B. adolescentis CCDM 368 (Bad368) and its EVs, shedding light on functional divergences and potentially contributing to a broader framework for identifying and characterizing Gram‐positive bacterial EVs.

Methods : Bacterial cultures were inoculated in MRS medium at a 1:8 ratio for 20 h at 37°C. The culture was centrifuged, and the supernatant filtered through a 0.22 µm filter. EVs were isolated via ultracentrifugation (3 h at 4°C and 150,000 × g ) and fractionated using chromatography methods. Lipidomic analysis was performed using LC‐MS with an ACQUITY UPLC BEH Shield RP18 column and a Xevo G2 spectrometer, with data processed through MS‐DIAL and MetaboAnalyst 6.0. For proteomic analysis, samples were lyophilized, resuspended in SDT buffer, incubated at 95°C, and centrifuged. Solubilized proteins underwent FASP and trypsin digestion, with peptides analysed via LC‐MS/MS on the RSLCnano+ ImpactII system.

Results : Correlation analysis revealed that one EV fraction exhibited a distinct protein profile, indicating differences in protein composition and potential functional roles. Lipid composition also varied between fractions and from the parent bacteria, although the main lipid groups in EVs were similar to those of Bad368.

Summary/Conclusion : This study highlights distinct proteomic and lipidomic profiles between Bad368 and its EVs, underscoring functional specializations that could influence their roles in host interactions. The findings not only advance our understanding of Bad368 biology but also offer insights into potential markers specific to Gram‐positive bacterial EVs, paving the way for improved identification and functional analysis of these vesicles. This work contributes to the foundational knowledge needed to harness bacterial EVs for therapeutic and diagnostic applications, with broader implications for understanding Gram‐positive EV biogenesis and their impact on host‐microbe communication.

Funding : Work supported by National Science Centre of Poland UMO‐2021/43/I/NZ7/00693

Leishmania infective forms release extracellular vesicles by a pH‐dependent mechanism that modulates its content and differs in the ability to induce the ligand CD200

Presenter: Deborah Brandt‐ Almeida

University of São Paulo, Brazil

Introduction : The neglected tropical disease leishmaniasis affects different mammals, including humans, and is caused by the intracellular protozoan Leishmania which presents two infective forms, the promastigote and the amastigote forms, presenting different strategies to evade the immune response. Amastigotes release extracellular vesicles (EVs), which induce CD200 from infected macrophages, a ligand that inhibits cellular activation, and the inducible nitric oxide synthase/nitric oxide (iNOS/NO) mechanism. Until now, no information is available on the capacity of promastigotes to induce CD200.

Methods : EVs were obtained by incubating the different L. amazonensis forms in their specific medium/temperature for 1 h. Supernatants were filtered through a 0.45‐mm filter, concentrated, ultracentrifugated, and used for the different assays. Nanoparticle tracking analysis (NTA) was included to verify the purity and quantity of EVs. Macrophage stimulation assays for CD200 induction with EVs were analysed by immuno‐precipitation/ western blot (IP/WB) or then analysed by immunofluorescence by using a polyclonal antibody against Leishmania‐EVs and anti‐CD200 antibody.

Results : The release of EVs from both promastigotes and amastigotes is similar in quantity and size, reaching a concentrated amount of EVs between 100 and 200 nm. More important, only EVs released by amastigotes induced CD200 in macrophages, analysed by IP/WB and IF. Since both forms are incubated at different pH (5.2 and 7.0), we test the effect of amastigote EVs obtained from different pH. When amastigotes are incubated in another pH different than 5.2, such as the pH of promastigotes culture (7.0), the same level of EVs quantity is obtained, but the capacity to induce CD200 is impaired and associated with a modification of the proteins/genetic content.

Summary/Conclusion : These results indicate that amastigotes of L. amazonensis are specialized forms to induce CD200 in infected cells, and the pH during infection could be critical for the release of effector content into EVs that modulate macrophage response, highlighting the unique factors that must be considered in studying parasite EVs.

Funding : FAPESP, CNPq, CAPES

TcVPS23 acts in the EV biogenesis pathway and in the immunopathology of Trypanosoma cruzi experimental infection

Nadjania Saraiva de Lira Silva 1 , Ana Claudia Trocoli Torrecilhas 2 , Sergio Schenkman 3

1 Universidade Federal de São Paulo, campus São Paulo 2 Universidade Federal de São Paulo, campus Diadema 3 Universidade Federal de São Paulo, campus São Paulo

Introduction : Endosomal Sorting Complex Required for Transport (ESCRT) plays a role in eukaryotic cell secretion. It consists of four multi‐subunit complexes: ESCRT‐0, I, II, and III. This complex can be found in protozoan parasites, which cause serious infectious diseases in humans. One of these is Chagas disease, which is caused by Trypanosoma cruzi infection and frequently leads to cardiac fibrosis in people. We envisioned examining whether the ESCRT complex affects extracellular vesicle (EV) secretion and the potential function of EVs in Chaga's disease pathogenesis.

Methods : We created parasites by replacing one copy of the TcVPS23 gene, a key element of the ESCRT‐I machinery, with the hygromycin resistance gene using CRISPR/Cas9 technology and evaluated the resulting parasites' EV generation, infectivity, immune response, and pathogenesis.

Results : TcVPS23 expression was reduced, resulting in altered surface proteins, increased soluble protein release, and decreased extracellular vesicle secretion by the parasite's infectious trypomastigote form. Mice infected with the mutated parasite lines showed reduced blood parasitemia and mast cell recruitment in the cardiovascular system during the acute phase of infection. Despite the high parasite load in heart tissue, they also showed reductions in IFN‐gamma and TGF‐β levels in the circulatory system and heart. Mice in the chronic stage showed lower levels of IL1‐β and IL‐6, fewer mast cells, lower levels of MCPI, KC, and IL6, and less cardiac fibrosis than the parental parasite line.

Summary/Conclusion : These findings show that TCVPS23 of ESCRT‐I component affects the release of extracellular vesicles and soluble molecules, altering the immune response and myocardial fibrosis in the T. cruzi‐infected host.

Funding : Supported by CAPES, CPNq, and FAPESP 2020/07870‐4, and 2021/12883‐0.

The parasite Schistosoma mansoni triggers the host cells to release extra‐cellular vesicles with immunomodulatory properties

Michael Thaler 1 , Lukas Neuninger 1 , Magdalena Wysmołek 1 , Agnieszka.Razim 1 , Anna Schmid 1 , Jan Dvořák 2 , Martin Horn 3 , Adrian Leontovyč 3 , Michael Mareš 3 , Christoph Nagl 4 , Muhammed Faruk Saglam 1 , Karin Hoffmann‐Sommergruber 4 , Viktor Černý 1 , Aleksandra Inic‐Kanada 1 , Ursula Wiedermann 1 and Irma Schabussova 1

1 Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. 2 Czech University of Life Sciences Prague. Prague, Czech Republic. 3 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic. 4 Department of Pathophysiology and Allergy Research Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

Introduction : Schistosoma mansoni uses proteases such as Cathepsin B1 (SmCB1) and SmCL3 to invade the host via skin and migrate through the lungs. Host‐derived extracellular vesicles (EVs), essential in cell‐to‐cell communication, play a role in parasitic infections and are characterized by the presence of tetraspanins.

Methods : Recombinant SmCB1 and SmCL3 were produced in yeast Pichia pastoris. Their proteolytic activity was evaluated at pH levels between 6 and 8.5. Human lung epithelial cells A549 cells were exposed to both active and E‐64‐inactivated SmCB1 or SmCL3. EVs were collected by ultracentrifugation and purified by size exclusion chromatography. Size and concentration were determined with a Zetasizer, and Western blot‐ting was employed to detect tetraspanins CD63, CD81, and CD9. Mice received intranasal treatments with active and E‐64‐inactivated SmCB1 or EVs from SmCB1‐treated A549 cells over three days, and lung cells were then analysed via flow cytometry.

Results : SmCB1 displayed protease activity at pH 7.0 or lower, while SmCL3 was active across all tested pH levels. We confirmed the presence of tetraspanins by Western blotting. A549 cells released EVs upon treatment with both active and E‐64‐inactivated SmCB1 and SmCL3, with EVs measuring approximately 100 nm in diameter. Intranasal administration of active SmCB1 reduced alveolar macrophages (AM) counts, and inactivation with E‐64 abolished this effect. SmCB1‐induced EVs also led to decreased AM and natural killer (NK) cell counts in the lungs compared to controls.

Summary/Conclusion : These findings demonstrate that SmCB1 induces the release of EVs, which exhibit significant immunomodulatory effects.

Funding : This work was funded by the Austrian Science Fund (FWF) under grant number P 34867 und by OEAD under grant number CZ 07/2023 and RS 08/2022

Subtopic EV Characteristics 2

Curvature patch model of biological membrane and its experimental evidence in animal and plant extracellular vesicle shape

Presenter: Veronika Kralj‐Iglic

University of Ljubljana, Slovenia

Introduction : A better understanding of basic physicochemical mechanisms of formation and stability of extracellular vesicles (EVs) is highly warranted in the development of methods based on EVs. Micro‐sized membrane‐enclosed vesicles without internal structure have been theoretically described by the area‐difference elasticity model which considers the membrane as an elastic laterally isotropic continuum whereas due to the small size of the EV, its membrane is sensitive to the shape of the membrane building blocks.

Methods : EV membrane was theoretically described using ensemble statistical physics methods. The membrane is represented by a surface divided into patches, each containing a very large number of constituents (inclusions) deriving from both closely coupled membrane layers. Free energy of a single inclusion takes into account curvature mismatch and in the case of anisotropic inclusions also the in‐plane orientation of the inclusion. The equilibrium shape of the membrane is obtained by minimization of the free energy obtained by summing up the contributions of all the inclusions. Minimization is performed by numerical solutions of Lagange‐Euler differential equations and Monte Carlo simulations. In the experimental part EVs were isolated from blood and tomato homogenate by differential centrifugation and observed by Scanning Electron Microscopy.

Results : Based on agreement of theoretical and experimental results we propose a model in which general properties of highly curved membranes are taken into account. In narrow necks and tubular parts, the inclusions undergo orientational ordering which stabilizes the shape. The model describes a variety of EV shapes and their possible transformations. Some of them (cuboid, discoid, dumbbell, and starfish) were imaged by SEM in isolates from blood and tomato homogenate.

Summary/Conclusion : The model explains the stability of nano‐sized membrane‐enclosed vesicles without internal structure which could not be explained by the area‐difference elasticity model relevant for mildly curved (micro‐sized or larger) structures. The results indicate that specific chemical composition is not a prerequisite for EV stability or its shape. The key parameter is the intrinsic shape of the inclusions, which reflects the properties of groups of molecules and their interactions.

Funding : Slovenian Research Agency: J3‐3066, P3‐0388, P2/0132, P2‐0232, J2‐4427, J2‐4447, EU Horizon 2020 MSC Staff Exchange: FarmEVs 101131175.

Utilizing microfluidic techniques to differentiate cancer patient samples based on extracellular vesicle profiles

Gréta Lilla Bányai 1 , András Merényi 2 , András Szabó 1 , Afrodité Németh 1 , Anikó Gaál 3 , Csaba Pongor 1 , Tamás Garay 1,2

1 Pázmány Péter Catholic University, Hungary; 2 Semmelweis University Hungary; 3 Research Centre for Natural Sciences, Hungary

Introduction : Liquid biopsies, including the analysis of circulating tumour cells, cell‐free DNA, and extracellular vesicles (EVs), offer promising potential for early and detailed diagnosis for cancer patients. EVs, might have a distinguished potential to provide insight into a patient's physiological and pathological state as they are secreted by all cell types. Microfluidic techniques enable characterization of dissolved particles based not only on their size distribution but accounting also for their mass, density and shape.

Methods : Blood samples were obtained from patients with prostate, pancreatic, and colorectal cancer. For each sample, the interval from blood draw to plasma isolation and hemoglobin levels were recorded. The microsomal fraction of plasma was isolated using size‐exclusion chromatography (SEC), and this fraction was subsequently characterized for particle size, size distribution, and concentration using nanoparticle tracking analysis (NTA). Total protein content was quantified using the Qubit protein assay, while lipid content was assessed using the SPV assay. Atto‐488 NHS ester‐stained samples were introduced into a microfluidic channel, where the distribution of protein‐bound green fluorescent signals was assessed across the channel diameter by capturing consecutive fluorescence microscopy images. Fluorescence intensity was quantified by fitting an asymmetric sigmoid function to both sides of each distribution curve, followed by determining the amplitude and growth rate of the averaged function.

Results : Our experiments did not demonstrate a significant correlation between hemoglobin levels and the time elapsed prior to sample processing. Additionally, lipid and protein concentrations lacked specificity across patient groups. Although nanoparticle tracking analysis (NTA) yielded uniform results among these groups, microfluidic analysis revealed distinct variations in the growth rates of fluorescence intensity curves as samples passed through the microfluidic channel. These differences enable robust differentiation among patients with unique oncological profiles.

Summary/Conclusion : This indicates that microfluidic techniques can offer a more comprehensive assessment of the extracellular vesicle (EV) fraction by leverageing diffusion characteristics alongside particle size distributions. Furthermore, the ability to differentiate between cancer patient samples using a robust, cost‐effective, and easily performed technique holds significant promise for clinical applications.

Funding : This research was supported by the National Research, Development and Innovation Office through the grant TKP2021‐EGA‐42.

Impact of the peptide treatment and storage on the protein corona of extracellular vesicles

Imola Cs. Szigyártó 1 , Tasvilla Sonallya 1 , Priyanka Singh 1 , Lilla Turiák 2 , László Drahos 2 , Zoltán Varga 1 , Tamás Beke‐Somfai 1

1 HUN REN Research Centre for Natural Sciences, Institute of Materials and Environmental Chemistry, Hungary. 2 HUN REN Research Centre for Natural Sciences, Institute of Organic Chemistry, Hungary.

Introduction : Identifying the spontaneously formed protein corona on the surface of nanoscale materials is of critical importance for the therapeutic and diagnostic development of nanoparticles. In order to explore the potential role in manipulating the surface of nanoparticles and also to progress towards surface engineering, cationic peptides with well‐characterized action mechanism were selected. Storage time and conditions were also investigated as they could influence the composition off protein corona.

Methods : Red blood cell‐derived extracellular vesicles (RBCEVs) were isolated, as they are easily produced in high concentrations, and have several unique advantages, such as the lack of DNA, prevention of immune clearance and extended circulation due to the presence of several integral membrane proteins. The interactions were followed using flow‐linear dichroism spectroscopy, flow cytometry and microfluidic resistive pulse sensing techniques. Nanoscale liquid chromatography coupled to tandem mass spectrometry was performed for characterizing protein corona composition.

Results : Based on results, peptide treatment has reduced the protein content of the treated REV fractions. TEM images clearly demonstrate a smooth vesicle surface lacking protein aggregates. The relative abundance of characteristic membrane and subcellular proteins remained mainly constant after treatment. The list of proteins found in treated sample was subtracted from the control protein list, which resulted in seventeen proteins, identified as protein corona members. These proteins include plasma membrane components or have cytosol, cytoplasm origin, which are likely to associate with the surface of EVs.

Summary/Conclusion : Our results envisage the applicability of peptides in modulating protein corona content, as a possible tool in specific surface engineering of nanoparticles from both synthetic and natural origin. Moreover, this study will create new opportunities in wound healing, tissue repair or biofilm inhibition as both EVs and host peptides could be present in the complex microenvironment.

Funding : This work was funded by the National Research, Development and Innovation Fund: 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021, TKP2021‐EGA‐31 and KKP_22 project no. 144180 and by Eötvös Loránd Research Network: SA‐87/2021 and KEP‐5/2021.

Comparing extracellular vesicles and liposomes from the protein corona perspective: a systematic review

Shan Wang, Qiubai Li

Union hospital, Tongji medical college, Huazhong University of Science and Technology, China

Introduction : Nanoparticles possess protein coronas that significantly affect their biological properties and in vivo behaviour. Extracellular vesicles(EVs) and liposomes are, respectively, natural and synthetic nanoparticles that resemble in size, morphology and structure. This systematic review comparatively analyses EVs and liposomes from the protein corona perspective, aiming to leverage insights from liposome corona research to enhance future exploration in the emerging field of EV corona research.

Methods : A comprehensive search was conducted using Web of Science, PubMed and Scopus to identify articles studying protein coronas of EVs and liposomes from 2010 to October 2024. Queries were derived from several related published articles and the MeSH Database. A total of 37 studies were included based on the PRISMA guidelines.

Results : Primary formation mechanisms of liposome corona—the Vroman effect and cooperative adsorption—partially apply to EVs, with cooperative adsorption observed in EV corona but the Vroman effect needing further validation. EVs can acquire corona proteins like ApoE and fibronectin intracellularly, showing a distinct difference in formation compared to liposome corona. Several studies identified immunoglobulins, fibrinogen, complement factors and apolipoproteins as universal corona components of EVs and liposomes, though their relative abundance differs. Albumin was consistently observed as a major component of liposome corona; however, studies reported conflicting findings on its presence in the EV corona. Biological significance of EV and liposome coronas were mainly assessed in the context of drug delivery regarding cellular uptake, circulation, and biodistribution, whereas EV corona also demonstrated potential as disease biomarkers for rheumatoid arthritis or ovarian cancer, as well as therapeutic applications in promoting tissue regeneration and wound healing. Furthermore, methods for isolating and characterizing protein coronas of EVs and liposomes were convergent. Yet, state‐of‐the‐art techniques for characterizing protein conformation, spatial arrangement, and affinity kinetics in EV corona require further exploration.

Summary/Conclusion : EV corona introduces a paradigm shift in EV research but is still undergoing early development. Integrating expertise from liposome corona research with the inherent strengths of EVs as natural nanoparticles may exert synergy effects, paving the way for new advancements in EV‐based nanomedicine for diagnosis, drug delivery, and therapeutics.

Funding : the National Key Research and Development Program of China (No. 2021YFA1101500)

Tspan8 and the Epigenetic Cargo of Extracellular Vesicles: Mapping DNA and Histone

Rihab Ksouri 1 , Stanka Matic 1 , Siobhan King 2 , Irina Nazarenko 1

1 Institute for Infection Prevention and Control, Faculty of Medicine, Medical Center—University of Freiburg, Germany 2 ONI, Oxford, England, United Kingdom

Introduction : Extracellular vesicles (EV) are nanoscale heterogeneous membrane structures released into the extracellular space by most cells and present in almost all biofluids. EVs are of remarkable importance due to their involvement in physiological and pathological processes; they contain diverse biomolecules, including proteins, lipids, metabolites and nucleic acids, including RNA and DNA. However, due to the small size and heterogeneity of EVs, it is still not known in detail which of the cargo is located inside the EV and which is associated with the EV surface, forming the so‐called EV biocorona, with DNA being the most prominent molecule identified as associated with EVs.

Methods : A fibrosarcoma cell model consisting of 3 cell lines was used: HT1080, HT1080‐V stably transfected with a control lentiviral vector T2A‐PuroR, and HT1080‐Tspan8 transfected with the hTSPAN8‐T2A‐PuroR vector and expressing Tspan8. Tangential Flow Filtration (TFF) was used to isolate EVs that retained the EV biocorona. To obtain a comprehensive DNA and histone profile, the whole cell secretome was separated into three fractions: largeEV, smallEV and EV‐depleted using TFF. The isolated fractions were characterised using nanoparticle tracking analysis (NTA) to determine particle concentration. Integrity and quality control of isolated fractions were assessed using microBCA protein assays and dot blot for EV biomarker's presence. Histone presence was assessed by Western blot, and DNA content and concentration were measured using a QIAamp DNA Mini Kit, followed by fragment length measurement using an Agilent Bioanalyzer.

Results : We observed that the particle concentration of small EV ranging between 50 and 150 nm, increased compared to large EV. Characterisation of small EV also showed the highest expression of EV biomarkers. increased DNA content was associated with small EV, with a variation of fragment sizes between 1500‐7000 bp. The presence of all histones was demonstrated by Western blot in our cell lines and small EV.

Summary/Conclusion : During this work, we were able to establish our method for the isolation and quantification of EV‐associated DNA and histones. Our future work will focus on the specific localisation of DNA and histones within EVs and the associated protein corona.

Dissecting the Roles of Extracellular Vesicles versus Their Corona Proteins in Modulating Cellular Behaviour

Presenter: Metoboroghene O. Mowoe

Karolinska Institutet, Sweden

Introduction : Extracellular vesicles (EVs) are increasingly recognized for their therapeutic potential, yet the role of the EV‐associated protein corona—functional and presumed variable—remains poorly understood. In this study, we aim to delineate the distinct roles of EVs versus their associated corona in cellular interactions and therapeutic applications.

Methods : To dissect the distinct contributions of EVs and their corona in cellular interactions, we treated fibroblasts with EVs stripped of their soft corona or corona proteins derived from 12 different cell sources. Doses ranged from 20 to 200,000 EVs per cell or 0.001 and 10 ng of protein per cell. Transcriptional effects were analysed using RNA sequencing.

Results : Unbiased global analyses revealed that while corona dose significantly influenced cell responses, cell source had minimal impact. Notably, EVs without their soft corona failed to elicit dose‐ or source‐specific effects, though high doses downregulated exocytosis. corona protein source‐specific dose‐dependence was more pronounced at low doses, likely reflecting increased cellular sensitivity to variations in corona composition when EV concentration is limited. At higher doses, this sensitivity diminished possibly due to the saturation of cellular pathways or receptor systems leading to a convergence in cellular responses regardless of cell source.

Summary/Conclusion : These findings underscore the critical importance of optimizing both EV source selection and dose determination for therapeutic applications, emphasizing the functional role of the EV corona in modulating cellular behaviour.

Subtopic EV Heterogeneity 2

Extracellular vesicles released from secretory lysosomes of Natural Killer contain NKG7 and differ from bona fide CD63+ vesicles

Yunjie Wu 1 , Miriam Aarsund 1 , Susanna Ng 2 , Sudworth A 1 , Marit Inngjerdingen 1

1 Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Norway 2 Institute of Experimental Oncology, University Hospital Bonn, University of Bonn, Bonn, Germany.

Introduction : The tetraspanin NKG7 is preferentially expressed in cytolytic granules of NK cells and T cells. Lack of NKG7 perturbs cytotoxic activity, and results in reduced control of tumours in vivo. We have previously shown that a subset of NK‐cell derived extracellular vesicles (EVs) contain NKG7. In this study we show that NKG7+ EVs form a distinct subset of EVs that likely originate from within cytolytic granules.

Methods : EVs were produced from the NK‐92 cell line grown in serum‐free media supplemented with IL‐15. The EVs were isolated via differential centrifugation and density‐gradient ultracentrifugation, followed by Izon qEV columns. Characterization of EVs was performed via BCA assays, NTA, Western blotting and flow cytometry. Proteomics profiling was done via LC‐MS/MS. EV subsets were isolated via immunoaffinity. NKG7 was knocked down via CRISPR, and cell morphology assessed via electron microscopy.

Results : Via transmission electron microscopy, we found that cytotoxic granules contain intraluminal vesicles positive for NKG7. Further, CRISPR‐mediated knockdown of NKG7 in the NK cell line NK‐92 led to accumulation of smaller‐sized cytotoxic granules, accompanied with fewer intraluminal vesicles in the granules. In contrast, multivesicular bodies appeared morphologically normal. Immunogold labelling indicated a higher amount of granzyme B in granules of NK cells lacking NKG7. EVs released from NK cells lacking NKG7 were smaller, but induced tumour cell apoptosis at a comparable level to EVs from control NK cells, indicating that vesicles from the endosomal system also may contribute to tumour cell apoptosis. To further understand the biology of NKG7+ EVs, we purified NKG7+ EVs and performed comparative proteomics profiling against bona fide CD63+ EVs. We found that NKG7+ EVs are enriched in granzymes, certain Rab proteins and proteins mediating vesicular fusion. These proteins were reduced in EVs derived from NK cells lacking NKG7.

Summary/Conclusion : The data indicates that NKG7 may play a role in cytotoxic granule biogenesis, and ILV generation. NKG7+ EVs may be a separate, specialized subset of EVs, possibly mediating an alternative pathway of targeting cancer cells.

Funding : The Research Council of Norway.

Identification of RNA‐binding proteins in extracellular vesicles that control mRNA sorting through binding to specific RNA motifs

Presenter: Ying Zhang

Tsinghua University, China (People's Republic)

Introduction : Extracellular vesicles (EVs) carry genetic materials, including RNA and DNA, to modulate gene expression in recipient cells, playing critical roles in both physiological and pathological processes. However, the mechanism underlying mRNA sorting into EVs—a key question in EV biogenesis—remains largely unknown.

Methods : Using HeLa cell‐derived EVs, we performed pull‐downs of poly(A)‐interacting proteins followed by proteomic analysis. From the identified proteins, several RNA‐binding candidates were selected for further investigation. By depleting these proteins in parental cells with CRISPR, we analysed changes in EV‐associated mRNAs using RNA sequencing and qPCR. Potential RNA motifs binding to RNA‐binding proteins were identified through bioinformatic analysis aided by AI algorithms. Additionally, RNA immunoprecipitation (RIP) was used to examine mRNA interactions with these proteins.

Results : Our study identifies nucleophosmin 1 (NPM1) as an RNA‐binding protein critical for mRNA sorting into EVs. NPM1 binds specific motifs, playing a pivotal role in the selective loading of functional mRNAs, such as EGFR, into EVs, which further translated into functional protein in recipient cells. Furthermore, the presence of one core motif identified in our study enhances the targeted selection of mRNA into EVs.

Summary/Conclusion : Our study not only enhances the understanding of the molecular mechanism underlying mRNA sorting into EVs, but also provides valuable insights for the potential development of EV mRNA transport methods.

Funding : This work was funded by the National Key R&D Program of China (no. 2023YFC3605400, 2021YFE0109300), National Natural Science Foundation of China (no. 22137004, 21977061), Beijing Natural Science Foundation (IS23100, IS23107).

The role of the miRNA processing complex in miRNA sorting into extracellular vesicles (EV)

Presenter: Yuzhou Zeng

University of Bonn, Germany

Introduction : Extracellular vesicles (EV), emerging as new mediators of cell‐cell communication in ubiquitous cell types, are capable of transporting RNA, including microRNA (miRNA), protein and lipid cargo to recipient target cells. In the nervous system, miRNAs can fine‐tune synaptic plasticity. They inhibit local mRNA translation at dendrites, and axons and subsequently downregulate synaptic proteins to enhance or reduce synapse formation and synaptic strength upon extracellular stimuli, known as synaptic plasticity. Based on our previous research, neurone‐derived EV can induce functional alterations during dendritogenesis and synapse formation in the recipient neurones via specific miRNA cargo.

Methods : In this study, we look into the mechanisms by which miRNAs are sorted for secretion in neuronal EV. During miRNA maturation, the RNA‐induced Silencing Complex (RISC)‐ loading complex (RLC), containing Dicer and associated proteins, mediate the cleavage of pre‐miRNAs, and loading onto the RISC complex, which mediates translational repression via the functional single‐stranded miRNA. We first examined the protein interactome of the RLC by mass spectrometry in rodent primary neurones. Then, we further assessed the role of candidate RLC‐interacting RNA binding proteins (RBPs) on EV‐miRNA secretion using immunoaffinity purification, as well as size exclusion chromatography with ultracentrifugation and rt‐qPCR. Moreover, we looked into the sub‐cellular localisation of RLC‐RBP interactions using high‐ and super‐resolution microscopy and proximity ligation assay.

Results : Our preliminary findings suggest that the RLC regulates the sorting of miRNAs to EVs through the binding of several RBPs previously implicated in recognizing motifs on miRNAs and directing specific miRNAs sorting into endosomes. Additionally, we observed that ER‐endosome membrane contact sites regulate the interaction between RLC and RBPs, hence the miRNAs could be incorporated into endosomes at those sites.

Summary/Conclusion : We illustrate a new sorting pathway of EV‐miRNAs, that may be important for the regulation of EV secretion in neurites and therefore regulate synapse development and synaptic plasticity.

Inhibition of Acid or Neutral Sphingomyelinases Differentially Impacts RNA and Protein Cargo Sorting to Extracellular Vesicles

Presenter: Juan‐Carlos Padilla

Institut de Recherches Cliniques de Montreal, Canada

Introduction : Extracellular vesicles (EVs) form through regulated biogenesis processes involving sphingomyelinases (SMases), enzymes that metabolize sphingomyelin to produce ceramide—a lipid influencing membrane rigidity and essential for EV generation.

Methods : This study explores the impact of inhibiting neutral SMase (NSM) and acid SMase (ASM) on the sorting of EV protein and RNA cargoes in human MCF7 cells.

Results : Our results revealed that NSM inhibition reduces EV nanoparticles and diminishes RNA and protein cargoes, including endosomal, spliceosomal, and translation‐related proteins. Conversely, ASM inhibition increased RNA‐binding proteins within and enhanced the expression of ribonucleoprotein complex‐associated RNA in released EVs, including several snRNAs and 7SL RNA. Intriguingly, ASM‐inhibited EVs enhanced the migration and translational activity of recipient MCF10A cells.

Summary/Conclusion : These findings suggest an important role for SMase‐dependent vesiculation in governing RNA and protein trafficking to the extracellular space, unveiling potential implications for cellular communication and function.

Funding : This research was made possible by grants awarded to E.L. from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Fonds de Recherche du Québec—Santé (FRQS). E.L. is a distinguished research scholar of the FRQS. J.C.A.P. was a recipient of doctoral scholarships provided by the FRQS, McGill University, and the IRCM.

LMTK3 regulates EV biogenesis and promotes packageing of immunomodulatory cargo into EVs though Rab7 phosphorylation

Presenter: Mark Samuels

Zhejiang Chinese Medical University, China (People's Republic)

Introduction : Breast cancer (BC) remains a leading cause of cancer mortality, with extracellular vesicle (EV)‐mediated communication between BC cells and the innate immune system playing a crucial role in all aspects of the disease. Lemur tail kinase 3 (LMTK3) is an oncogene frequently upregulated in BC, driving therapy resistance, invasion and metastasis, ultimately worsening patient outcomes. Building on preliminary data linking LMTK3 to endosomal trafficking and immunomodulation, this study investigates the roles of LMTK3 in EV biogenesis and EV‐mediated communication between BC cells and the innate immune system.

Methods : EVs were collected from control and LMTK3‐overexpressing BC cells using differential ultracentrifugation, then their proteins and physical characteristics were thoroughly characterised on a single particle and population level. Biochemical assays and microscopy techniques were used to identify how LMTK3 affects EV biogenesis. EVs were also used in 3D coculture models to assess how LMTK3‐dependent EV cargo alters monocyte infiltration, macrophage polarisation and anti‐cancer activity. Bioinformatics analysis of TCGA datasets validated results using clinical data.

Results : LMTK3 upregulation induces the production of large EVs enriched in oncogenic and immunoregulatory proteins. Single particle analysis revealed changes in the proportions of different EV subpopulations as measured by the abundance of tetraspanins. We identified Rab7 as an in vivo substrate of LMTK3 where its phosphorylation results in altered late endosomal trafficking, inhibiting degradation of cargo and favouring release of EVs. Functionally, EVs from LMTK3‐overexpressing but not control BC cells caused metabolic changes in monocytes, shifting macrophages towards and M2‐like polarisation and reducing monocyte infiltration.

Summary/Conclusion : LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. This may partly be due to macrophage M2 polarisation and exclusion of M1 macrophages from the tumour microenvironment, mediated by LMTK3‐dependent alterations in EV cargo. Furthermore, we demonstrate a novel mechanism of Rab7 regulation by LMTK3, impacting EV cargo selection and multivesicular body trafficking dynamics.

The role of LAMP2A in Endosomal Protein Composition: Implications for Membrane Identity, Endosomal Maturation and Exosome Biogenesis

Presenter: Joao Ferreira

NOVA Medical School, Portugal

Introduction : The endolysosomal network plays a crucial role in preserving cellular and tissue homeostasis, either by endocytosis, whereby endosomes undergo maturation to form endolysosomes for subsequent degradation, or by producing and releasing exosomes that facilitate intercellular signalling. We have previously described an ESCRT‐independent mechanism known as exosomal LAMP2A Loading of Cargo (eLLoC), where endosomal LAMP2A selectively directs proteins containing a targeting pentapeptide, the ExoSignal, to exosomes. However, LAMP2A knockout led to alterations in exosome composition that were not limited to changes in the levels of ExoSignal‐containing proteins.

Methods : In this study, we used mass spectrometry, as well as biochemistry and microscopy techniques, to analyse exosomal and endosomal fractions and investigate the influence of LAMP2A on endosome protein profiles.

Results : We found evidence of the involvement of LAMP2A in endosomal processes such as cortical actin association, RAB27A and PI(4,5)P2 regulation of membrane tethering for plasma membrane fusion, and enhanced RAB7 association and endolysosomal maturation. The experimental validation confirmed that LAMP2A knockout (KO) in endosomal compartments leads to significant alterations in Rab protein profiles, including increased levels of Rab27B and decreased levels of Rab27A, as well as enhanced actin dissociation from endosomes. Additionally, LAMP2A KO resulted in increased endolysosomal acidification and activity, elevated Cathepsin D activity, and changes in phosphoinositide dynamics, with reduced PI(4,5)P2 and increased PI4P colocalization with endosomes.

Summary/Conclusion : This study highlights the broader impact of LAMP2A on endosomal function and suggests a role in defining the unique identity of a specific subset of endosomes essential for eLLoC.

Funding : This work is supported by Portuguese Foundation for Science and Technology, COVID: “Programa Operacional Regional de Lisboa— FEDER/ Project 02/SAICT/2020/072552”, iNOVA4Health—UIDB/Multi/04462/2020 and UIDP/Multi/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia / Ministério da Educação e Ciência, through national funds and TransNet project 2022.8649. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work is also supported by the European Union, EVCA Twining Project (Horizon GA n° 101079264).

PI3K/Rab signalling regionally and conditionally regulates large exosome biogenesis

Shuo Wang, Yosuke Tanaka

The University of Tokyo, Japan

Introduction : Sonic hedgehog (SHH)‐containing large exosomes are a new type of large EVs. They may be equivalent to ‘ART‐EVs’ that contain SHH and Rab18 proteins (Coulter et al., Cell Rep 2018); and the hMSC‐derived ones relevant for post‐myocardial infarction recovery (Mackie et al., Circ Res 2012). Here we show that PI3K/Rab signalling facilitates SHH‐containing large exosome release from hMSCs. In our previous study of polydactyly mice (Wang et al., Dev Cell 2022), we detected an FGFR/PI3K signalling dependency of a posterior‐to‐anterior SHH protein gradient formation in developing limb buds. Regional regulation of SHH‐containing large exosome release may contribute as an underlying mechanism, of which rate was significantly augmented by FGFR/PI3K signalling in the peripheral layer. In the present study, we have investigated precise molecular and cellular mechanisms of PI3K‐induced large exosome release.

Methods : We stimulated human MSCs and mouse fibroblasts with PI3K agonists and Rab18‐modulating compounds and/or constructs. Then we measured the changes in the large exosome releasing cascade by immunocytochemistry, protein biochemistry/immunoblotting, NTA, TEM, and live imaging. In addition, we investigated the physiological relevance of the large exosomes using HUVEC cell assay for in vitro angiogenesis.

Results : PI3K agonists significantly stimulated hMSCs to release large exosomes of 0.6–1.5 um large, bounded by lipid bilayers, and highly enriched by SHH protein. The EVs from stimulated hMSCs showed 4 folds higher angiogenic capacities on HUVEC cells than control EVs. Rab18 GTPase may act as a downstream target of PI3K signalling that critically facilitates the membrane trafficking of large exosome components. We show evidence that PI3K signalling increases the GDP‐bound form of Rab18 GTPase, which was preferentially accumulated in the cell centre according to the microtubule‐mediated intracellular trafficking. SHH‐containing large exosomes were then directly released from this perinuclear endosomal compartment, probably by the function of Rab18‐GDP effector proteins.

Summary/Conclusion : This study describes a PI3K/Rab‐mediated regulation mechanism of SHH‐containing large exosome production. It will provide cell biological insights on how the noncanonical secretion drives large exosome release, which will be of high clinical values for regenerative medicine.

Elucidating the Role of Mitochondrial Extracellular Vesicles in Cancer Drug Resistance through Single‐Particle Analysis Using Nano‐flow Cytometry

Xu Xiao, Cheng Lu, Yunyun Hu, Xiaomei Yan

Xiamen University, China

Introduction : Mitochondrial quality control (MQC), critical for cellular metabolism and dynamics, encompasses mechanisms such as fusion, fission, and mitophagy. Recently, mitochondrial extracellular vesicles (mitoEVs) have emerged as novel mediators of MQC. While links between alterations in mitoEVs and various diseases, including cancer drug resistance, are established, their specific roles and characteristics in tumour chemoresistance are not well understood. This study aims to develop and apply a high‐throughput, multi‐parameter single‐EV level technique using nano‐flow cytometry (nFCM) to characterize mitoEVs, shedding light on their biogenesis and interactions with cellular organelles in drug‐resistant cancer cells.

Methods : EVs were isolated from both chemosensitive and chemoresistant cancer cell lines (MCF‐7 and MCF‐7/Dox) with or without doxorubicin (DOX) treatment through differential ultracentrifugation. These EVs were analysed at the single‐particle level using an nFCM equipped with a 532‐nm laser or a dual‐laser system (488 and 647 nm). Parameters such as size, purity, particle concentration, DOX content, and co‐localization with mitochondrial and other cellular organelle markers were measured. MitoEVs were further enriched from MCF‐7/Dox cells through high‐resolution iodixanol density gradient centrifugation. Key proteins involved in the biosynthesis and secretion pathways of mitoEVs in MCF‐7/Dox cells were targeted using inhibitors or RNA interference (RNAi).

Results : Our high‐throughput, multi‐colour single‐particle analysis by nFCM revealed that drug‐resistant MCF‐7/Dox cells secreted a higher abundance of mitoEVs and DOX‐loaded EVs than chemosensitive MCF‐7 cells. Notably, mitoEVs and DOX‐loaded EVs co‐localized within the same density layers. Using inhibitors and RNAi against ATP‐binding cassette (ABC) transporters, an increase in drug expulsion via mitoEVs was observed in MCF‐7/Dox cells, suggesting a novel compensatory mechanism of MQC. Furthermore, mitoEVs predominantly interacted with autophagosomes before being released into the extracellular space, especially when mitochondrial function was impaired in MCF‐7/Dox cells.

Summary/Conclusion : Through single‐EV analysis by nFCM, this study demonstrates the critical role of mitoEVs in MQC during cancer progression. Our findings not only improve methods for isolating mitoEVs but also provides valuable insights into their biogenesis and secretion mechanisms, highlighting their potential as biomarkers and therapeutic targets for overcoming cancer drug resistance.

Funding : This research was supported by the National Natural Science Foundation of China (Grants 21934004 and 32450337).

Plasma‐Derived Exosomal miRNAs Reveal Unique Expression Patterns in Non‐Small Cell Lung Cancer Patients

Presenter: Kamal Baswal

All India Institute of Medical Sciences, India

Introduction : Non‐small cell lung cancer (NSCLC) remains a major health challenge worldwide, accounting for a significant portion of cancer‐related deaths. Early detection and diagnosis are crucial for improving patient outcomes; however, reliable biomarkers are still limited. Exosomes—small extracellular vesicles secreted into bodily fluids—have emerged as promising candidates for biomarker discovery due to their role in cell communication, cargo protection and disease specificity. This study aims to identify exosomal miRNA signatures in NSCLC patients, providing insights into novel biomarkers for NSCLC diagnosis.

Methods : Exosome isolation was performed on blood samples using ultracentrifugation. The isolated exosomes were validated through the expression of characteristic markers CD63, ALIX, and CD9, and morphological confirmation was achieved using transmission electron microscopy (TEM). RNA extraction from exosomes was conducted and next‐generation sequencing (NGS) was subsequently performed to profile miRNAs across all samples. Differential expression analysis identified specific miRNAs of interest, with miR‐16‐5p used as a housekeeping gene due to its stable expression across samples. Validation of differentially expressed miRNAs, was carried out using quantitative PCR (qPCR). In addition, expression levels in plasma‐derived exosomes were compared to those in plasma alone to evaluate miRNA exclusivity to the exosomal fraction.

Results : The sequencing analysis revealed hundreds of miRNAs across the patient and control samples, with a subset displaying differential expression between the NSCLC and control groups. Notable miRNAs, including hsa‐miR‐7b‐5p, hsa‐miR‐486‐5p, hsa‐miR‐122‐5p, and others, were significantly upregulated or downregulated in the NSCLC samples. qPCR validation confirmed these expression trends, underscoring their potential as diagnostic markers. Additionally, comparison of plasma versus plasma‐derived exosome miRNA profiles revealed specific miRNAs, that were exclusively present in the exosomal fraction, further suggesting a role for these miRNAs in NSCLC pathophysiology.

Summary/Conclusion : we identified several differentially expressed miRNAs, including hsa‐miR‐7b‐5p, hsa‐miR‐486‐5p, and hsa‐miR‐122‐5p, that show promise for diagnostic applications. The exclusive presence of certain miRNAs within the exosomal fraction, as compared to plasma alone, further highlights the diagnostic specificity of exosomal miRNAs. These findings provide a foundation for developing minimally invasive, blood‐based diagnostic tools for NSCLC, which could significantly improve early detection and patient outcomes.

Funding : INDIAN COUNCIL OF MEDICAL RESEARCH, NEW DELHI

Diameter, protein‐to‐lipid ratio, lipid signature, and Lamp1 as defining parameters of ectosomes, exosomes, and exomeres

Presenter: Sayam Ghosal

Semmelweis University, Hungary

Introduction : Recent advancements in extracellular particle (EP) research have unveiled novel non‐vesicular extracellular particles (NVEPs), such as exomeres and supremeres, yet much remains unknown about their biogenesis and physiological roles. This study investigates the properties of NVEPs, isolated from a 167k g pellet (167k‐NVEP), and compares them to well‐characterized extracellular vesicle (EV) subtypes: small EVs (100k‐sEVs) and large EVs (14k‐lEVs) isolated from 100k g and 14k g pellets respectively. Our goal was to explore the biogenesis, molecular composition, and structural diversity of these EPs, thereby contributing to a deeper understanding of EP dynamics.

Methods : We employed an integrative approach, combining an image meta‐analysis from various published studies on intraluminal vesicles (ILVs) across species and kingdoms, with extensive characterization of 167k‐NVEPs and EV subtypes. Characterization techniques included transmission electron microscopy (TEM), biochemical assays, Raman spectroscopy, lipidomic profiling via mass spectrometry, high‐resolution flow cytometry and super‐resolution microscopy. These methods enabled us to identify structural, biochemical, and molecular distinctions among the EP subtypes.

Results : TEM image meta‐analysis revealed that ILVs within multivesicular bodies (MVBs) consistently measured with a mean ± SD diameter of 109.60 ± 36.77 nm. This size range closely aligns with sEVs but not lEVs. The maximum observed ILV size was 197.00 nm, showing that lEVs exceeding 200 nm in diameter may be of ectosomal origin but not derived from the exosome biogenesis pathway. 167k‐NVEPs demonstrated a significantly higher protein‐to‐lipid ratio and unique lipid composition, marked by cholesteryl ester enrichment. Raman spectroscopy revealed a similar trend of significantly high protein‐to‐lipid ratio in 167k‐NVEP compared to 100k‐sEV and super‐resolution microscopy showed strikingly stronger Lamp1 positivity.

Summary/Conclusion : Our findings suggest that EPs >200 nm likely do not originate from the exosomal pathway. Elevated Lamp1 expression in 167k‐NVEPs indicates a lysosomal association, suggesting distinct functions and physiological roles from traditional EVs.

Funding : EU's Horizon 2020 Research and Innovation Programme (739593), OTKA FK 147023, The Higher Education Excellence Program (FIKP) and the Therapeutic Thematic Programme (TKP2021‐EGA‐23), National Cardiovascular Laboratory Program (RRF‐2.3.121‐2022‐00003) and 2019‐2.1.7‐ERA‐NET‐2021‐00015, NVKP_16‐1‐2016‐0004 grant of the Hungarian National Research, NKFIH, VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, EKÖP‐2024‐237 and Stipendium Hungaricum Scholarship 2021.

Collagen alpha‐1 (VI) chain in Urinary Extracellular Vesicles as a Panacea to Anti‐ageing: from Diagnostics to Therapeutics

Olusanya Olasehinde 1,2,3* , Joshua Seun Olajide 4 , Helen B. Binang 5,6 , Rosephine Enadeghe 7 and Fuquan Yang 1,2

1 Institute of Biophysics, Chinese Academy of Sciences, China. 2 University of Chinese Academy of Sciences, China. 3 University of Benin, Nigeria. 4 Chinese Academy of Agricultural Sciences, China. 5 University of New South Wales, Australia. 6 Ingham Institute for Applied Medical Research, Australia. 7 University of Saskatchewan, Canada.

Introduction : Cellular senescence is a hallmark of ageing and contributes to the progression of age‐related diseases. Understanding protein complexes involved in senescence could provide insights into the ageing process and potential therapeutic approaches.

Methods : This study analysed protein complexes in small urinary extracellular vesicles (suEVs) isolated from human urine. Isolation was achieved using differential ultracentrifugation (DUC), membrane filtration, and 1D blue native polyacrylamide gel electrophoresis (1D‐BN‐PAGE). Protein complexes and protein‐protein interactions (PPIs) were further confirmed using molecular docking and molecular dynamics simulations.

Results : Seven distinct protein bands were identified in suEVs, revealing a variety of proteins and their isoforms that form complexes through PPIs. Notably, the collagen alpha‐1(VI) chain, encoded by the COL6A1 gene, was detected, suggesting a role in cellular and metabolic processes related to ageing and diabetes. These protein complexes could be key players in pathways that regulate cellular senescence and ageing‐related diseases.

Summary/Conclusion : This study highlights the significance of suEV protein complexes in understanding ageing mechanisms and identifies potential therapeutic targets, particularly in ageing and diabetes management. Findings emphasize the importance of suEV protein complexes in regulating cellular functions and suggest their potential in age‐related therapeutic applications.

Funding : 1. The National Key R&D Program of China (2018YFA0507801 and 2018YFA0507103) 2. Chinese Academy of Sciences (CAS) and The World Academy of Sciences (TWAS).

Standardized Analyses of Extracellular Vesicles and the Cells that Shed them Across Five Model Tissues

Presenter: Cristina Zivko

University of Bern, Switzerland

Introduction : Extracellular Vesicles (EVs) are released by cells across all biological domains, playing crucial roles in many intercellular interactions under physiological and pathological conditions. They are thus increasingly studied for diagnostic and therapeutic purposes, some already reaching clinical trials.

Methods : EVs were isolated from five distinct human or bovine model tissues (hepatic, ureteral, intervertebral disc, umbilical cord mesenchymal, and induced pluripotent stem cells). Isolation was performed by standardized ultracentrifugation followed by size exclusion chromatography to ensure high purity and minimal protein aggregation. The resulting EV populations were thoroughly characterized in terms of yield, size distribution, and surface charge. For all samples, membrane blotting of the purified EVs was performed to recover proteins for untargeted shotgun proteomics analysis. Proteins were also isolated from the cells that shed them.

Results : EV isolation yielded 10⁸–10 11 particles per sample depending on the origin, indicating successful recovery but also differences across tissue types. Nanoparticle tracking analysis confirmed heterogeneous but consistent vesicles’ size distribution profiles, averageing 180±100 nm. Measurements of zeta potential indicated a mean surface charge of ‐29±11 mV, suggesting colloidal stability and a negatively charged membrane, which aligns with typical EV properties. Proteomics analysis revealed a highly distinctive yet reproducible protein signature for each tissue‐derived EV population. It was powerful enough to also discriminate between different treatment conditions within the tissue of interest. Cells proteomics were clearly discriminated from EVs. Comparative analysis between EVs and their respective parent cells uncovered selective protein enrichment patterns, supporting the hypothesis that EVs actively package specific biomolecules rather than merely reflecting cellular protein content.

Summary/Conclusion : We provide reproducible evidence that EVs are not passive cellular byproducts but functionally specialized particles. Results were consistent across 5 tissues of origin, contributing to the growing body of knowledge on EV heterogeneity, highlighting the potential of tissue‐specific EVs for translational applications. By establishing a solid foundation for future clinical/biotechnological applications, our study advances the standardized understanding of EV biology and paves the way for their integration into therapeutic strategies.

Funding : Center for Extracellular Vesicles Research (EVR) at the University of Bern, the Proteomics and Mass Spectrometry, the Flow Cytometry and Cell Sorting Core Facilities.

Comparison of spontaneously released and experimentally induced red blood cell‐derived extracellular vesicles

Zsuzsanna Adamecz 1 , Attila Tiba 2 , Mária Péter 3 , Gábor Balogh 3 , Zsolt Török 3 , Edit I Buzás 1,4,5 , Evelyn Orsó 1

1 Semmelweis University, Hungary, 2 University of Debrecen, Hungary, 3 HUN‐REN Biological Research Centre, Hungary, 4 HCEMM SU Extracellular Vesicles Research Group, Hungary, 5 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Hungary

Introduction : Red blood cells (RBCs) are anucleated cells that obligatorily consume glucose and lack an extensive intramembranous system. Eryptosis, an apoptosis‐like process in RBCs, is regarded as the main source of red blood cell‐derived extracellular vesicles (RBC‐EVs). This study aimed to compare different populations of RBC‐EVs generated spontaneously and in response to various in vitro stimuli, and to understand the mechanisms underlying RBC‐EV biogenesis.

Methods : RBCs were isolated from EDTA‐anticoagulated whole peripheral blood collected from healthy volunteers. While erythrocytes undergo spontaneous, time‐dependent vesicle formation, this process can be stimulated in vitro by exposure to A23187 calcium ionophore or to cold stress (4°C). Vesicle formation was modulated by the administration of calcium ions and glucose. RBC‐EVs were isolated by differential centrifugation and characterized using flow cytometry, nanoparticle tracking analysis (NTA), lipidomics and transmission electron microscopy (TEM) combined with morphometry and bioinformatic analysis.

Results : The released RBC particles were annexin‐V positive, indicating phosphatidylserine externalization, and expressed CD235a (glycophorin‐A), confirming their RBC origin. The vesicular nature of these particles was validated by their sensitivity to Triton‐X 100 detergent. TEM images revealed vesicular structures with a heterogeneous size distribution. At least two distinct types of RBC‐EVs were identified: populations with low and high electron density. We hypothesize that the RBC‐EVs with high electron density contain hemoglobin. Bioinformatic analysis of TEM images further supported differences in EVs released spontaneously and EVs induced by different stimuli. The mean diameter of RBC‐EVs, assessed by NTA and TEM morphometry, varied across treatment groups, with both methods yielding comparable results. Mass spectrometric lipid profiling showed that A23187‐induced RBC‐EVs were enriched in conical, membrane curvature‐promoting lipid species (e.g., phosphatidic acid and ceramide) compared to intact RBCs. Additionally, we found that exposure to A23187 and calcium ions significantly enhanced RBC‐EV release, while glucose partially inhibited vesicle formation, suggesting a regulatory role of glucose in RBC‐EV production.

Summary/Conclusion : In conclusion, we have established and characterized an in vitro system to study eryptosis and regulate RBC‐EV production. Our findings highlight differences between spontaneously released and experimentally induced RBC‐EVs, which should be considered in future research.

Funding : NVKP_16‐1‐2016‐0004, VEKOP‐2.3.2‐16‐2016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003, 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 No. 739593.

The cell adhesion protein ALCAM controls the cargo composition of EVs

Presenter: Javier Ramirez‐Ricardo

Vanderbilt University, USA

Introduction : ALCAM is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily of cell adhesion proteins. It is expressed in various cell types, including cancer cells, regulating the dynamic turnover of adhesions to contribute to cell motility and metastasis. Given the critical emerging roles of extracellular vesicles (EVs) in regulating cancer cell migration and metastasis, we asked whether ALCAM might impact EV biogenesis, composition, or function to control cancer cell migration or invasion.

Methods : To study the effects of ALCAM on cancer cells, we generated ALCAM‐deficient UMUC3 bladder tumour cell lines using CRISPR‐Cas9. EVs were isolated from control and ALCAM knockout cell culture supernatants by ultracentrifugation and size exclusion chromatography (SEC) and characterized by nanoparticle tracking (ZetaView), western blots (WB), flow cytometry with dimensional reduction analysis of single EVs after staining with Di8‐ANEPPs lipid dye (“EV fingerprinting”), and proteomics.

Results : EV fingerprinting identified select enrichment of subpopulations of EVs in ALCAM‐deficient UMUC3 cells, an invasive bladder tumour cell line. Proteomics of EVs demonstrated abundant cargos related to cell‐cell adhesion, cell migration, ECM‐receptor interaction, focal adhesion, and signalling pathways in cancer. This analysis identified an increase in alpha 2 integrin (ITGα2) and a decrease in alpha 3 integrin (ITGα3) in EVs from ALCAM‐deficient cells, which was confirmed by western blot and single vesicle flow cytometry. Consistent with these changes, we detected higher levels of laminin in wild‐type EVs, which contained higher levels of ITGα3, a receptor for this extracellular matrix protein. Changes in integrin cargos in UMUC3 cell EVs reflected changes in protein content in the parental cells.

Summary/Conclusion : Our data indicate that ALCAM affects the protein composition of the EVs secreted from the UMUC3 cancer cell line. Future studies will explore the functional impacts of these EVs on bladder cancer cell behaviour.

Funding : NIH grant (R01CA249424)

Systematic in vivo Investigation of Fecal Extracellular Vesicles Using a Controlled Colonization model

Presenter: Hajar Yaakoub

Nantes Université, France

Introduction : Early‐life gut microbiota is a key determinant of lifelong health, influencing intestinal function and mediating interactions with distant organs, a process increasingly linked to bacterial extracellular vesicles (BEVs). However, fecal extracellular vesicles (fEVs) remain understudied, with no standardized extraction protocol, limiting insights into microbiota‐host interactions. Using a simplified gut microbiota murine model, this study aims to characterize fEVs by examining their origin, contributions of G‐ and G+ bacteria, and metabolite profile, while assessing the impact of gut colonization type, sample type, sex, and age.

Methods : Two controlled colonization models of 7‐week‐old mice were used: one group monocolonized with a G‐ bacterium (Escherichia coli) and another bicolonized (dixenic) with both G‐ (E. coli) and G+ (Bifidobacterium longum) species. Control groups included germ‐free (axenic) and naturally colonized mice. A 15‐week‐old dixenic group was included to assess age effects. EVs were extracted from cecum, colon, feces, and plasma using an optimized protocol. In addition, culture‐derived BEVs were used for comparison. Characterization involved protein quantification, nanoparticle tracking analysis, dot blotting, mass spectrometry, and transmission electron microscopy (TEM).

Results : Cecum samples contained half a log more fEVs than colon or feces, with higher protein enrichment and larger size, making them the optimal sample for fEV studies. Colonization model had a slight effect on fEV abundance, with the dixenic group showing the lowest values. However, fEVs were significantly smaller in axenic mice and highly protein‐enriched in the normal group, highlighting colonization's impact on fEV size and production. The fEV properties were similar, irrespective of age and sex. Dot blotting of fEVs confirmed the presence of BEVs and host‐derived EV components. Data suggest bioactive molecule enrichment in culture‐derived BEVs, with ongoing analyses determining whether this extends to fEVs. TEM observations confirmed the integrity of fEVs.

Summary/Conclusion : This study establishes a framework to explore fEV production, origin, composition, and circulation. The findings presented here spur further investigational efforts and pave the way for more comprehensive research, potentially advancing research on their impact on health and disease.

Membrane‐associated vesicles generated by mesenchymal stem/stromal cells—are they distinguished subtype of extracellular vesicles?

N.A. Basalova 1, O.A. Grigorieva 1 , U.D. Dyachkova 1 , M.A. Vigovsky 1 , D.V. Bagrov 2 , O.S. Sokolova 2 , E.S. Novoseletskaya 1,3 , A.Yu. Efimenko 1

1 Centre for Regenerative Medicine, Medical Research and Educational Institute, Lomonosov MSU, Moscow, Russia. 2 Faculty of Biology, Lomonosov MSU, Moscow, Russia. 3 Shenzhen MSU‐BIT University, Shenzhen, China.

Introduction : Mesenchymal stem/stromal cells (MSCs) are important regulators of tissue homeostasis and regeneration mostly due to the secretion of bioactive factors, including molecules within extracellular vesicles (EV). Recently, a new type of EVs has been discovered—membrane‐associated vesicles (MAV). However, their certain characteristic and role in the regulatory effects of MSCs, as well as reliable methods of isolation, have not been previously studied.

Methods : For the isolation of MAV from MSCs (ASC52telo, ATCC), we proposed methods based on disruption of MAV from cell membranes—the use of TritonX100; trypsin; hyaluronidase and proteinase K. Free‐secreted EV, isolated from MSC conditioned medium by ultrafiltration(300 kDa), were used as control. The isolated fractions were characterized according to MISEV2023 criteria by size (SEM, NTA), morphology (SEM) and markers (WB‐CD81, CD9, CD73, HSP70, Alix—positive marker, H2Ax—negative marker for cell contamination verification). Since one of the important homeostatic functions of MSCs is ability to suppress fibrosis, the functional activity of the isolated fractions in equal concentration was tested on the TGFβ‐induced fibroblast‐to‐myofibroblast differentiation model (WB; ICC–αSMA).

Results : We have shown that the optimal protocol for isolating MSC‐MAV includes hyaluronidase treatment, which allows obtaining the highest yield of particles homogeneous in size and save cells in a native state. Isolated particles have a morphology and average size similar to EV (MAV 133±69 nm, EV 139±64 nm). However, the peak concentration of MAV is significantly lower ‐ 110±5 nm vs 135±10 nm for EV. Alix is not expressed on MAV, unlike EV, which contain all studied positive markers. In model of fibroblast‐to‐myofibroblast differentiation MAV suppresses the expression of αSMA 30 times less than EV.

Summary/Conclusion : Among the tested approaches for the separation of MAV from MSCs we suggest the use of hyaluronidase. The obtained MAV have the characteristic morphology of EVs, but their size is smaller than EV and they do not carry Alix. MAV are able to suppress the differentiation of fibroblasts into myofibroblasts, but less effectively than EV from the same MSCs, which may indicate MAV distinguished cargo and function.

Funding : Support by Interdisciplinary Scientific and Educational School of Moscow State University “Molecular Technologies of Living Systems and Synthetic Biology”,#24‐Ш04‐14.

Study on the promotion of extracellular vesicle secretion by CT in spheroid culture of MSCs and its therapeutic effect in skin injury

Presenter: Ting Lan

School of Medicine, Southeast University,China (People's Republic)

Introduction : Human umbilical cord mesenchymal stem cells (hUC‐MSCs) are pluripotent stem cells with significant clinical potential. The extracellular vesicles (hUC‐MSC‐EVs) secreted by these cells carry RNA and proteins beneficial in various disease models. However, traditional two‐dimensional culture methods for hUC‐MSCs face limitations, including restricted growth area and inconsistent cell quality. Scaffold‐free spheroid culture offers a more physiologically relevant microenvironment, preserving cell characteristics and optimizing culture space. Despite its advantages, challenges such as spontaneous spheroid formation and EV yield remain. This study explores how CT enhances MSC spheroid formation and the role of secreted EVs in treating skin injuries.

Methods : (1) The impact of CT on MSCs was evaluated using CCK8 assays and paracrine functional tests. (2) MSCs underwent scaffold‐free spheroid culture with CT intervention, with morphology documented over time. (3) Second‐generation transcriptome sequencing elucidated mechanisms underlying CT's effect on MSC spheroid formation. (4) PCR assessed EV production‐related gene expression post‐CT intervention; cell supernatants were collected for EV isolation and NTA detection. (5) CT‐3D MSC‐EVs were utilized in HUVEC scratch and tube formation assays. (6) A mouse skin injury model assessed the efficacy of CT‐3D MSC‐EVs.

Results : CCK8 assays indicated that CT promotes MSC proliferation and upregulates paracrine factors. After 24 h of spheroid culture, CT enhanced MSC spheroid formation and elevated EV‐related genes (CD9, CD81, CD63, Alix) and stemness genes (Nanog, Sox2, Oct4). Transcriptome sequencing confirmed CT's role in promoting spheroid formation via upregulation of adhesion molecules. Scratch assays showed CT‐3D MSC‐EVs significantly improved HUVEC migration compared to 2D‐MSC‐EVs, and tube formation assays further validated their superior efficacy. In vitro experiments demonstrated the effectiveness of CT‐3D MSC‐EVs in repairing mouse skin injuries.

Summary/Conclusion : This study highlights that CT promotes MSC spheroid formation by enhancing intercellular adhesion molecules and EV secretion, improving therapeutic effects in skin injury treatment.

Exploring Extracellular Vesicles from Kombucha Microbiome: Composition, Diversity, and Health Potential

Presenter: Thirunavukkarasu Ramasamy

National council research (CNR), Italy

Introduction : Recent emerging studies have been consistently demonstrated that microbes secrete the extracellular vesicles (EVs), are lipid bilayer‐bound nanoparticles that act as a major communication channel and facilitate the transfer of biomolecules between intra‐ and inter species. Fermented tea called kombucha has become very popular because of its possible health advantages, which are mostly linked to its probiotics, bacteria and yeasts. Though their use does not carry the risk of infections from microbes, it has recently been proposed that probiotics derived EVs may also provide health advantages. The purpose of this study was to pinpoint the microbial diversity and functional traits found in kombucha. Furthermore, aimed to separate and describe EVs from kombucha and its predominant microorganisms to study the biological activities of EVs in an in vitro model, with particular attention to their functions in immune‐regulation, intercellular communication, and therapeutic potential.

Methods : Metagenomic analysis of kombucha involved high‐throughput DNA sequencing and data processing to characterize microbial diversity, composition, and functional capabilities throughout fermentation. To isolate EVs, we used differential centrifugation, followed by density gradient ultracentrifugation. Further, EVs were characterized by Qubit Protein Assay, SDS‐PAGE analysis, and Interferometric light microscopy.

Results : Dominant bacterial species belonged to cellulose and acetic acid producers (Acetobacteraceae and Lactobacteriaceae), while identified yeasts were from Zygosaccharomyces during kombucha fermentation. The protein profiles of kombucha‐derived EVs revealed bands with varying intensities and dissimilar banding patterns suggest different protein profiles across the fermentation process, indicating a variety of EV protein sizes and expressions over multiple species. At first attempt, EVs were isolated from komagataeibacter xylinus which is one of the most dominant strains in kombucha. SDS‐PAGE image suggests that contain multiple protein species with different molecular weights. The intensity and distribution of the bands can provide insights into protein composition of K. xylinus derived EVs.

Summary/Conclusion : Production of EVs was successfully confirmed with the isolation and observation of EVs in k. xylinus culture, as well as in kombucha. Results may help to understand the central role of microbial‐interactions and open new perspectives on human health significance.

Funding : Work was supported by SCOBY‐DO project (HORIZON‐MSCA‐2022‐PF‐01) under Horizon Europe Framework Programme, grant agreement N.101109583.

Quantitative and spatial tissue and serum pharmacokinetics of EVs from diverse sources using high content single vesicle imaging

Maria Jaritsch 1 , Tanja Plank 1 , Melanie Schürz 1,2 , Joachim Danmayr 1 , Cristian T. Matea 1 , Vesna Stanojlovic 1,2 , Eva Klinglmayr 1,2 , Patricia Hrasnova 1,3 , Heloisa Melo Benirschke 1 , Sarah Schönleitner 1 , Anna Müller 1 , Jana Kiefer 1 , Laurens Kober 1 , Petra Reinthaler 1 , Angelika Sales 1 , Martin Wolf 3,4 , Dirk Strunk 3,4 , Mario Gimona 2,3 , Eva Rohde 2,3,5 , Irma Schabussova 6 , Valentina Perova 1 , Gunda Üblagger 1 , Theresa Neuper 1 , Silja Wessler 1 , Wolf Heusermann 7 , Martin Hintersteiner 7 , Nicole Meisner‐Kober 1,2

1 Paris Lodron University of Salzburg, Austria; 2 Ludwig Boltzmann Institute for Nanovesicular Precision Medicine at PLUS, Austria; 3 Paracelsus Medical University Salzburg, Austria; 4 Austrian Red Cross Research, Austria; 5 University Hospital of Salzburg, Austria; 6 Medical University of Vienna, Austria; 7 EvoBiotiX SA, Switzerland;

Introduction : Interest in extracellular vesicles (EVs) as therapeutics or for drug delivery is steadily increasing, but data on quantitative in vivo pharmacokinetics/dynamics, bioavailability and biodistribution—especially a systematic comparison between EVs isolated from different sources—are still limited. Since EVs are heterogeneous in size and composition, normalization of the labelling and detection strategies are challenging. This study presents a strategy for normalization and a side‐by‐side biodistribution map of vesicles from different sources.

Methods : We investigated EVs from different sources (bovine, goat and human milk, umbilical cord MSCs, HEK293 cells, H.pylori and E.coli) by labelling with FRET‐pair fluorophores in the NIRF‐range, quantifying endotoxin levels and administering them intravenously into mice. To quantify EVs directly in serum and PFA‐fixed tissue cryosections, we developed a high‐content imaging workflow using automated high‐resolution widefield imaging and EVAnalyzer2. For normalisation of whole organ fluorescence, we investigated cow milk EVs labelled at different EV‐to‐dye stoichiometries and developed a normalisation strategy using single vesicle imaging.

Results : Covalent labelling of EVs with NIRF‐dyes allowed the detection of single vesicles and provided the best signal‐to‐noise in serum and tissue, but we observed that for different EV‐sources the same labelling stoichiometry leads to different brightness and number of labelled vesicles. To overcome this heterogeneity, we have developed a normalisation strategy that enables a cross‐comparison of EV‐sources. In general, we observed a uniformLy short serum half‐life (< 5 min) of all EV‐sources in mice. This suggests rapid extravasation into tissue which was confirmed by cryosections of selected organs. In the liver, high EV signals were observed within cells after 5 min, followed by a decrease and redistribution to other organs at later timepoints. In addition, we observed differences in organ distribution between the EV‐sources, supporting the concept of a differential tropism linked to the EV‐source.

Summary/Conclusion : We successfully established a workflow for quantifying EVs from diverse sources in serum and cryosections using NIRF‐dyes and automated high‐resolution widefield imaging. In addition, by normalizing the whole organ fluorescence data based on single vesicle imaging, we were able to compensate for the different labelling of the EV‐sources thereby allowing to link pharmacokinetics, biodistribution and spatial biology of EVs in vivo.

Extracellular vesicles farming: how to get the best from tomato

Gabriella Pocsfalvi 1 , Veronika Kralj‐Iglič 2 , Kim Kwang Pyo 3 , Seppo Vainio 4 , Maneea Moubarak 5 , Fabrizio Cillo 6 , Ani Barbulova 1 , Wilma Sabetta 1 , Kim Dae Ho 3 , Immacolata Fiume 1 , Ramila Mammadova 4 , Anna Romolo 2 , Ales Iglič 2

1 National Research Council of Italy, Institute of Biosciences and BioResources, Italy, 2 University of Ljubljana, Slovenia, 3 Kyung Hee University, Republic of Korea, 4 University of Oulu, Finland, 5 Damanhour University, Egypt, 6 National Research Council of Italy, Institute for Sustainable Plant Protection, Italy

Introduction : Plant‐derived nanovesicles (PDNVs) isolated form plant homogenate are studied for their nutritional and biotechnological potentials. Natural variability in crop composition, presence of plant pathogens and the inherent heterogeneity of PDNVs make them less suitable for pharmaceutical applications. Recently, the production of extracellular vesicles (EVs) through in vitro plant cultures is raising interest. Here we aimed to setup cell suspension culture (CSC) of tomato (Solanum lycopersicum L.) to produce EVs and to compare them to tomato PDNVs.

Methods : Cell culture was characterized by cell numbers, packed cell volume, growth curve, metabolic activity and morphological features. EVs were harvested either from the conditioned cell culture media (CCM) or from the pelleted cells treated with cell wall degrading enzymes. PDNVs and EVs were isolated by density gradient ultracentrifugation. Nanostructures were visualized by cryogenic transmission electron microscopy and characterized for particle size distribution, particle concentration, protein/nucleic acid content, SDS PAGE protein profile and protein contents.

Results : We have established and optimized a CSC‐based EV‐farming platform. Plant EVs can be successfully harvested from CSCs. Size, size distribution (100‐200 nm diameter particles) and morphological features of EVs and PDNVs were similar, the yield of EVs from CSC was lower that PDNVs’ yield. Protein biocargo of tomato CSC EVs were different from PDNVs. Membrane proteins including different enzymes (plasma membrane ATPases, ceramidase, pectate lyases, hydrolyses of phospholipids and ceramides), transporters (aminoacid and ABC transporters), abiotic stress tolerance PLAT domain protein and 14‐3‐3 domain‐containing protein were identified.

Summary/Conclusion : CCM‐derived plant EVs bypass the structural complexity of the plant organism and could overcome some of the inherited drawbacks of PDNVs, like low reproducibility, presence of viruses, etc. The scalability of CSC, the high rate of cell growth and the good reproducibility make suspension‐cultured cells suitable for EV production. Importantly, the EV‐farming can foster the in vitro studies of EV biogenesis and uptake and the roles of EVs in plant biology.

Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs,” grant agreement N. 101131175 and the “Italian Ministry of Foreign Affairs and International Cooperation” EV‐C@p grant number PGR02032.

Comparative Efficacy of Extracellular Vesicles and Soluble Fractions of Ovine Fetal MSC and Chondrocyte Secretomes for in vitro Osteoarthritis Treatment

Presenter: Maria Belen Arteaga

Veterinary Medicine University of Vienna, Austria

Introduction : The secretome of a cell reflects its intrinsic capacity to modulate the surrounding microenvironment, offering equivalent therapeutic potential shaped by the donor cell's age, type, and differentiation status. However, it remains unclear whether the secretome's components act synergistically or redundantly, and whether the therapeutic effect requires the entire secretome, isolated extracellular vesicles (EVs), or solely soluble factors. This study thus evaluates the therapeutic efficacy of secretome fractions derived from fetal MSCs and chondrocytes on inflamed adult ovine chondrocytes in vitro.

Methods : Conditioned medium (NCM) from ovine fetal umbilical cord MSCs (fMSCs) and fetal chondrocytes (fCCs) was collected daily for 6 weeks ( n = 4 biological replicates each) from serum‐free cultures maintained in hollow fibre bioreactors and pooled per donor. EVs were isolated using tangential flow filtration (300 kDa cut‐off), and the remaining EV‐free medium was concentrated using crossflow filtration (CCM, 3 kDa pore size). EV characterization included particle size, concentration, and protein markers. The therapeutic effects of NCM, isolated EVs (1x10⁹ particles/mL), and EVs combined with CCM were assessed on inflamed (1 ng/mL TNF‐α and IL‐1β) chondrocytes in 2D and 3D cultures. Healthy, inflamed untreated and inflamed dexamethasone‐treated chondrocytes served as controls. Multi‐omics analyses (RNA‐Seq, miRNA‐Seq, Mass Spectrometry proteomics and metabolomics) were employed as readouts.

Results : Treatment with EVs and combined CCM‐EVs significantly reduced inflammation at 24 h post‐treatment, on par with dexamethasone, while NCM showed lower efficacy. Ingenuity pathway analysis indicated fCC‐ and fMSC‐derived EVs prominently inhibited “neutrophil degranulation”, followed by fCC‐NCM. However, fCC‐NCM yielded the greatest suppression across inflammatory pathways, including “macrophage, fibroblast, and endothelial cell roles in rheumatoid arthritis” and “S100 family signaling,” while enhancing “chaperone‐mediated autophagy”, indicating a stronger therapeutic effect of fCC‐ compared to fMSC‐derived secretome and fCC‐CCM‐EVs versus fCC‐EVs alone. These effects were confirmed by proteomics and metabolomics, which revealed the most substantial differential abundance of proregenerative and immunomodulatory protein and metabolite sets in fCC‐derived NCM and EVs, compared to fMSC fractions.

Summary/Conclusion : Results highlight donor cell differentiation and secretome fraction as critical determinants of therapeutic efficacy, with the combination of fCC‐derived soluble proteins and EVs yielding the best anti‐inflammatory and proregenerapive effects.

Funding : wings4innovation and KHAN‐I technology transfer fund

Lifespan extension of human MSCs with hTERT does not change the functional properties of cells and secreted EVs

Presenter: Alessia Brancolini

Evercyte GmbH, Austria

Introduction : Mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) have shown promise as safe and effective therapeutic agents, exhibiting regenerative, immunomodulatory, and anti‐inflammatory properties comparable to those attributed to parent cells. Donor heterogeneities, limited replicative life span, and alterations in cellular phenotype throughout in vitro cultivation, however, continue to be significant obstacles for adequate cell expansion required for scalable EV manufacturing. This study investigates the use of hTERT immortalized (‘telomerized’) Wharton's Jelly (WJ)‐derived MSCs as source for efficient, standardized, reliable MSC‐EVs production by comparing primary WJ‐MSCs and the secreted EVs to their telomerized counterparts.

Methods : Primary WJ‐MSCs (WJ‐MSC273) were immortalised by non‐viral overexpression of the catalytic subunit of human telomerase (hTERT). hTERT immortalized cells (WJ‐MSC/TERT273) and primary cells were compared in terms of morphology, cellular doubling time, surface/senescence marker expression, and tri‐lineage differentiation ability. Primary and telomerised WJ‐MSCs‐derived EVs were enriched from xeno ‐free conditioned medium by tangential flow filtration, and characterized for morphology, size, EV‐marker expression and miRNA cargo composition. The biological activity of primary and telomerised WJ‐MSC‐derived EVs was assessed by anti‐inflammatory, anti‐fibrosis and wound healing in vitro assays. Primary and telomerized WJ‐MSCs, as well as secreted EVs were characterized for their safety profile.

Results : hTERT overexpression enabled stable and extended in vitro proliferation of WJ‐MSC/TERT273 cells, while ensuring cellular morphology and doubling time comparable to their primary counterparts. Telomerized WJ‐MSCs did not show increased SA‐ß‐gal activity at late population doublings and retained the canonical surface marker expression and tri‐lineage differentiation potential of WJ‐MSC273 cells. Furthermore, the immortalization process did not impact particle release, EV characteristics or biological activities. Notably, no increase of hTERT DNA, mRNA or activity was measurable in WJ‐MSC/TERT273‐derived EVs, and telomerized WJ‐MSCs showed no in vitro tumourigenic potential.

Summary/Conclusion : MSC immortalization by ectopic expression of hTERT generates cell lines capable of continuous MSC‐EV production without compromising neither key cellular and EV functionalities nor their safety profiles. This approach suggests that telomerization of human cells from single donors is a promising strategy to overcome some of the limitations of using primary MSCs as EV cell factories, while supporting stable EV production.

Subtopic Cross Kingdom 2

The milk EV—bacterial EV—host health triad

Janos Zempleni, Peerzada T. Mumtaz

University of Nebraska‐Lincoln, USA

Introduction : Gram‐negative and ‐positive bacteria communicate with the environment through outer membrane vesicles (OMVs) and cytoplasmic membrane vesicles (CMVs), respectively. We have reported that 50% of an oral dose of milk EVs (MEVs) escape absorption and alter bacterial communities in mice. We tested the hypotheses that 1) gut bacteria internalize MEVs, 2) MEVs alter the secretion and cargo of OMVs and CMVs, and 3) OMVs and CMVs are bioavailable.

Methods : Gram‐negative and ‐positive bacteria were cultured in MEV‐free media (MEF) or MEV‐supplemented media (MES). Internalization of MEVs was assessed using reporter plasmid‐loaded bacterial EVs. OMV and CMV secretion was assessed by NTA. Cargo was assessed using RNA‐seq and mass spectrometry. Bioavailability was assessed using HiLyte750‐labelled and Cre‐loaded OMVs and CMVs in C57BL/6J and reporter mice.

Results : REscherichia coli and Bifidobacterium infantis internalized MEVs. E. coli and B. infantis grew 2% and 19% faster in MES compared to MEF cultures, respectively. The secretion of OMVs by E. coli was 7% lower in MES compared to MEF cultures. The secretion of CMVs by B. infantis was 8% higher in MES compared to MEF cultures. RNA annotation is ongoing. We identified 1199 proteins in B. infantis that were not detected in CMVs, and we identified 217 proteins and 141 and metabolites in CMVs from B. infantis that were not detected in B. infantis. We identified 1,448 proteins in E. coli that were not detected in OMVs, and we identified 110 proteins and 141 metabolites in OMVs from E. coli that were not detected in E. coli. HiLyte750‐labelled CMVs and OMVs, administered by oral gavage, were detected primarily in the lung, followed by accumulation in heart, kidneys, spleen, and liver. Similar patterns were observed when B. infantis were engineered to express Cre recombinase in CMVs and the CMVs were administered to Cre reporter mice.

Summary/Conclusion : Bacteria internalize MEVs, and MEVs promote bacterial growth. OMVs and CMVs harbor a diverse cargo. MEVs decrease the secretion of OMVs by E. coli and increase the secretion of CMVs by B. infantis. Mice absorb both OMVs and CMVs.

Funding : NIH GM154608 , NIFA 2023‐67017‐39056, USDA Hatch NEB‐036‐096 and W5002. JZ co‐owns Minovacca (no COI).

Novel insights into interkingdom communication in neuroinflammation: effects of honeybee extracellular vesicles on microglial membrane mechanics and inflammation

Gabriela Zavala 1 ; Pablo Berríos 2 ; Felipe Sandoval 1 ; Sebastian Aguayo 2,3 ; Christina Schuh 1

1 Centro de Medicina Regenerativa, Facultad de Medicina, Clínica Alemana‐Universidad del Desarrollo, Santiago, Chile 2 School of Dentistry, Faculty of Medicine, UC Chile. 3 Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, UC Chile

Introduction : Neuroinflammation is caused by everyday noxae such as air pollution, trauma or SARS‐COV‐2, among others. Continuous exposure results in the activation of microglia, leading to changes in membrane fluidity, increased motility and secretion of pro‐inflammatory cytokines, which can result in neuronal damage. Recently, our group discovered EVs in Royal Jelly (RJEVs)‐ a bee product with known anti‐neuroinflammatory properties. Until now, it is not known how Royal Jelly exerts its anti‐inflammatory effects. Aim of this study was to investigate the effects of RJEVs on LPS‐activated microglia, focusing on mechanical properties, motility and modulation of the inflammatory response.

Methods : RJEVs (isolation: Vivaspin (100 kDa), ultracentrifugation) were characterized with NTA, TEM and proteomics. RJEV uptake into ctrl and LPS‐stimulated HMC‐3 microglia were assessed with inhibitors and flow cytometry. Secretion of pro‐inflammatory cytokines (10‐2500 RJEV/cell) was measured with ELISA. Young's modulus of HMC‐3 cells was probed with living‐cell atomic force microscopy force spectroscopy. Motility was assessed with a PHIOcell watcher, tracing HMC‐3 cells for 24 h after stimulation.

Results : Proteomics of RJEV (130 nm, mean size) revealed the presence of bee‐derived proteins such as MRJP‐1, Jelleins, Defensins, apolipoproteins, CD63, and Synthenin. LPS exposure altered uptake mechanics: 33% more RJEVs were internalized and the main mechanism changed to clathrin‐dependent endocytosis and macropinocytosis, shifting away lipid raft endocytosis or membrane fusion. RJEVs significantly decreased IL1b, IL6 and TNFa in a dose‐dependent manner. LPS decreased the Youngs modulus by 44% and RJEVs by 28%, indicating a more flexible cell membrane. RJEVs in LPS‐stimulated HMC‐3 cells increased Youngs modulus to ctrl levels. Also, RJEVs decreased LPS‐induced motility in a dose‐dependent manner.

Summary/Conclusion : Honeybee RJEVs are able to integrate into human microglia and exhibit significant anti‐inflammatory effects, decreasing pro‐inflammatory cytokines, motility, and altering membrane mechanics. These findings highlight the potential of RJEVs to modulate neuroinflammatory pathways and cell mechanics, offering insights into interkingdom communication with therapeutic implications.

Funding : Funding: ANID FONDECYT 1220803

Effect of Candida‐derived extracellular vesicles to the epithelial‐mesenchymal transition of oral squamous cell carcinoma

Éva Veres 1 , Zóra Szilovics 1 , Dóra Adamecz 1 , Gergő Svorenj 1 ,Krisztina Buzás 2 , Mónika Kiricsi 1 , Attila Gácser 1

1 University of Szeged, Hungary; 2 Biological Research Centre (BRC), Hungary.

Introduction : Oral squamous cell carcinoma (OSCC) represents about 90% of oral cancer cases globally. In the presence of a tumour, the normal human microbiota often shows alterations, which may increase susceptibility to infections such as oral candidiasis. Previous research of our Laboratory demonstrated that Candida albicans can accelerate OSCC progression in vitro and in vivo by activating tumour‐associated pathways, enhancing oncometabolite production, and increasing matrix metalloproteinase (MMP) activity. Despite these insights, the exact factor causing these effects remains unclear. Our current research investigates the role of extracellular vesicles (EVs) derived from Candida albicans in OSCC progression, as we hypothesize that EVs are essential mediators in cell‐to‐cell communication and may be pivotal in host‐pathogen interactions, potentially affecting cancer progression.

Methods : For this purpose, we isolated EVs from both the yeast and hyphal forms of Candida albicans using differential ultracentrifugation. The characterization of the EVs was performed by nanoparticle tracking analysis and transmission electron microscopy. The uptake of fungal EVs by HSC‐2 human OSCC tumour cells was analysed using flow cytometry and confocal microscopy. We also examined the influence of Candida‐derived EVs to the epithelial‐mesenchymal transition (EMT) in OSCC cells, a crucial step in cancer metastasis formation. Specifically, we examined the migration of the tumour cells using a live cell imaging system, as well as MMP activity, cytokine production, and EMT‐related gene expression.

Results : Our results showed that tumour cells can internalize fungal EVs through an energy‐dependent mechanism. Furthermore, our findings reveal that exposure to Candida‐derived EVs significantly alters tumour cell migration and morphology. Candida EV treatment increases MMP activity and expression in the tumour cells. Additionally, it also increases cytokine production of the OSCC cells.

Summary/Conclusion : These findings suggest that Candida EVs play a significant role in promoting EMT and facilitating OSCC progression. By enhancing tumour cell motility and invasivity Candida EVs may contribute to metastasis formation.

Fasciola hepatica infection modulates the production and proteomic profile of milk‐derived extracellular vesicles in cattle

Christian M. Sánchez‐López 1,2 , Aránzazu González‐Arce 1 , Mari Cruz Manzaneque‐López 3 , Marta González‐Warleta 4 , Jose Antonio Castro‐Hermida 4 , Mercedes Mezo 4 , Roberta Gasparro 5 , Carla Soler 2,6 , Alba Cortés 1 , Dolores Bernal 7 , Pedro Pérez‐Bermúdez 3 , Antonio Marcilla 1,2

1 Àrea de Parasitologia, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot (Valencia), Spain. 2 Joint Research Unit on Endocrinology, Nutrition and Clinical Dietetics, Health Research IIS La Fe‐Universitat de València, Valencia, Spain 3 Departament de Biologia Vegetal, Universitat de València, Burjassot (Valencia), Spain. 4 Labouratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, AGACAL‐Xunta de Galicia, Abegondo, A Coruña, Spain 5 Department of Biomedicine, Neurosciences, and Advanced Diagnostics (Bi.N.D), Section of Biology, Via Divisi 83, University of Palermo, Palermo, Italy. 6 Instituto de Ciencia de los Materiales, Parque Científico, Universitat de València, Burjassot, Valencia, Spain. 7 Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Burjassot, Valencia, Spain

Introduction : Fasciola hepatica infection, a major concern in European dairy cattle, is known to impact milk yield and composition, although studies addressing these effects are limited. Among milk components, milk‐derived extracellular vesicles (mEVs) have garnered attention due to their roles in immune regulation, antioxidant activity or pathophysiological processes. This study explores the presence of F. hepatica EVs (FhEVs) in milk and examines how F. hepatica infection influences the abundance and proteomic content of mEVs.

Methods : Milk samples were collected from dairy farms in Galicia, Spain. The presence of specific antibodies against F. hepatica infection was assessed using the MM3‐SERO ELISA test. mEVs were isolated through a combination of differential centrifugation, EDTA treatment, ultracentrifugation and size exclusion chromatography (SEC). mEVs were characterized through NTA, TEM and LC‐MS/MS and label free semi‐quantitative proteomics.

Results : mEVs were successfully isolated from the milk of both infected and uninfected cows. Notably, the quantity of mEVs recovered was significantly higher in samples from infected animals compared to uninfected ones, while both mEVs preparations exhibited similar particle/protein ratios. A total of 338 proteins were identified in mEVs through LC‐MS/MS, with 46 proteins showing significantly lower levels, and 30 proteins displaying higher levels in mEVs infected animals compared to those from uninfected animals. No proteins from F. hepatica were detected. The absence of FhEVs was confirmed by ELISA, using PAb against FhEVs.

Summary/Conclusion : F. hepatica infection significantly alters the abundance and proteomic composition of mEVs in dairy cattle, indicating their potential as biomarkers for infection status and milk quality. While infection affects the host response, the parasite may not shed EVs into the milk. Further research is needed to clarify the functional implications of these mEV changes on the health and productivity of infected cows.

Funding : Work supported by Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Spain (Grants PID2019‐105713GB‐I00 and PID2023‐146116NB‐I00 and ERDF/EU), and Conselleria d'Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Valencia, Spain (Grants PROMETEO/2020/071 and CIPROM/2023/54). C.M.S‐L is the recipient of a Postdoctoral fellowship funded by AEI /10.13039/501100011033.

Regulation of cancer‐derived EV secretion and contents by an immune‐activating cytokine in metastatic progression

Presenter: Dokyung Jung

Kyungpook National University, Republic of Korea

Introduction : Cancer Seed and soil hypothesis EV Cytokines

Methods : Cell lines and cell culture Isolation of EVs Nanoparticle tracking analysis (NTA) Cell viability assay Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) Western blot assay Immunofluorescence staining Protein profile analysis Animal study H&E staining EV miRNA data analysis Kaplan‐Meier survival analysis Statistical analysis.

Results : 1. Discovery of a cytokine for inhibition of EV secretion 2. Cytokine receptor‐dependent regulation of EV secretion in breast cancer 3. An immune‐activating cytokine reduces lung metastasis mediated by cancer‐derived EVs 4. An Immune‐activating cytokine modulates cancer cell‐derived EV miRNA profiles 5. Correlation between immune‐activating cytokine expression in human patient survival.

Summary/Conclusion : This study highlights this immune‐activating cytokine as a promising therapeutic agent with anti‐metastatic effects, capable of regulating EV secretion and modulating EV characteristics. These findings underscore its potential utility as both a prognostic biomarker and a novel therapeutic strategy for metastatic cancer, paving the way for its broader application in clinical settings.

Funding : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT).

Impact of bacterial extracellular vesicles on the reprogramming of the cancer cells and tumour microenvironment to promote metastasis in colorectal cancer

Alireza Shamsian, Farzaneh Malekian, Van Manh Hung Le, Biljana Gigic, Christoph Kahlert

Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany

Introduction : Colorectal cancer (CRC) progression is significantly influenced by interactions within the microbiome and the tumour microenvironment (TME). It is observed that dysbiosis imbalance in bacterial proportions) is potentially contributing to disease progression. We hypothesize that bEVs from bacterial species associated with CRC contribute to reprogramming macrophages and fibroblasts, promoting a pro‐tumourigenic environment and facilitating metastasis, especially to nearby organs like the liver. Specifically, macrophages exposed to bEVs may adopt an immunosuppressive phenotype that supports tumour growth, while hepatic stellate cells (HSCs) could undergo changes indicative of epithelial‐mesenchymal transition (EMT), enhancing CRC's metastatic potential.

Methods : Bacterial EVs (bEVs) were isolated from cultures of Fusobacterium nucleatum, Bacteroides fragilis, and Enterococcus faecalis using size exclusion chromatography (SEC). Characterization of these bEVs is performed by detection of bacterial origin markers like lipopolysaccharide (LPS) and outer membrane protein A (OmpA) through flow cytometry and Western blotting, along with size distribution analysis via nanoparticle tracking analysis (NTA). LPS activity was further evaluated using toll like receptor 4 (TLR4) reporter cells and the Limulus Amebocyte Lysate (LAL) assay. Macrophages (THP‐1 stimulated with phorbol 12‐myristate 13‐acetate (PMA)), and LX‐2 as fibroblasts were treated with the isolated bEVs. Tranwell migration assay and scratch assay was perofemed with HCT116 cell line.Gene expression in macrophages and hepatic stellate cells (HSCs) was analysed, focusing on immune polarization and EMT markers.

Results : Preliminary findings indicate that macrophages treated with bEVs display polarization towards a pro‐tumourigenic, M2‐like phenotype, with upregulation of TNF‐α, TGF‐β and markers of tumour‐associated macrophages (TAMs). Additionally, bEV exposure seems to activate cancer‐associated fibroblasts (CAFs) in a manner consistent with EMT. Migration of cancer cells also reinforcing the overall capacity to support metastasis. These data suggest bEV‐mediated signalling as a promoter of a pro‐metastatic environment in CRC.

Summary/Conclusion : This study highlights bacterial EVs’ role in reprogramming key TME components, contributing to CRC progression and metastasis. By inducing M2 polarization in macrophages and promoting EMT in fibroblasts, bEVs foster a TME conducive to tumour growth and spread. Furthermore, evaluation of the difference of total microbiome of metastatic CRC patients with CRC patients could be a leading point to promote the tumour to metastasis.

Messenger and Messages: Uncovering the role of extracellular vesicles in the arbuscular mycorrhizal symbiosis

Alice Boussaroque 1 , Samuel Holland 1 , Zongyu Gao 1 , Serena Qiao 1 , Mary Woodward 2 and Ronelle Roth 1

1 University of Oxford, Oxford, UK. 2 Vrije Universiteit Amsterdam, The Netherlands

Introduction : The importance of extracellular vesicles (EVs) in modulating beneficial plant‐fungal associations such as the arbuscular mycorrhizal (AM) symbiosis remain largely unknown. Conversely, in plant pathogen interactions, EVs mediate cross‐kingdom communication by transporting defence‐related molecular cargoes such as mRNA, stress related proteins and small RNAs (sRNA) that attenuate microbial virulence. AM symbiosis is an ancient mutualistic interaction that co‐evolved with land plants around 450MYA and today persists in over 70% of terrestrial plants. Plants benefit nutritionally from the symbiosis by obtaining essential minerals such as phosphate from the fungus. In return plants provide the fungus with organic carbon. As a fatty acid auxotroph the fungus relies entirely on host lipids to complete its life cycle. Symbiotic exchange occurs in highly branched, short‐lived fungal arbuscules that form in roots and is underpinned by an intimately regulated plant‐fungal dialogue. Initial evidence that EVs might function in AM symbiosis came from transmission electron microscopy that showed EVs accumulate in the shared apoplast between arbuscule and host membranes. Here we present compelling evidence that EV encapsulated host sRNAs modulate symbiotic function.

Methods : Using differential ultracentrifugation, EVs were purified from apoplastic wash fluids from AM fungal colonised and mock inoculated roots followed by LC‐MS/MS and RNAseq analyses. The Roth lab uses a combination of reverse genetics, molecular, biochemical and imaging approaches to analyse EV cargo and gene expression and to determine their role in AM symbiosis.

Results : Protein and small RNA plant cargo are enriched in EVs compared to whole cell lysate. Nuclease protection assays show host sRNAs are encapsulated within EVs. Temporal analysis of sRNA target gene expression coincide with mature arbuscules and the expression of the EV biomarker Tetraspanin8. sRNAs targets include fungal genes that regulate fungal lipid utilisation.

Summary/Conclusion : Our results provide evidence for plant‐to‐fungal cross‐kingdom RNA interference during AM symbiosis, likely mediated by EVs. We observed attenuation of sRNA target genes corresponding to lipid utilisation that coincide with arbuscule maturation. This points to a dynamic and highly regulated role for an EV‐encapsulated sRNA in modulating symbiotic function by rationing lipid utilisation to curb AM fungal growth.

Funding : Royal Society URF. BBSRC DTP. Diamond Light Source

Subtopic Extracellular particles 2

What are we looking at: mast cell‐derived extracellular vesicles or granules?

Krisztina V. Vukman 1 , Kelsey Fletcher 1 , Tamás Visnovitz 1,2 , Péter Lőrincz 3 , Tünde Bárkai 1 , Dorina Lenzinger 1 , Edit I.Buzás 1,4,5

1 Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Nagyvárad tér 4. 1089 Budapest, Hungary; 2 ELTE Eötvös Loránd University, Department of Plant Physiology and Molecular Plant Biology, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary 3 ELTE Eötvös Loránd University, Department of Anatomy, Cell and Developmental Biology, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary 4 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Nagyvárad tér 4. 1089 Budapest, Hungary; 5 HCEMM‐SU Extracellular Vesicle Research Group, Hungary, Nagyvárad tér 4. 1089 Budapest, Hungary

Introduction : Mast cells are best known for their ability to release granules during allergic reactions upon cross‐linking of Fcε‐bound IgE molecules by allergens. On the other hand, mast cell‐derived extracellular vesicles (EVs) were among the first EVs shown to carry RNA molecules (Valadi H et al., 2007).

Methods : The aim of our study was to clearly distinguish, for the first time, different types of mast cell‐derived extracellular particles (EPs) including extracellular granules (EGs) and EVs. We studied cultured mast cells from mouse bone marrow, mouse peritoneal mast cells, and the mouse mast cell line (MC/9). Mast cells were cultured in the presence and absence of IgE‐DNP, LPS, and Ca++ ionophore A23187. To separate and characterize the released EPs, we employed differential centrifugation, density gradient centrifugation, size‐exclusion chromatography, immunoaffinity techniques, high‐resolution flow cytometry, nanoparticle tracking analysis, confocal and electron microscopy.

Results : We observed differences in EP secretion patterns during Th1 responses (upon LPS stimulation) as well as in IgE‐dependent and IgE‐independent (Ca++ ionophore A23187‐induced) mast cell degranulation. Our data suggest that mast cells predominantly secrete EVs during Th1 responses, while produce mainly EGs during Th2‐associated degranulation. Surprisingly, during IgE‐independent degranulation, mast cells secreted a mixed population of both EVs and EGs. Since the calcium ionophore A23187 is routinely used in both mast cell research to study degranulation and in EV research to induce EV secretion, our findings indicate a need to reinterpret previous results in both fields. Importantly, we successfully identified molecular markers (such as histamine) that can help distinguish and separate mast cell‐derived EP populations.

Summary/Conclusion : In summary, this study represents an important step toward understanding the distinct roles of mast cell‐derived EP types (EGs and EVs) in the complex immune‐orchestrating roles of mast cells.

Funding : NVKP_16‐1‐2016‐0004 grant of the Hungarian National Research, Development and Innovation Office (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, the Therapeutic Thematic Program TKP2021‐EGA‐23. RRF‐2.3.121‐2022‐00003 (National Cardiovascular Laboratory Program), 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 Research and Innovation Program (No. 739593) and János Bolyai Research Scholarship of the Hungarian Academy of Sciences. National Research, Development and Innovation Fund ( FK138851 ), Hungarian Academy of Sciences (LP2022‐13/2022), Eötvös Loránd University Excellence Fund (EKA 2022/045‐P101‐2)

Impact of the production method on the properties of erythrocyte‐derived extracellular particles: a quantitative and qualitative evaluation

Presenter: Maria Chiara Ciferri

University of Genova, Italy

Introduction : The absence of DNA, their abundance, and ease of acquisition make erythrocytes an excellent source for EV production. Accordingly, erythrocyte‐derived extracellular particles (E‐EPs) possess many benefits in healthcare applications representing a simple and powerful platform for drug delivery. The influence of various production methods on their properties remains poorly investigated nowadays.

Methods : We compared three main types of E‐EVs: (i) those naturally released during the blood bag storage (EryEs), (ii) those produced artificially through RBC sonication (NanoEs), and (iii) those released after RBC chemical stimulation, in this case with calcium ionophore (VI‐EryEs). We evaluated the characteristics of the different subtypes and the efficiency of membrane functionalization by copper‐free click chemistry. Concentration and protein amount were analysed by NTA and BCA, respectively, while transmission electron microscopy (TEM) was employed to evaluate size distribution and morphology. The expression of both EV‐ and erythrocyte‐associated markers was evaluated by flow cytometry and western blot while proteomic analyses estimated the differences in total protein content.

Results : As expected, all three subpopulations were able to be produced on a large scale, with NanoEs being the ones with the highest concentration, polydispersity, and largest dimension. TEM analysis revealed not only the traditional EV‐associated rounded shape but also the presence of a “tubular” shape. Tetraspanins were universally absent while all three subpopulations expressed Alix, Flotillin‐1 and Tsg101. On the other hand, Glycophorin A was expressed ubiquitously while BAND 3 was uniquely found in NanoEs and EryEs. In addition, the analysed subpopulations showed three distinct proteomic signatures and a significant amount of differentially expressed proteins that indicated distinct enrichment profiles of molecular functions. In vitro experiments on both a human fibroblast cell line and THP‐1 macrophages revealed that internalization efficiency was not significantly influenced by the subpopulation type. Finally, NanoEs turned out to be the most efficient in surface functionalization with almost 90% of Click‐EPs.

Summary/Conclusion : Our data suggest that the isolation method can significantly impact both the qualitative and quantitative properties of E‐EPs. Among the three subpopulations examined, NanoEs turned out to be the one suiting better the role of targeted drug delivery vehicles.

The functional role of hepatocellular carcinoma derived exomere‐CLTA in tumour microenvironment

Presenter: Yi Xu

The University of Hong Kong, Hong Kong

Introduction : Hepatocellular carcinoma (HCC), the most frequent neoplasm among all primary liver cancer, is currently the third leading cause of cancer related deaths worldwide. Exomere is an extracellular nanoparticle with a diameter less than 50 nm. Clathrin light chain A (CLTA) is one of the three subunits of the light chain of clathrin.

Methods : CLTA expression was overexpressed and suppressed in metastatic HCC cell lines, respectively. Exomeres from supernatant of these cells were isolated by ultra high speed centrifugation method. The diameter, morphology as well as specific biomarker were further identified. The non‐metastatic HCC cells including HLE and Huh7 were pre‐treated with different cell derived exomere. Afterwards, cell colony forming ability, migratory and invasive potential were tested. Education mouse model was used to confirm the in vitro data.

Results : As a result, exomere from stably transfected HCC cells were isolated and identified. We found that the cell aggressiveness was positively correlated with the level of CLTA in pre‐treated exomere. In addition, animal study indicated the cell growth promoting role of exomere‐CLTA. What's more, education study demonstrated that exomere‐CLTA could remodel tumour pre‐metastatic niche in vivo. s pre‐treated with exomere.

Summary/Conclusion : The study reveals the role of HCC derived exomere‐CLTA in tumour microenvironment. Inhibition of CLTA and its mediated pathway may illuminate a new therapeutic strategy for HCC patients.

Funding : National Natural Science Foundation of China (grant number 81872340, 82072626, and 81902431); Hong Kong Scholars Program (grant number XJ2020012 and 2020–036); Marshal Initiative Funding of Harbin Medical University (grant number HMUMIF‐22008); Natural Science Foundation of Heilongjiang Province (grant number LH2023H043).

Cancer cells release ubiquitinated PCNA via extracellular vesicles

Nofar Cohen, Etti Azulay, Ishai Luz, Tomer Cooks

Ben Gurion University, Israel

Introduction : Proliferating Cell Nuclear Antigen (PCNA) is a nuclear protein that enhances DNA polymerase processivity during DNA replication. It primarily resides in the nucleus, where it interacts with various proteins involved in DNA replication and repair. Here, we show that PCNA is also present in extracellular vesicles (EVs) produced by cancer cells. Given the involvement of EVs in intercellular communication with neighboring cancer cells and cells from the tumour microenvironment (TME), this study is focused on the role of exosomal PCNA shed by cancer cells. The presence of PCNA in EVs is therefore investigated to delineate whether PCNA carried by EVs and taken up by TME cells (particularly the immune component) might further contribute to tumourigenesis.

Methods : EVs from various cancer cell line including A549 (lung carcinoma), JimT1 (breast carcinoma), PANC1 (pancreatic carcinoma) and Jurkat (T cell leukemia) were isolated using ultracentrifugation techniques abiding MISEV guidelines. After isolation, the EVs underwent a thorough characterization; their size distribution and concentration were determined using nanoparticle tracking analysis (NTA). We tested for the presence of exosomal PCNA using a unique antibody and western blotting. Subsequently, we examined the uptake of these EVs by the A549 cell line, Jurkat T cell line, and NK cells, and assessed their impact on cell activity.

Results : We found that PCNA is present on the surface of extracellular vesicles (EVs) derived from lung, breast, pancreatic, and T cancer cells. We discovered that PCNA is in a ubiquitinated form in Lysine 164. A549 cancer cells and Jurkat T cells exhibited uptake of these EVs compared to NK cells, as observed in a time‐lapse examination for a 24‐h period. NK cells were co‐cultured with PCNA‐containing EVs, we did not observe significant changes in NK cell activity markers (such as CD107a) as measured by flow cytometry.

Summary/Conclusion : The presence of ubiquitinated PCNA on EVs derived from cancer cells suggests a potential mechanism for immune modulation or as a marker for DNA damage. Further investigation is needed to fully unravel the role of exosomal Ub‐PCNA in the context of cancer, immune modulation and DNA damage response.

Transfer of ADAM 10 and ADAM17 proteolytic activity via EV induces substrate shedding on distant cells

Presenter: Maide Zehra Gencali

Friedrich‐Alexander Universität Erlangen‐Nürnberg, Germany

Introduction : HEK WT cells express the proteases ADAM10 and ADAM17, whereas HEK dKO cells lack these proteases. Located on the cell membrane, ADAM10/17 can shed the inactive form of the cell surface metalloproteinase Meprin β. This study aims to demonstrate the proteolytic activity of ADAM10/17 in HEK WT cells and show that this activity can be transferred through EVs to HEK dKO cells. Additionally, we investigate the release of ADAM10/17 on EV in dependency of bacterial OMV from THP‐1 macrophage cells.

Methods : EVs generated from HEK WT cells were transfered to HEK dKO cells that had been transfected with Meprin β. The shed Meprin ß was assessed by Western Blot. Meprin ß shedding from EVs was assessed on EV derived from HEK WT cells expressing Meprin ß. The proteolytic activity of ADAM10/17 present on EVs was additionally measured with a quenched fluorescent peptide substrate. Furthermore, THP‐1 cells were stimulated with Gram‐negative bacteria derived OMV or OMV with reduced LPS surface expression (clear coli). We then characterized THP‐1 cell released EV for ADAM10/17 content.

Results : Our results show that HEK WT cells transfected with Meprin β exhibit significant shedding of Meprin β due to the activity of ADAM10/17. This shedding also occurs on EVs derived from such Merpin ß expressing HEK WT cells and when EVs containing ADAM10/17 are added to HEK dKO cells. Notably, ADAM10 is preferentially released onto HEK WT cell derived EVs. To investigate the release of ADAM10 and 17 in a more meaningful setting, we stimulated THP‐1 cells with OMV and determined the release pattern of both proteases onto EV.

Summary/Conclusion : Using ADAM10/17 deficient cells, we can now show that ADAM10 proteolytic activity is present on EV and can be transferred to other cells. This basic finding opens new avenues for mechanistic studies capitalizing on this finding. IT will be of interest to investigate the ratio between ADAM10 and 17 released on EV in different physiological and pathological conditions and how transfer of these proteases to distant cells will change cell behaviour and properties.

Nano‐flow cytometry‐based discrimination of extracellular vesicles and non‐vesicular particles: insights into extracellular carriers of specific biomolecules

Yunyun Hu, Haonan Di, Ye Tian, Xiaomei Yan

Xiamen University, China

Introduction : Recent advances in isolation and analytical methods have highlighted the complexity and diversity of extracellular particles (EPs). Among these, non‐vesicular extracellular particles (NVEPs) are distinct from extracellular vesicles (EVs) as they lack lipid bilayer membranes, and are more abundant in extracellular spaces and body fluids. NVEPs exhibit higher pro‐metabolic activity and hold significant promise in clinical diagnostics. However, traditional EP separation methods are often labour‐intensive and inefficient, and many characterization techniques lack the specificity to differentiate EVs from smaller NVEPs. Developing a rapid and robust approach to discriminate EVs and NVEPs is therefore critical for identifying extracellular carriers of specific biomolecules and understanding their biological functions.

Methods : EPs were isolated from colorectal cancer cell lines using differential ultracentrifugation (UC), followed by high‐resolution iodixanol density gradient centrifugation for separation of EVs and NVEPs. A Laboratory‐built nano‐flow cytometer (nFCM) with two spatially arranged lasers was applied for single‐particle characterization of size, purity, particle concentration, DNA content, lipid membranes, and specific proteins for EVs, NVEPs, and mixed EP samples. Side scattering and fluorescence detection were performed after SYTO 16, di‐8‐ANEPPS, Cy5‐NHS, and/or immunofluorescent staining. Additionally, EPs obtained via UC were processed using UC, ultrafiltration (UF), and size exclusion chromatography (SEC) to evaluate their efficacy in recovering NVEPs.

Results : Significant differences in morphology (via TEM), biophysical properties, and molecular compositions were observed between EVs and NVEPs. DNA staining together with enzyme treatment enabled precise single‐particle discrimination of EVs and NVEPs. Through multiparameter analysis of DNA and protein/lipid membrane, NVEPs lacked EV‐specific protein markers and lipid bilayers. However, NVEPs could labelled with Cy5‐NHS and sensitive to proteinase K, suggesting NVEPs are protein‐nucleic acid complexes distinct from EVs. Enhanced centrifugation force and duration in UC, as well as SEC, were effective for improving NVEP recovery.

Summary/Conclusion : This study demonstrates that nFCM is a powerful platform for distinguishing EVs from NVEPs through DNA analysis on single EPs. Multiparameter analysis of DNA and proteins provide deeper insights into the intra‐subtype heterogeneity of NVEPs and EVs, paving the way for further fundamental and translational research on EP subtypes.

Funding : This research was supported by the National Natural Science Foundation of China (Grants 21934004)

Modulation of extracellular vesicle production in primary bacterial colonizers of the newborn gut microbiota exposed to stressful conditions

Presenter: Anaïs Halbert

Nantes Université, France

Introduction : The gut microbiota (GM) plays an essential role in immune, metabolic and digestive functions, especially during early life. After birth, the establishment of the gut microbiota is influenced by various factors such as the environment, the diet, and the antibiotherapy. According to the Developmental Origins of Health and Disease (DOHaD) concept, early disruptions can affect long‐term health. Antibiotics can disrupt the GM balance by reducing bacterial diversity and promoting dysbiosis, which is associated with an increased risk of chronic diseases. Moreover, GM is exposed to specific gut conditions, including bile salts, which can stress gut bacteria and alter their biological activities. This study investigates the effects of antibiotics and bile salts on bacterial extracellular vesicles (BEVs) production by two major beneficial bacteria, Bifidobacterium longum and Lactiplantibacillus plantarum, in the newborns gut microbiota.

Methods : Bifidobacterium longum subsp. longum ATCC 15707 and Lactiplantibacillus plantarum ATCC 14917, two Gram‐positive strains, were studied B. longum was grown under anaerobic conditions. Amoxicillin, the most commonly prescribed antibiotic in neonates, and a bile salt cocktail simulating intestinal stress conditions were used at sub‐inhibitory concentrations. BEVs from B. longum (Bl‐EVs) and L. plantarum (Lp‐EVs) were extracted using a protocol combining clarification, concentration, and size exclusion chromatography to optimize purification and ensure integrity. Vesiculogenesis and BEVs integrity were examined by transmission electron microscopy (TEM).

Results : Bl‐EVs and Lp‐EVs were successfully purified and their integrity was confirmed by TEM. The BEVs release mechanisms seems to be different between these two Gram‐positive bacteria. While BEVs size did not differ significantly between the species and conditions, antibiotic exposure strongly increased BEVs production by both bacteria. In contrast, bile salts reduced BEVs production in B. longum but had no effect on L. plantarum. These results indicate that intestinal stress conditions, such as antibiotic exposure and bile salts, differentially modulate BEVs production of first colonizers.

Summary/Conclusion : These findings suggest that BEVs could play a role in the microbiota‐host dialogue and contribute to the physiological maturation of the newborn. Further research is needed to identify the specific components modulated under intestinal stress and to clarify their functional impact on host development.

Identification of EV‐specific vaccine candidates for Oesophagostomum dentatum through lipidomic and proteomic profiling

Anna Sophia Feix 1,2 , Bärbel Ruttkowski 1 , Anja Joachim 1

1 University of Veterinary Medicine Vienna, Austria, 2 Aarhus University, Denmark

ABSTRACT UNAVAILABLE.

Investigation of Boric acid effect on miR127‐5p Expression in Human Adipose Tissue‐derived Mesenchymal Stem Cell Exosomes via Canonical Wnt/β‐catenin Signalling Pathway

Presenter: Zarifa Ahmadova

Heidelberg University, Germany

Introduction : Boric acid is known for its antiproliferative and antiapoptotic properties in cancer cells; however, its effects on mesenchymal stem cells and exosomes remain largely unexplored. MiR‐127‐5p is a significant regulator that impacts processes such as cell growth, apoptosis, and metastasis in cancer, and it plays a crucial role in the proliferation and differentiation of chondrocytes. This study aimed to investigate how boric acid affects miR‐127‐5p in mesenchymal stem cells derived from adipose tissue, focusing on the Wnt‐β signalling pathway, which is essential for growth and differentiation.

Methods : We assessed the effect of boric acid on cell viability using the MTT assay. Mesenchymal stem cells were incubated with boric acid, followed by the isolation of exosomes. Exosomes were characterized using flow cytometry and electron microscopy. The expression levels of miR‐127‐5p, WNT2, AXIN, and CTNNB1 were analysed through qPCR.

Results : Boric acid treatment resulted in increased expression of miR‐127‐5p and WNT2, while the expression of AXIN and CTNNB1 was downregulated.

Summary/Conclusion : Our findings suggest that boric acid, which we obtain daily from water, fruits, and vegetables, plays an important role in the proliferation of mesenchymal stem cells. Further in vivo studies and advanced molecular techniques are necessary to support these results.

Subtopics Microbes, parasites, and plants 2

The outer membrane of Fusobacteria nucleatum: a key to unlocking intercellular junctions and activating innate immune responses in Oral Cavity Squamous Cell Carcinoma (OSCC)

Jamie Bojie Chen 1 , Zigui Chen 2 , Andreas Möller 1 , Jason Y K Chan 1

1 Department of Otorhinolaryngology, Head and Neck Surgery, The Chinese University of Hong Kong, Hong Kong S.A.R., China 2 Department of Microbiology, The Chinese University of Hong Kong, Hong Kong S.A.R., China

Introduction : Fusobacterium nucleatum is widely recognized as a cancer‐promoting bacterium in several solid tumours. It is found to be enriched in oral cavity squamous cell carcinoma (OSCC), but surprisingly associated with lower tumour stage and better patient survival. Several studies suggest that its outer membrane vesicles (OMVs) contribute to the activation of innate immune responses that might cause anti‐tumour immunity. This study explores the roles of F. nucleatum OMVs in modulating immune responses in OSCC.

Methods : Healthy human gingival keratinocytes (HGK12), and OSCC cell lines (SAS and HSC3) were used. OMVs were extracted using ultracentrifugation. The morphology and size distribution were measured using transmission electron microscopy and nanoparticle tracking analysis. Cell proliferation, migration, and invasion were examined respectively. Trans‐epithelial electrical resistance was measured to evaluate barrier integrity across a transwell insert.

Results : The median particle size of F. nucleatum OMVs was 115 nm (quartiles: 85, 145 nm), and a typical double‐membrane structure was observed. In presence of OMVs, no significant change in OSCC cell proliferation was observed but a decrease in HGK12 was noted ( p = 0.035). The oral epithelial barrier was disrupted following a 3‐day intervention of OMVs (Day 3 p = 0.022). After 2‐day co‐culture with OMVs, cells were more susceptible to bacteria colonization at an MOI 1:100 (HGK12 p = 0.030, SAS p = 0.011, HSC‐3 p < 0.001). OMVs accelerated cell migration (HGK12 p = 0.004, SAS p < 0.001) and enhanced OSCC cell invasion (SAS p = 0.049, HSC‐3 p = 0.045). In vivo, cancer mass volumes were significantly smaller in the presence of F. nucleatum OMVs when compared to PBS control group from Day 7 onwards (Day 7 p = 0.003, after Day 7 p < 0.001). Interestingly, increased cytokine release of IL‐6 ( p < 0.001) and CXCL10 ( p = 0.011) in the F. nucleatum OMV group indicated stimulation of pro‐inflammatory and anti‐tumour M1 macrophage responses.

Summary/Conclusion : F. nucleatum OMVs contribute to cancer progression by promoting bacterial invasion into cells and enhancing tumour migration and invasion, likely by disrupting intracellular adhesion. However, they can also stimulate innate immune response resulting in suppressed cancer growth, highlighting their context‐dependent, dual role in cancer biology.

Persistence and transmission of antibiotic resistance genes associated with bacterial membrane vesicles in wastewater treatment

Presenter: Hongyue Zhang

George Washington University

Introduction : Antibiotic overuse has made wastewater a major reservoir for antibiotic‐resistant bacteria (ARB) and antibiotic resistance genes (ARGs). The growing demand of wastewater reuse further expands the spread of ARBs and ARGs, posing risks to public health. ARGs can be encapsulated in bacterial membrane vesicles (BMVs) released during ARB growth and apoptosis, enhancing their environmental persistence and potential for bacterial transformation compared to free ARGs. However, the presence, prevalence, and transmission of BMV‐associated ARGs was largely underexplored. Additionally, isolating and purifying BMVs from complex environments like wastewater suffer from a low yield and selectivity, which requires further advancement.

Methods : Immunomagnetic selection was adopted for isolating BMVs from wastewater, by using antibodies targeting lipoteichoic acid (LTA) and lipopolysaccharides (LPS) that are biomarkers of Gram‐positive and Gram‐negative bacterial membrane vesicles, respectively. Wastewater samples were collected from various stages of the treatment process at a local wastewater treatment plant (WWTP). RT‐qPCR and metagenomics sequencing were applied to evaluate the presence and abundance of ARGs throughout the treatment process.

Results : We cultured Methicillin‐resistant Staphylococcus aureus (MRSA) and ARG‐loaded E. coli, and pelleted down BMVs through ultracentrifugation. BMVs were spiked into wastewater, and the recovery rate was 51.8% and 32.4% respectively for Gram‐positive and Gram‐negative BMVs. Minimal impurities were co‐isolated from the wastewater, including free DNAs (in the form of plasmids) and viruses. Transmission electron microscopy and nanoparticle tracking analysis confirmed that the isolated specimen were BMVs. During WWTP surveillance, BMV‐associated kanamycin resistance gene (KanR) reached up to 5.49 log10 gene copies per litre, contributing to 11.9% of total KanR. The removal efficiency of total KanR was 3.95 log10 through all treatment chains, while BMV‐associated KanR showed a lower removal of only 2.35 log10. The ratio of BMV‐associated KanR to the total KanR increased from < 1% to 11.9% through treatment chains.

Summary/Conclusion : Our study introduced a new approach to isolate BMVs from complex environment that can outperform conventional methods. This isolation method was successfully applied for BMV isolation and purification from wastewater. BMVs can facilitate ARG transmission due to increased persistence of ARGs.

Construction and membrane vesicle production of rodZ‐deficient mutant of Escherichia coli Nissle 1917

Yoshihiro Ojima 1 , Toru Sawada 1 , Yuhei O. Tahara 2 , Makoto Miyata 2 , Masayuki Azuma 1

1 Graduate School of Engineering, Osaka Metropolitan University, Japan 2 Graduate School of Science, Osaka Metropolitan University, Japan

Introduction : Escherichia coli Nissle 1917 (EcN) is probiotic strain and is marketed in several countries as the drug product “Mutaflor” (Ardeypharm, Germany); it is widely used to treat enteric diseases, including infectious diarrhea and inflammatory bowel disease. Furthermore, membrane vesicles (MVs) of EcN are known as an immunoactivator. We previously reported a hypervesiculating Escherichia coli K‐12 strain constructed by the deletion of rodZ gene, which plays an important role in peptidoglycan synthesis and determination of rod cell shape. In this study, we constructed a rodZ‐deficient mutant of EcN and evaluated MV production.

Methods : The rodZ mutant strain of EcN was constructed by P1 transduction using a rodZ mutant of K‐12 strain as a template. Wild‐type (WT) and rodZ mutant of EcN were cultured in LB for 24 h at 37°C. After removing the cells by centrifuge, MVs in supernatant were collected by ultracentrifuge (109,000 × g ). The MVs was quantified with a lipophilic dye FM4‐64. The cell volume was determined by qNano system and cells were observed by Quick‐freeze, deep‐etch replica electron microscopy (QFDE‐EM).

Results : The rodZ mutant of EcN was successfully constructed by P1 transduction and showed a spherical and larger cell morphology in the same manner as K‐12 strain. The cell growth of EcN was not inhibited by the deletion of rodZ. MV production only increased by two times whereas the rodZ mutant of K‐12 showed approximately 100 times higher MV production than WT. In QFDE‐EM observation, budding of MVs from the spherical cell surface was not observed, as was seen with the k12 strain. Since it is reported that the budding of MVs from rodZ mutant of K‐12 strain was triggered by osmotic pressure, EcN strain is considered to be highly osmotic tolerant.

Summary/Conclusion : It was found that the effect of deletion of the rodZ gene on MV production was different between K‐12 and EcN strains. Although the cells of rodZ mutant of EcN became spherical and large in the same manner as K‐12 strain, MV production was not greatly promoted in EcN due to the its high osmotic tolerance.

Funding : JST FOREST Program (JPMJFR230T).

Vesicle‐cloaked virus clusters in the environment and their impact on public health

Presenter: Danmeng Shuai

George Washington University, USA

Introduction : Vesicle‐cloaked virus clusters (also known as viral vesicles) are emerging environmental pathogens. Compared to free viruses, viral vesicles are more infectious, environmentally persistent, and resistant to disinfection. Norovirus is the leading cause of worldwide gastroenteritis, and thus the presence and environmental transmission of norovirus vesicles raise significant public health concerns. In this study, we explore the inactivation of rotavirus and norovirus vesicles under ultraviolet light B (UVB) and peroxide treatment, which represents typical wastewater disinfection and natural attenuation processes for controlling the spread of waterborne pathogens.

Methods : Norovirus vesicles were isolated from murine norovirus 1 stock via immunomagnetic selection. TIM4 protein has a strong and selective binding towards phosphyltidylserine in the vesicle membrane but it does not bind free noroviruses. Norovirus vesicles were lysed by a non‐detergent lysis buffer to release free viruses. Both norovirus vesicles and free noroviruses were subjected to UVB exposure and peroxide treatment, and viral infectivity loss was quantified via the integrated cell culture‐reverse transcription‐quantitative polymerase chain reaction. The damage to viral biomolecules and lifecycle was determined.

Results : Norovirus vesicles were more resistant to UVB disinfection when compared to free noroviruses, especially under a low viral load. This could be due to en bloc transmission of viruses in vesicles, increased multiplicity of infection, and complementary and cooperative effects between viruses for successful infection. Neutral peroxide, i.e., peracetic acid, penetrated through the vesicle lipid membrane and inactivated noroviruses inside the vesicles. In contrast, negatively charged peroxides, i.e., peracetate and peroxymonosulfate, did not inactivate viruses inside vesicles. Both UVB and peroxide damaged vesicle/viral proteins but not viral genomes, and they did not compromise vesicle integrity. UVB oxidized a tyrosine residual in the receptor binding domain of the murine norovirus viral protein 1, resulting in reduced virus binding and infectivity. Peroxides oxidized sulfur‐containing amino acid residuals and impaired vesicle/viral internalization into host cells and corresponding infectivity.

Summary/Conclusion : Our study discovered that norovirus vesicles were more resistant to UVB and peroxide disinfection in wastewater treatment and natural attenuation in surface water than free viruses. It advocates for future advancement of water disinfection strategies to protect public health.

Funding : NSF and USDA‐NIFA

Molecular insights into the immune modulation by membrane vesicles of Lacticaseibacillus rhamnosus CCM7091

Miriam Sandanusova 1,2 , Gabriela Ambrozova 2 , Eva Pechackova 3 , Veronika Skockova 2 , Lukas Kubala 1,2,4 , Kristyna Turkova 2

1 Faculty of Science, Masaryk University, Czechia 2 Institute of Biophysics of the CAS, Czechia 3 Center for Regenerative Medicine, University for Continuing Education Krems, Austria 4 International Clinical Research Center, St. Anne's University Hospital Brno, Czechia

Introduction : Membrane vesicles (MVs) produced by probiotic bacteria Lactobacillus rhamnosus CCM7091 hold significant potential in modulating immune responses. However, their precise molecular effects on immune cells are not fully understood. Here, we aimed to isolate and comprehensively characterize L. rhamnosus‐derived MVs and examine their immunomodulatory properties, focusing on the molecular basis of the interaction. Understanding these interactions could provide critical insights into host‐microbe communication and support the development of new therapeutic strategies for immune modulation.

Methods : The MVs were isolated from the late‐stationary growth phase of the maternal culture by multiple centrifugation, filtration, and ultracentrifugation steps, followed by purification on a sucrose cushion. Subsequently, the MVs were characterized (according to MISEV2023 guidelines) by MADLS, NTA, Cryo‐EM, and western blotting. To assess their functional capacity, we evaluated the MVs’ effects in vitro on RAW264.7 macrophage cell line. Additionally, to explore the molecular mechanisms underlying L. rhamnosus MVs’ immunomodulatory effects, we used HEK293 cells overexpressing various pattern recognition receptors. To study the involvement of the inflammasome pathway in the interaction, we used primary murine bone marrow‐derived macrophages (BMDMs) and detected the production of Caspase 1, IL‐1beta, and IL‐18.

Results : Our findings revealed that L. rhamnosus‐MVs elicited a robust immune response in macrophages, as shown by increased production of TNFα, IL‐6, IL‐10, and reactive species of nitrogen. At the molecular level, we observed that the L. rhamnosus‐MVs induce TLR2 signalling pathways. Moreover, the MVs treatment of BMDM showed that the MVs may interfere also with the inflammasome signalling.

Summary/Conclusion : In summary, our results indicate that MVs derived from L. rhamnosus CCM7091 activate TLR2 and inflammasome pathways in macrophages, significantly enhancing cytokine and inflammasome‐associated responses. These results highlight their potential role as potential modulators of innate immune pathways.

Funding : Supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104)—Funded by the European Union—Next Generation EU.

Gut symbiont Bifidobacterium adolescentis CCDM373‐ derived extracellular vesicles exert in vitro immunomodulatory effects

Angelika Sysak 1,3 , Dominika Kozakiewicz 1 , Dagmar Šrůtková 2 , Martin Schwarzer 2 , Sabina Górska 1

1 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Poland. 2 Institute of Microbiology, Czech Academy of Sciences, Czech Republic. 3 Department of Pharmacology and Toxicology, Wroclaw University of Environmental and Life Sciences, Poland.

Introduction : Allergic diseases are becoming increasingly common across the globe, making them the most prevalent chronic illnesses in children. Probiotic bacteria represent a highly promising approach for primary prevention, with significant research confirming their potential. Several recent studies indicate that probiotics might help lower the risk of allergic conditions by modifying the natural microbiota, affecting infants' immune function. In addition to whole bacteria, researchers are exploring their components and bioactive molecules to develop new preventive and therapeutic strategies. Recently, there has been growing interest in extracellular vesicles (EVs) released by bacterial cells. This study aimed to investigate the physicochemical characteristics and immunomodulatory effects of Bifidobacterium adolescentis CCDM373 (Bad 373) derived EVs.

Methods : Anaerobic bacteria cultures of B. adolescentis CCDM373 were used for EVs isolation. After centrifugation, supernatant filtration, and concentration with the pressure‐based Amicon device, EVs were isolated by ultracentrifugation (3 h at 4°C and 150,000 × g ). Obtained EVs were dissolved in 500 µL of sterile HEPES in 0.9% NaCl and purified on the Izon size‐exclusion column. Properties of EVs were investigated by Dynamic Light Scattering (DLS) method using Zetasizer (Malvern Panalytical), and transmission electron microscope (TEM, JEOL JEM F‐200). Three size‐based fractions of EVs were further investigated on human embryonal kidney cells (HEK293), stably transfected with TLR receptors, mouse bone marrow‐derived dendritic cells (BMDCs), and mouse splenocytes to determine their immunomodulatory properties. The influence of EVs on the production of cytokines was determined by the ELISA method. Flow cytometry analysis was used to investigate the effects of EVs on the expression of co‐stimulatory molecules (MHCII, CD40, CD80, CD86) in BMDCs.

Results : DLS analysis revealed the batch‐to‐batch similarity of isolated EVs. In addition, they stimulated the TLR2 receptors in HEK293 cells, caused increased expression of co‐stimulatory molecules in BMDCs, and modulated the production of cytokines in mouse splenocytes.

Summary/Conclusion : The results obtained allowed us, for the first time to our knowledge, to carry out physicochemical characterisation of Bad 373‐derived EVs. Furthermore, their immunomodulatory properties confirmed their valuable potential in the search for new preventive and therapeutic approaches for allergic diseases.

Funding : This study was supported by the National Science Centre of Poland (UMO‐2021/43/I/NZ7/00693).

Interkingdom communication between Staphylococcus aureus and Candida species at the level of extracellular vesicles

Zóra Szilovics 1 , Éva Veres 1 , Krisztina Buzás 2 , Attila Gácser 1

1 University of Szeged, Hungary. 2 Biological Research Centre, Hungary.

Introduction : The human oral cavity is colonized by more than 700 microbes, such as bacteria, viruses, fungi, known as the oral microbiota. As a result of environmental effects, such as smoking or infections, the microbial composition may change, which can result in dysbiosis that may lead to diseases, such as oral candidiasis. Oral candidiasis is most commonly caused by Candida albicans, which can alter the bacterial diversity. To examine the nature of such fungal‐bacterial interactions, we aim to investigate the interaction between Candida species‐ and oral pathogenic bacteria at the level of extracellular vesicles (EV).

Methods : For our experiments we used the C. albicans SC5314 and C. parapsilosis CLIB214 strains, along with Staphylococcus aureus as pathogenic bacterial counterpart. We optimized the fungal and bacterial EV isolation protocol from solid media. The characterisation of the EVs by transmission electron microscopy and NanoSight showed round shaped particles with diameters between 50 and 250 nm. We examined the effects of EVs released by C. parapsilosis and the yeast and hyphae form of C. albicans on the growth and biofilm formation efficiency of S. aureus and vica versa.

Results : As a results, we found that EVs from C. albicans and C. parapsilosis had different effects on the growth and biofilm formation efficiency of S. aureus. Regarding the effect of bacteria, the S.aureus EV treatment can induce the hyphae formation of C. albicans cells.

Summary/Conclusion : Altogether these results suggest the presence of an active interaction between fungal and bacterial cells at the level of EVs.

This abstract is about the role of syntenin during the secretion of a non coding RNA fo dengue virus in the mosquito saliva

Presenter: Florian Flo Rachenne

University of Montpellier, France

Introduction : The dengue virus (DENV) is an arbovirus transmitted by Aedes mosquitoes through saliva during a bite on a vertebrate host, playing a key role in viral infection. Dengue affects nearly 390 million people annually and with the absence of an effective treatment, understanding saliva's role in transmission could reveal new therapeutic targets. My research team identified extracellular vesicles (EVs) in Aedes aegypti saliva. These EVs contain subgenomic flavivirus RNA (sfRNA), a non‐coding RNA from the degradation of viral genomic RNA by a cellular enzyme. sfRNA interacts with immune proteins in human.

Methods : Using molecular biology and proteomics, I showed that syntenin in the yellow fever mosquito is involved in the secretion of DENV sfRNA in saliva. Using RNA affinity chromatography, I isolated proteins from the Aag2 cell line involved in extracellular vesicle (EV) biogenesis, focusing on syntenin. I confirmed this interaction via immunoprecipitation and quantitative PCR. Western blot and PCR assessed the virus's impact on EV secretion, while nanoparticle tracking analysis (NTA) quantified EV size and number during infection. Finally, syntenin knock‐down in infected Aedes aegypti mosquitoes confirmed syntenin's role in sfRNA secretion.

Results : Syntenin and DENV sfRNA interaction: I showed that syntenin from the yellow fever mosquito interacts with DENV sfRNA. Using RNA affinity chromatography, syntenin was isolated with in vitro‐produced sfRNA. Immunoprecipitation from infected Aag2 cells revealed enrichment of both sfRNA and viral genomic RNA. Impact of DENV infection on EV secretion: I demonstrated that DENV infection increases secretion of two EV markers (syntenin and CD63 orthologue) into Aag2 cell supernatant, correlating with larger and more numerous EVs. Syntenin levels were also elevated in the carcasses and salivary glands of infected mosquitoes. Finally, syntenin knock‐down in infected female mosquitoes reduced DENV sfRNA secretion in saliva, suggesting syntenin's role in sfRNA secretion.

Summary/Conclusion : In conclusion, my thesis highlights syntenin's role in dengue virus sfRNA secretion in mosquito saliva. My results show an interaction between syntenin and sfRNA, explaining how sfRNA is internalized into extracellular vesicles (EVs). Synhenin knock‐down in vivo reduced sfRNA secretion. This work enhances our understanding of how viruses hijack EV biogenesis in mosquitoes to boost transmission to humans.

Outer membrane vesicles and nanotubes in Francisella tularensis—characterization, role in host‐pathogen interaction and protective potential

Jana Klimentova, Ivona Pavkova, Jan Bavlovic, Jiri Stulik

Military Faculty of Medicine, University of Defense, Czech Republic

Introduction : Francisella tularensis is a gram‐negative facultative intracellular bacterium causing a severe disease called tularemia. Being one of the most infectious pathogenic bacteria and because there is no licensed vaccine, it is classified as a potential biological warfare agent. Francisella releases vesicles of unusual tubular shape highly enriched in immunomodulatory proteins and virulence factors.

Methods : The vesicles were isolated from the culture supernatants of F. tularensis subsp. holarctica fully virulent or mutant strains by ultrafiltration followed by ultracentrifugation and purified by density gradient centrifugation. For their characterization we used TEM, SEM and proteomic analyses. In in vitro studies we used the isolated vesicles to treat murine primary bone marrow macrophages and evaluated their response by antibody microarrays, cytokine ELISA, TEM and fluorescence microscopy. The release of vesicles from the bacteria during the interaction with macrophages was visualized by TEM. In in vitro studies mice were immunized by the isolated vesicles and then challenged by the lethal dose of the parent strain. The sera of the immunized animals were probed for cytokine and antibody response by the antibody microarrays and western blot. All animal experiments were approved by the Animal Care and Use Committee of the Military Faculty of Medicine, University of Defense.

Results : The vesicles entered rapidly into macrophages and accumulated inside. They were non‐toxic for the macrophages, slightly prolonged their viability and elicited strong proinflammatory and antimicrobial response. The nanotubules were formed mainly during the entry of the bacterium into the host cell. In mice the in vivo protective effect was significant but the survival time was only slightly prolonged. We have found several hypervesiculating strains among mutants with disrupted O‐antigen.

Summary/Conclusion : Due to their origin and content and regarding their rich interaction with the host cells, the membrane vesicles of Francisella present an interesting material from the point of view of their protective potential. Currently, we also search for mutant strains, especially the hypervesiculating ones and those with enhanced protectivity, which could serve as a source of vesicles more suitable for the protective studies.

Funding : The work was supported by the Ministry of Defense of the Czech Republic (project no: DZRO‐FVZ22‐ZHN II).

The effects of extracellular vesicles isolated from the protozoa parasite that causes Chagas Disease (Neglected Tropical Disease) on human cardiomyocytes

Mariana Ottariano and Ana Claudia Torrecilhas

Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários.

Introduction : Chagas disease (CD), which is caused by Trypanosoma cruzi, is an important problem for public health in Latin America. Chagas cardiomyopathy, which the most severe form of the disease, impacting approximately a third of the patients. EVs from T. cruzi contain molecules to modulate invasion mechanisms, regulatory processes and impact on immune response. The goal is to understand the interaction of T. cruzi EVs with host cardiomyocytes.

Methods : Invasion assays used EVs from parasites pre incubation with AC‐16 and after infection with trypomastigote. EVs were labelled with PKH26 Dye Solution to track their interactions with AC16 cells during 1, 6, 24, and 48 h, cells were fixed with PFA4% and incubated with anti‐trans‐sialidase antibody for 2 h, incubated with anti‐mouse IgG PE‐Texas Red secondary antibody and DAPI for 1 h. The interaction cells‐EVs was examined using Confocal Microscope and Amnis Image Stream Cytometer (Leica XMk II).

Results : Our results shown that EV increase parasite invasion into host cells, implying a function for EVs in T. cruzi‐induced cardiac disease. Observed that cardiomyocytes uptake T. cruzi EVs with a significant increase in the percentage of AC16 cells positive for PKH26‐labelled EVs and endocytosed EVs over time. These EVs transport active trans‐sialidase to the cell cytoplasm, an important virulence factor of T. cruzi. After 1 h of uptake, EVs migrate from the plasma membrane of cardiomyocytes to the region close to the cell nucleus. Some populations of EVs (PHH26+ and TS‐PE‐Texas Red+) co‐localize with the nuclear membrane that T. cruzi EVs deliver important factors to nucleus of recipient cells, where they regulate fundamental cellular functions.

Summary/Conclusion : EVs play an essential function in the pathophysiology of Chagas cardiomyopathy by promoting T. cruzi invasion of cardiomyocytes. The results support the suggestion that EVs influence important parasite‐host interactions and increased parasite invasion. Additionally, the co‐localization of EVs with TS location close to the nuclear membrane point to a specific intracellular mechanism that may affect cellular processes and the development of disease. These results improve the understanding of how T. cruzi affects host cell responses and open the pathway for novel therapies for treating Chagas cardiomyopathy.

Funding : Support by FAPESP 2020/07870‐4, CNPq and CAPES.

Human Monocyte‐to‐Macrophage Differentiation and NF‐κB Activation by Extracellular Vesicles shedding from Trypanosoma cruzi

Juliana Fortes Di Iorio 1 , Anna Julia Pietrobon 2 , Nadjania Saraiva de Lira Silva 1 , Maria Notomi Sato 2 , Ana Claudia Torrecilhas 1

1 Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. 2 Laboratory of Dermatology and Immunodeficiencies, LIM‐56, Department of Dermatology, Tropical Medicine Institute of São Paulo, University of São Paulo Medical School, São Paulo, Brazil

Introduction : Chagas disease is caused by T. cruzi affects approximately 6 to 7 million people in America. Diagnosis, treatment, and control of the disease offer significant difficulties to eliminating Neglected Tropical Diseases in endemic areas. T. cruzi shed EVs at different phases of the life cycle, that affect the innate immune via TLR2 in host cells. Monocytes and macrophages play an important role in starting innate as well as adaptive immune responses during T. cruzi infection.

Methods : THP‐1 cells incubated with EVs‐Tcruzi for 24 and 48 h, fixed and using scanning electron microscopy (SEM). The EVs labelled with PKH26 and after interaction with THP‐1 cells at 0, 5, 15, 30 min, 1, 24, and 48 h (confocal microscopy and flow cytometry‐Amnis ImageStream). Human monocyte isolated from peripheral blood PBMCs and treated with EVs‐Tcruzi and performed by flow cytometry to detected markers (CD14, CD16, CD80, CD86, CD69, HLA‐DR). The invasion assay, monocytes incubated with or without EVs and used to two distinct parasite concentration and measured the number of infected cells and intracellular parasites.

Results : EVs‐Tcruzi enhance intracellular parasites and differentiation of monocytes in macrophages. In parallel, we found NF‐kB translocation using confocal microscopy, as demonstrated by endogenous GFP expression. NF‐kB pathway activation observed 30 min after interaction, which was consistent with ImageStream's results. We found an established connection between EV uptake and NF‐kB activation as the cells. We detected that monocytes uptake more EV‐Tcruzi than lymphocytes after 24 h. In parallel, we observed changes in the monocyte activation profile and increased expression of CD14, CD16, CD80, CD86, CD69 and HLA‐DR performed by flow cytometry.

Summary/Conclusion : Our results demonstrate that T. cruzi‐derived EVs interact with monocytes, activating the NF‐kB pathway and stimulating an increase in cell area and complexity that results in monocytes differentiation into macrophages. These data provide new evidence that EVs shed by T. cruzi interact with host cells, acting as a booster in infection, modulating the immune response.

Funding : Support by CAPES, CPNq e FAPESP 2020/07870‐4

Effects of Dasatinib (Src/Abl and Serine/Threonine Kinase Inhibitor) on Extracellular Vesicle shedding by Trypanosoma cruzi

Gabriela Villa Marin, Ana Claudia Torrecilhas

Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários.

Introduction : Trypanosoma cruzi is the protozoan that causes Chagas Disease (CD). The medications used to treat CD, Benznidazole (BZ) and Nifurtimox, remain to cause side effects at different phases of the disease. There is still a need for additional study into the parasite's molecular structure to develop new therapies and improved comprehension of T. cruzi's interaction mechanisms during its life cycle in both invertebrate and vertebrate hosts. Our research has previously shown that T. cruzi shed EVs during its life cycle. These particles play a critical role in host communication, interaction, and immune response regulation.

Methods : The main aim of this study was to examine the effect of Dasatinib (DAS) on EV release by T. cruzi. DAS was initially developed to inhibit the dual Src/Abl tyrosine kinase, although it can also inhibit several serine/threonine kinases. A published study found that DAS suppressed the growth of parental amastigotes but not those with TcK2 depletion, indicating that it could be a promising lead for developing CD treatments that target TcK2. Trypomastigote forms were cultured at 37°C at varying time intervals (0, 15, 30 min, 1, 2, and 4 h) and concentrations of DAS, BZ, and a control (no compounds). After incubation, the cultures were centrifuged, and EVs were purified to evaluate particle the quantity and parasite viability using NTA and Cytoflex.

Results : The results showed variations in the concentration of vesicles produced by parasites treated with DAS and BZ. The parasite produced a smaller amount as the concentration of the chemical increased. However, the size of EVs obtained from the parasite was not significantly different from the control.

Summary/Conclusion : DAS action on TcK2 kinase, may influence parasite communication processes involving EVs, creating more prospects for the development of CD therapies. The stability of EV size under treatment situations indicates that the substances are selective for release without impacting EV structure, emphasizing the importance of additional study of these mechanisms for therapy.

Funding : CAPES, CPNq e FAPESP (2020/07870‐4)

Optimization of EV production from Ascaridia galli to preserve immunomodulation

Anna Sophia Feix 1 , Konstantinos Papanikolaou 1 , Rikke Brødsgaard Kjærup 1 , Suranga Kodithuwakku 2 , Alireza Fazeli 2 , Chanaka Premathilaka 2 , Anders Miki Bojesen 3 , Carolina Corral Yélamos 3 , Tina Sørensen Dalgaard 1

1 Aarhus University, Denmark, 2 Estonian University of Life Science, Estonia, 3 Copenhagen University, Denmark

Introduction : Ascaridia galli is a parasitic roundworm that primarily infects poultry, especially chickens, causing the disease ascaridiosis. Economically, it is significant because infections lead to reduced growth rates, lower egg production, and higher immunosuppressive effects in poultry, affecting both small‐scale and commercial farms worldwide. In vitro cultures of A. galli are essential as they provide a controlled environment to study the nematodes life cycle, physiology, and test potential treatments in accordance with the 3Rs. Research on EVs from A. galli has gained importance, as these vesicles potentially play a role in inter‐species communication, possibly influencing the host's immune response and infection outcomes. The aim of this preliminary study was to optimize in vitro EV production from the parasite, enabling their future assessment in functional immunoassays and studies of host‐pathogen interactions.

Methods : Adult A. galli, which were further sorted by sex into male and female groups, were collected from chickens, washed with NaCl and cultured in vitro for EV collection. A. galli eggs were collected from female adult worms, embryonated, cultured and hatched in vitro, allowing for the collection of larvae. To determine the optimal time‐point for maximal EV release, supernatant was collected at different time points. EV purification was compared using two methods: size exclusion chromatography and ultracentrifugation. Characterization of isolated EVs was conducted via nanoparticle tracking analysis and transmission electron microscopy, with effective refractive indices calculated to enhance measurement accuracy. Functional properties of the EVs were further assessed using functional immune assays involving macrophage phagocytosis and T‐cell proliferation.

Results : The data revealed a time‐dependent EV secretion pattern, with distinct profiles for larvae and adult male and female worms, respectively. SEC produced purer EV populations, while ultracentrifugation yielded higher EV concentrations, with sizes ranging from 50–500 nm. Notably, EVs from larvae were significantly smaller than those from adult worms. Calculating the effective refractive index refined particle size and concentration measurements, enhancing data comparability across isolation methods.

Summary/Conclusion : The combined results enhance our understanding of EV production in A. galli and enabled optimization of in vitro EV production for downstream studies of host‐parasite interactions.

Immunological and Apoptotic Effects of Trypanosoma cruzi Extracellular Vesicles on Macrophages: Implications in Chagas Disease Pathogenesis

Náthani Gabrielly Silva Negreiros, Nadjania Saraiva, Ana Claudia Torrecilhas

Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários.

Introduction : Chagas disease (CD) caused by Trypanosoma cruzi (protozoa parasite), affecting more than 6‐7 million people worldwide. There are many factors are involved in the development the pathogenesis of CD; virulence, strains of parasite and host immune system. EVs shedding by T. cruzi trypomastigotes are involved in cell‐cell communication and can induce inflammation and activated host immune response. The goal was to evaluate the various additions of EVsTcruzi in macrophage to determine their pro‐ and anti‐inflammatory properties.

Methods : Functional assays were conducted to determine the interaction of EVsT cruzi with macrophage. The THP‐1 cells differentiated into macrophages used 100 nM of phorbol 12‐myristate 13‐acetate (PMA) by 24 h. In parallel, we performed the interaction assay EVsTcruzi with macrophage was evaluated by confocal microscope and measure the number of EVs‐uptake during 1, 24, 48, and 72h. Cell viability was determined using the 7‐AAD and Annexin V stain by cytometry, and relative expression of cytokines (pro and anti‐inflammatory) mRNA and the levels of cytokines and cell culture supernatants were measured by RT‐PCR and Cytometric Bead Array‐CBA respectively.

Results : Results showed that T. cruzi EVs interact with macrophages, and particles uptake by the cells over time, migrating to the nucleus after 1 h of incubation with EVs. After 72 h of EVs and macrophages interaction, the increase the number of particles and cell death via apoptosis. In addition, early exposure to EVsT.cruzi induce pro‐inflammatory cytokines (TNF‐alpha, IL‐6 and IL‐1) during 48, but after 72 h the profile were anti‐inflammatory response. T. cruzi‐secreted EVs interact with macrophages modulate the host immune response.

Summary/Conclusion : T. cruzi‐secreted EVs interact with macrophages modulate the host immune response. These findings showed that internalization of T. cruzi EVs implications for immunological activation and control, opening the way for novel therapeutic targets and biomarkers in Chagas disease.

Funding : Supported by CAPES, CNPq e FAPESP 2020/07870‐4.

Immunomodulatory effect of extracellular vesicles secreted by the free‐living amoeba Naegleria fowleri over microglia

Presenter: Lissette Retana Moreira

Universidad de Costa Rica, Costa Rica

Introduction : Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, an acute and rapid infection of the central nervous system with a fatal outcome in >98% of cases. Due to the infrequent report of cases, and the low possibility of recovering this free‐living amoeba from clinic samples, pathogenesis‐related investigations are scarce. However, the secretion of cytolytic molecules is proposed as a factor involved in the progression of the infection; several of these molecules could be included in EVs. In this work, we evaluated the role of EVs secreted by two clinic isolates of N. fowleri in modulating the immune response using in vitro models.

Methods : Characterization analyses of EVs secreted by trophozoites, including electron microscopy, dynamic light scattering and nanoparticle tracking analyses were performed and proteomes were compared. Then, gene transcription analyses of primary cultures from mouse cell microglia and BV‐2 cells incubated with EVs for different time points were performed. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions.

Results : Increased expression of proinflammatory cytokines IL‐6, TNF‐α, and IL‐23, and the regulatory cytokine IL‐10 was observed in primary cultures of microglia, while increased expression of IL‐13 was observed in BV‐2 cells. Morphologic changes from homeostatic microglia to a more amoeboid morphology were also identified after the incubation of these cells with EVs. Specific N. fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions.

Summary/Conclusion : These results confirm the immunomodulatory effects of EVs of N. fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.

Funding : This research was funded by the “Vicerrectoría de Investigación”, Universidad de Costa Rica, through the project C‐2600: “Secreción de vesículas extracelulares por Naegleria fowleri y evaluación de su potencial rol inmunomodulador en un modelo in vitro”.

Tiny messengers with big impact: malaria parasites use extracellular vesicles to prepare for transmission

Tosin Opadokun, Sophie Chauvin‐Bose, Petra Rohrbach

McGill University, Canada

Introduction : Plasmodium falciparum infected red blood cells (iRBCs) release extracellular vesicles (EVs) during their asexual stages (ring, trophozoite, schizont). These EVs carry biomolecular cargo that influence parasite development. We hypothesize that EVs from stage specific‐iRBCs carry cargo that induces gametocytogenesis, the differentiation of asexual parasites into sexual stages. We isolated stage specific EVs from conditioned media and tested their effect on gametocyte development in P. falciparum cultures.

Methods : Stage‐specific EVs were isolated from P.falciparum blood stages using differential centrifugation. Nanoparticle tracking analysis was used to measure EV concentration and size, and transmission electron microscopy confirmed EV size and purity. Western blotting detected stage‐specific proteins, including Band 3, Glycophorin A, flotillin 1 and 2. Proteomic analysis was performed by LC‐MS/MS, with peptides analysed using a Thermo Orbitrap Fusion mass spectrometer at 120,000 resolution (MS1) and HCD sequencing (15,000 resolution) at top speed for peptides with a charge of 2+ or greater. Cell culture assays assessed gametocyte development after stage specific EVs were added to infected RBC cultures.

Results : We identified eight proteins potentially involved in gametocytogenesis, including GEXP02, GEXP12, plasmepsin IV, RESA, and StAR‐related lipid transfer protein. To assess EV function, we treated P. falciparum cultures with stage specific EVs on various days. The highest gametocyte production occurred 72 h post‐treatment. The priming effect of EVs was further confirmed by treating uninfected RBCs with EVs prior to adding them to parasite cultures. RBCs primed in this manner produced similar levels of gametocytes to those treated directly with EVs, indicating that EVs do not necessarily interact directly with the parasite but rather prime the RBCs, enabling them to support parasite invasion and differentiation into sexual stages.

Summary/Conclusion : Treating cultures with P. falciparum‐derived EVs induced gametocyte production, demonstrating that these EVs carry cargo capable of triggering gametocytogenesis. Uninfected RBCs primed with EVs also facilitated gametocyte development, suggesting that EVs prime RBCs for parasite invasion and differentiation into gametocytes. These findings have significant implications for malaria transmission and potential intervention strategies. Future studies will focus on mechanistic insights and MS to compare protein profiles of EV‐treated cultures.

Funding : Natural Sciences and Engineering Research Council of Canada (NSERC).

Physical and functional properties of Anisakis spp. extracellular vesicles: shedding light on human‐parasite routes of communication

Ilaria Bellini 1 , Bradley J Whitehead 2 , Claudia Chiovoloni 1 , Silvia Rondón 1 , Agostina Pietrantoni 3 , Anna Kashkanova 4 , Federica Rinaldi 5 , Maria Gioia Fabiano 5 , Eleonora D'Intino 5 , Ilaria Rago 6 , Luca Cecchini 6 , Stefano D'Amelio 1 , Peter Nejsum 2 and Serena Cavallero 1

Serena Cavallero and Peter Nejsum are co‐senior authors

1 Department of Public health and infectious diseases, Sapienza University of Rome, Italy. 2 Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark. 3 Core facilities, Istituto Superiore di Sanità, Rome, Italy. 4 Max Planck Institute for the Science of Light, Erlangen, Germany. 5 Department of Drug Chemistry and Technology, Sapienza University of Rome, Rome, Italy. 6 Department of Physics and IFNF, Sapienza University of Rome, Italy.

Introduction : Helminth extracellular vesicles (EVs) in parasitic diseases represent a growing area of interest. However, challenges in parasite EVs characterization hamper the understanding of their properties. This research investigated the physical and functional features of Anisakis EVs, a zoonotic nematode known to cause allergic reactions and gastrointestinal inflammation in humans, with a potential link to cancer. EVs were isolated using size exclusion chromatography (SEC) and characterized using orthogonal optical approaches: Nanoparticle Tracking Analysis (NTA), Interferometric NTA (iNTA)‐ applied to helminth EVs for the first time‐ Dynamic Light Scattering (DLS); Atomic, Scanning and Transmission Electron Microscopy (AFM, SEM, TEM, CRYOTEM). Biological effects were evaluated on human peripheral blood mononuclear cells (PBMCs) and complement activation assays.

Methods : 1000 Anisakis third stage larvae (L3) were collected from fish and cultured in RPMI with 1% of pen/strep, media were collected daily. EVs were analysed for size, concentration, inner water content, zeta potential, and protein amount. Proteinase K treatment was used to explore the influence of furry coat on EV properties. Functional assay used human serum treated with increasing concentrations of Anisakis and HEK293 EVs followed by ELISA to quantify levels of C5a. Human PBMCs were treated with Anisakis EVs and analysed for TNF production.

Results : Anisakis EVs showed 10 10 particles/mL concentration and an average diameter of 150 nm. Following “shaving” treatments, DLS showed a mild reduction in size, suggesting the presence of a furry coat, confirmed by microscopy. iNTA revealed that 5–20% of EVs' internal content was non‐aqueous, suggesting the presence of a bioactive cargo. Anisakis but not human EVs dose dependently activated the complement pathway resulting in production of the anaphylatoxin C5a. Furthermore, Anisakis EVs induced production of TNF by human PBMCs in a mechanism that was augmented by the presence of human serum.

Summary/Conclusion : Anisakis EVs revealed biological properties able to expand the knowledge in host‐parasite dynamics. Additionally, the exploration of the parasitic EVs procedures are essential for progress, particularly regarding their applications in in vitro and in vivo models and clinical trials.

Paeonia lactiflora Pall‐derived exosomes repair skin photodamages and prevent photoageing by mitigating senescence and oxidative damage of skin repair cells

Presenter: Fang Ma

Carrier Biomed (Suzhou) Co.,Ltd., China (People's Republic)

Introduction : UV exposure‐induced photoageing accounts for over 80% of facial ageing, resulting in apparent damage to HDFs and HaCaTs. Excessive ROS is observed in these damaged cells, leading to serious oxidative damage and senescence. Plant‐derived exosomes have garnered significant interest due to their inherent bioactivities, excellent biocompatibility, low immunogenicity, and cost‐effectiveness. Paeonia lactiflora Pall (PLP), a traditional herbal with pain‐relieving, anti‐inflammatory, and anti‐oxidant properties, has been widely used in skin administration. However, the role of PLP‐derived exosomes in repairing skin photodamages and preventing photoageing has not been explored.

Methods : First, exosomes from fresh PLP (FP‐Exo), dried PLP (DP‐Exo), and decocted dried PLP (DPd‐Exo) were obtained by differential centrifugation. Second, the morphology and biological functions of FP‐Exo, DP‐Exo, and DPd‐Exo were evaluated to determine the most suitable extraction method. Third, the anti‐photoageing and ROS scavenging efficacy of DP‐Exo was investigated in UV‐damaged HDFs and HaCaTs. Next, the acute and chronic photoageing of rodent models and human volunteers were created to evaluate its photodamage repairing and photoageing preventing performances.

Results : TEM images and SDS‒PAGE analysis revealed that FP‐Exo and DP‐Exo maintained intact exosomal membranes, whereas DPd‐Exo exhibited noticeable membrane rupture and protein loss due to the boiling process. All exosomes showed particle sizes of about 200 nm. DP‐Exo demonstrated superior biological functions, including enhanced cell migration, ROS scavenging, and apoptosis reduction in both UV‐induced cellular models. Furthermore, DP‐Exo enhanced the expression of collagen I and reduced the secretion of MMP‐1. In vivo studies revealed that mice treated with DP‐Exo experienced substantial improvement in erythema and skin lesions, with epidermal thickness and inflammation levels comparable to those of healthy controls. Additionally, a cream formulation incorporating DP‐Exo demonstrated pronounced anti‐photoageing effects and skin‐improvement benefits in human volunteers.

Summary/Conclusion : Collectively, we identify an optimal extraction method for PLP‐derived exosomes and underscores the therapeutic efficacy of DP‐Exo across cellular assays, murine models, and human trials. DP‐Exo exhibits promising anti‐photoageing effects through mechanisms including ROS scavenging, anti‐inflammatory activity, extracellular matrix remodelling, and anti‐senescence. This study shed light on the potential application of DP‐Exo in repairing skin photodamages and preventing photoageing.

Characteristics of extracellular vesicles isolated from protoplast cultures

Csenge Szász 1 Ani Barbulova 2 , Kameswari Priyanka Devarakonda 2 , Immacolata Fiume 2 , Angela Mary Joseph 2 , Veronika Kralj‐Iglic 3 , Anna Romolo 3 , Lilla Turiák 4,5 , Mirjam Balbisi 4,5 , Ádám Vannay 1,6 Gabriella Pocsfalvi 2

1 Pediatric Center, MTA Center of Excellence, Semmelweis University, Hungary. 2 Institute of Biosciences and BioResources, National Research Council of Italy. 3 University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Slovenia. 4 MTA‐TTK Lendület (Momentum) Glycan Biomarker Research Group, Institute of Organic Chemistry, Hungary 5 HUN‐REN Research Centre for Natural Sciences, Hungary. 6 HUN‐REN—SU Peditarics and Nephrology Research Group, Hungary

Introduction : Extracellular vesicles (EVs) from plant cell cultures can be advantageous over prokariotic and mammalian cell lines as therapeutic agent or drug delivery system. The plant cell wall is a firm barrier that restricts the exchange of metabolites between the cell and the extracellular space thereby also limiting EVs production. Therefore the aim of this study was to investigate the effect of cell wall removal on the yield and characteristics of EVs isolated from BY‐2 protoplast culture, a model species in plant cell biology.

Methods : Protoplast isolation circumstances, including digestion time, enzyme concentration and media composition from BY‐2 cells were optimized, viable protoplasts were purified and cultured for three days. The state of the cultures was followed by cell counting and viability measurement using fluorescein diacetate. EVs were isolated from the culture supernatant using ultracentrifugation and resuspended in phosphate buffered saline. The protein concentration, protein profile, morphology, particle number and size distribution were determined by Qubit assay, SDS‐PAGE, cryo‐electron microscopy and Videodrop analysis, respectively.

Results : In our experiment conducted in three biological replicates the average protoplast yield was 10.4% determined by cell counting. The protoplasts showed high viabilty, round morphology and loss of cell‐cell connections during the whole course of the experiment. 10 mL of protoplast culture yielded 5.25 µg of protein and 1.11×109 particles on average. On the electron microscopic images of the isolated EVs, an intermediate density plasma and the surrounding phospholipid bilayer is visible. The median diameter was 260.2, 272.9 and 340 nm in the three samples.

Summary/Conclusion : We were able to obtain and culture viable protoplasts from BY‐2 cell suspension culture and successfully isolate and characterize EVs from their culture supernatant. The optimization of EVs isolation from plant resources might provide multiple opportunities both for basic and translational research fields.

Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs” grant agreement N. 101131175 and the National Research, Development and Innovation Office, K‐142728; EKÖP‐2024‐53, EKÖP‐2024‐160, EKÖP‐2024‐162 New National Excellence Program of the Ministry for Culture and Innovation; Semmelweis University, TKP2021‐EGA‐24; Hungarian Academy of Sciences, János Bolyai Research Scholarship.

First insights into the molecular cargo and secretion mechanism of extracellular vesicles from Nicotiana tabacum Bright Yellow‐2 cell suspension culture

Kameswari Priyanka Devarakonda 1 , Csenge Szász 2 , Ani Barbulova 1 , Angela Mary Joseph 1 , Immacolata Fiume 1 , Veronika Kralj‐Iglic 3 , Anna Romolo 3 , Lilla Turiák 4 , Mirjam Balbisi 4 , Gabriella Pocsfalvi 1

1 National Research Council of Italy, Institute of Biosciences and BioResources, Italy. 2 Pediatric Center, MTA Center of Excellence, Semmelweis University, Hungary. 3 University of Ljubljana, Slovenia. 4 HUN‐REN Research Centre for Natural Sciences, Hungary.

Introduction : Plant cell suspension culture (CSC) derived extracellular vesicles (EVs) less studied than mammalian EVs in vitro cultures. The outstanding plasticity of plant cells makes plant EVs an interesting system to study from the point of view of EV secretion. Here, we optimised the EV isolation from the model Nicotiana tabacum Bright Yellow‐ 2 (BY2) plant CSC to determine the molecular cargo and improve understanding on the biogenesis of EVs.

Methods : The cellular growth of BY2 cells was studied by counting, fluorescein diacetate (FDA) based metabolic viability measurements and packed cell volume. EVs were harvested from conditioned cell media at initial exponential phase of growth at maximum metabolic activity. BY2 EVs isolation was performed by differential ultracentrifugation by optimising different buffers and conditions to achieve maximum EV yield. The isolate was purified by size exclusion chromatography or tangential flow filtration. BY2 EVs were separated into BY2 EVs and EVs fractions by Density gradient ultracentrifugation (DGUC) later were characterised by protein content, particle size and distribution by interferometric light microscopy, morphology by cryoTEM. Mass spectrometry based proteomic analysis and bioinformatic analyses were performed to identify proteins.

Results : Out of four different buffers and 50 mM citrate pH 5.8 resulted in good yield and less copurifying extracellular matter. Three different scales of BY2 isolation resulted in lowest volume scale (5 mL starting volume) with the highest EV yield. DGUC led to two main EV populations: the low density at 1.06‐1.08 g/mL and high density at 1.12‐1.13 g/mL. Initial proteomic analysis identified 50 proteins. Interestingly most of the proteins were enzymes, amongst these, top ranking proteins were beta‐xylosidase, tetrahydrocannabinolic acid synthase‐like and subtilisin‐like protease SBT1.8.

Summary/Conclusion : Three different scales of BY2 isolation will facilitate different aspects of plant EV structural, biological and molecular studies. We speculate on possible secretion mechanisms based on the putative roles of the protein content on the EVs. The optimised conditions let us to isolate BY2 EV at 1ug/mL with 2.33x108particles/mL at the lowest scale. Proteomic and bioinformatics studies let us to hypothise interesting insights into the EV secretion.

Funding : This work was supported by the European Union's Horizon 2020 “nutriEVs” grant agreement N. 101109583.

Apoplastic vesicles from in vitro grown cells of Lycopersicon esculentum L. obtained through enzymatic cell wall degradation

Ani Barbulova 1 , Immacolata Fiume 1 , Veronika Kralj‐Iglic 2 , Ales Iglic 2 , Anna Romolo 2 , Maneea Moubarak 3 , Gabriella Pocsfalvi 1

1 National Research Council of Italy, Institute of Biosciences and BioResources, Italy, 2 University of Ljubljana, Slovenia, 3 Damanhour University, Egypt

Introduction : In vitro plant cell cultures are increasingly exploited for extracellular vesicles (EVs) farming due to their higher reproducibility, scalability and bio‐sustainability on long term scale compared to mammalian systems. Generally, the nanoparticles are isolated from the conditioned culture medium (CCM) and are considered as EVs since they are secreted in the medium during the cells life cycle. Recent studies report a relatively low yield of plant EVs from CCM. Plant cells grown in the CCM could be considered as an alternative source of apoplastic vesicles (AVs), generally isolated from plant tissues or organs applying vacuum infiltration and differential centrifugation. We have established a novel protocol that employs cell wall degrading enzymes to release AVs from in vitro grown cells. These AVs together with the EVs isolated from the CCM of the same suspension culture could open new perspectives to study plant EVs from in vitro cultures.

Methods : CCM and cells from suspension cultures of tomato in the log phase of their growth were used for EVs and AVs isolation using differential centrifugation. Isolation of AVs was performed using a mix of cell degrading enzymes. Treatment conditions and enzyme composition were optimized. Obtained AVs and EVs were characterized for morphology, particle concentration, size distribution and protein profile, using cryotem, videodrop and SDS‐PAGE.

Results : We optimized a method for AVs isolation from cells grown in suspension cultures of tomato using cell wall degrading enzymes. The morphology, size and size distribution of the obtained AVs were similar to that of EVs derived from the CCM of the same suspension culture, showing doble membrane layer and size of 50‐250 nm diameter. The yield and the protein content of the AVs were significantly higher than the CCM‐derived EVs.

Summary/Conclusion : The developed enzymatic method for release and isolation of AVs from cells in suspension culture after treatment with cell wall degrading enzymes could be a step towards an increased EVs yields. This could further boost their exploitation for agricultural, biotechnological and one health applications.

Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs,” grant agreement N. 101131175.

Deciphering the Molecular Messages and Bioactivity of Plant Extracellular Vesicles in Plant‐Fungi Interactions

Monica De Palma 1 , Michele Bifolco 2 , Olga Cannavacciuolo 1 , Emanuele Rosa 2 , Marisa Conte 2 , Elisa Cappetta 2 , Francesca Mensitieri 3 , Carmine Del Regno 2 , Adriana Sacco 4 , Liberata Gualtieri 4 , Maria Moros 5 , Nunziatina De Tommasi 2 , Fabrizio Dal Piaz 3 , Alfredo Ambrosone 2

1 National Research Council (CNR), Institute of Biosciences and BioResources (IBBR), Italy, 2 Department of Pharmacy, University of Salerno, Italy, 3 Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Italy, 4 National Research Council (CNR), Institute for Sustainable Plant Protection (IPSP), Italy, 5 Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC‐Universidad de Zaragoza, Spain

Introduction : Plant extracellular vesicles (EVs) are key components in plant‐microbe interactions, facilitating complex intercellular communication and defense mechanisms. However, the molecular composition and functional roles of EVs in these processes remain largely unexplored. This study explores the plant “secrEVome”, the array of molecules carried by EVs that transmit molecular messages to both beneficial and pathogenic fungi.

Methods : We investigated a model of plant‐fungi interactions, employing tomato (Solanum lycopersicum) as a host system to elucidate the role of EVs in the beneficial associations with Trichoderma spp and in the interaction with the phytopathogen Botrytis cinerea. More in detail, Trichoderma spores were inoculated at the roots, while B. cinerea was applied to the leaves. EVs were collected from different sources: leaf apoplast EVs (apoEVs) were purified using vacuum infiltration, and root exudate EVs (reEVs) were obtained from a split‐root hydroponic floating system designed to prevent co‐purification of fungal EVs. Additionally, hairy roots (HRs) were utilized as a biotechnological platform to produce EVs (hrEVs) under control and elicited conditions. All EV types were isolated using differential ultracentrifugation. EV size and concentration were assessed by nanoparticle tracking analysis. Ultrastructural imaging was conducted using a transmission electron microscope (TEM). Metabolomic profiling was performed by mass spectrometry (MS), while proteomic and miRNome analyses are ongoing by MS and next‐generation sequencing, respectively.

Results : apoEVs, reEVs and hrEVs exhibit similar size (150‐200 nm, mean). HR cultures yielded the highest of EVs, likely due to their high metabolic activity, while apoEVs showed the lowest concentration, limited by apoplastic space. TEM confirmed the presence of intact round‐shaped EVs, indicative of consistent isolation quality. Metabolomic analysis identified tomatine and tomatidineol as primary bioactive compounds, suggesting EVs' potential in plant defense, especially against fungal pathogens, by enabling targeted distribution of antimicrobial molecules.

Summary/Conclusion : By characterizing the molecular composition of plant EVs and integrating multi‐omics data, this study aims to elucidate how EVs regulate defense responses, modulate host‐pathogen interactions, and impact the surrounding microbiome, unveiling fascinating evolutionary dynamics. These findings pave the way for novel agronomic strategies that exploit EV‐mediated pathways to enhance plant resilience and cope with phytopathogenic challenges.

Funding : SecrEVome project (Grant number: 202224M943)—European Union‐ NextGeneration EU within the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.1 ‐ National Research Programme Fund (NRP) and Research Projects of National Relevant Interest (PRIN), CUP: D53D23011500006, B53D23017030006.

Proteomic analysis of exosome‐like nanoparticles obtained from panax ginseng and red ginseng

Presenter: Kwang Pyo Kim

Kyung Hee University, Republic of Korea

Introduction : Exosome‐like nanovesicles (ELNs) obtained from plant are natural nano‐sized membranous vesicles containing bioactive molecules. ELNs consist of a bilayer of lipids that can effectively encapsulate hydrophilic and lipophilic drugs in bioegineering. Panax ginseng, one of the most widely used herbal medicines worldwide, has been studied comprehensively in terms of the chemical components and pharmacology. Red ginseng (RG), steamed and dried ginseng root, is considered as an effective herbal medicine for anti‐inflammatory and antioxidant activity. The proteins from ginseng are of great importance for both nutrition value and the mechanism of secondary metabolites. Previous studies have reported the proteomic analysis of Panax ginseng and ginseng‐derived ELNs, however, the proteome of RG and RG‐derived ELNs has been still unclear. The objective of this study is to detail characterize the proteome of ELNs obtained from Panax ginseng and Red ginseng.

Methods : In this study, extracellular vesicles (EVs) derived from ginseng juice and nanovesicles (NVs) from homogenized red ginseng were isolated using sucrose density gradient ultracentrifugation (DGUC). Morphological characteristics were analysed via transmission electron microscopy (TEM) and dynamic light scattering (DLS). For proteomics, proteins were extracted, digested with suspension trapping (S‐Trap), labelled with tandem mass tag (TMT), and quantified using mass spectrometry.

Results : In the proteomic data analysis, a total of 905 proteins were identified by searching the database of Panax ginseng and Apiales. The proteomic data of four groups including Panax ginseng, ginseng‐derived EVs, RG, and RG‐derived NVs were well separated in the principal component analysis (PCA) and hierarchical clustering analysis (HCA). Differentially expressed proteins (DEPs) were also identified among four groups. In particular, 55 proteins were identified and quantified as DEPs between ginseng‐derived EVs and RG‐derived NVs.

Summary/Conclusion : Proteomic analysis was effectively applied to detail charaterize the DEPs of four groups including Panax ginseng, ginseng‐derived EVs, RG, and RG‐derived NVs. Particularly, in bioengineering, the effective use of ginseng and RG will be possible by characterizing 55 DEPs between ginseng‐derived EVs and RG‐derived NVs.

Combining deep proteomic analysis of apoplastic washing fluid‐derived EVs and bioinformatic analyses to build up a compendium of bona fide reference markers for genuine Plant EVs, providing their degree of phylogenetic conservation

Rodríguez de Lope, Miriam M. 1,2 ; Sánchez‐Pajares, Ibone R. 1,2 ; Herranz, Estela 1,2 ; López‐Vázquez, Cristina M. 3 ; González‐Moro, Ainara 1,2 ; Rivera‐Tenorio, Alan 1,2 ; González‐Sanz Carlos 4,5 , Soledad Sacristán 4,5 , Chicano‐Gálvez, Eduardo 3 ; de la Cuesta, Fernando 1,2 .

1 Department of Pharmacology, School of Medicine, Universidad Autónoma de Madrid, Spain. 2 Instituto de Investigación Hospital Universitario La Paz (IdiPaz), Madrid, Spain. 3 IMIBIC Mass Spectrometry and Molecular Imaging Unit, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Reina Sofia University Hospital, University of Cordoba (UCO), Córdoba, Spain. 4 Centro de Biotecnología y Genómica de Plantas (CBGP, UPM‐INIA/CSIC), Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain. 5 Departamento de Biotecnología‐Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid (UPM), Madrid, Spain.

Introduction : Although the field of Plant EVs is experimenting an exponential growth, rigorous characterization complying with MISEV guidelines has not been yet implemented due to the lack of bona fide reference markers. The ISEV Plant Task Force is working on this standardisation, among other goals, and will benefit of deeper and broad proteomic dataset mining.

Methods : In this work, we have paved the way for the standardization of genuine Plant EV markers, providing the most profound proteomic data so far from apoplastic washing fluid‐derived EVs, a sample enriched in genuine extracellular vesicles from plant tissue, as opposed to methods involving tissue disruption; of two reference plant species: Arabidopsis thaliana (Arath‐EVs) and Brassica oleracea (Braol‐EVs). Additionally, we have conducted an exhaustive analysis of the proteomic data available so far from genuine EVs from any plant species, evaluating current potential markers, together with those found in our proteomic analyses. Next, we analysed the presence of orthologues and their degree of conservation throughout plant taxa, as well as in 2 reference species from the animal kingdom: human and mouse. Their degree of conservation was compared with that of current EV markers used in animal‐derived EVs: CD63, CD81 and CD9.

Results : Our results provide evidence supporting the potential use of the following families as Plant EV markers: fasciclin‐like arabinogalactan proteins (FLAs), patellins, germin‐like proteins, aquaporins, heat shock 70 proteins, 14‐3‐3 like proteins, Rab proteins, and vacuolar‐type ATPase complex subunits. Among these, 3 were found to be plant‐specific: FLAs, patellins and germin‐like proteins. FLA proteins, in particular FLA1, FLA2 and FLA8, were recurrently identified in previous EV datasets from different plant sources showing great abundance.

Summary/Conclusion : This work contributes to addressing more rigor and defining the utility of the Plant EV markers proposed so far, also providing novel potential markers to the field. Besides, FLAs arise as a novel family of plant‐specific GPI‐anchored proteins with great potential as Plant EV markers, since they have orthologues across the different plant subdivisions and display a similar phylogenetic distribution in plants to that of currently used markers in animals.

Funding : Comunidad de Madrid 2023‐5AIND‐28938 and Ministerio de Ciencia e Innovación (PID2021‐126274OB‐I00; CNS2022‐135368).

Yam‐Derived Exosome‐Like Nanovesicles and Their Role in Osteoblast Function

Presenter: Do‐kyun Kim

Jeonbuk National University, Republic of Korea

Introduction : Several studies have reported that substances extracted from yam (diosgenin and dioscin) promoted osteoblast activation in osteogenic MC3T3‐E1 cells. Accordingly, we hypothesized that yam‐derived exosome‐like nanovesicles (YNVs) would play a pivotal role in osteoporosis preventive activity. In this study, an isolation of PENs to prepare YNVs containing RNAs, proteins, and lipids is optimized. Next, YNVs‐mediated osteogenic differentiation is demonstrated with a mechanism study on BMP‐2/p‐p38‐dependent Runx2 pathway in mouse primary osteoblasts and osteogenic MC3T3‐E1 cells. Finally, therapeutic potentials of YNVs are evaluated in ovariectomized (OVX)‐induced osteoporotic mice, in addition to the demonstration of biodistribution and biosafety in naive mice.

Methods : 1) Isolation and purification of Yam‐derived exosome‐like nanovesicles 2) Nanoparticle tracking analysis 3) Negative‐stain transmission electron (TEM) microscopy and Cryo‐EM 4) OVX‐induced osteoporotic mouse model 5) Micro‐CT analysis 6) Bone histomorphometry.

Results : # yam‐derived exosome‐like nanovesicles (YNVs) 1) YNVs are taken up by osteoblastic MC3T3‐E1‐cells, which stimulate osteoblast differentiation and mineralization 2) YNVs improves osteoblast proliferation, differentiation, and mineralization 3) YNVs show higher osteogenic activation than diosgenin and dioscin 4) YNVs modulate p38 MAP kinase pathway 5) YNVs promote osteogenesis in OVX‐induced mice.

Summary/Conclusion : In summary, YNVs were successfully isolated, purified, and characterized, which osteogenic activity and mechanisms were first identified. In detail, MTT assay, immunoblot/RT‐qPCR, and ALP/von Kossa assays demonstrated that YNVs improved proliferation, differentiation, and biomineralization of osteoblast cells, respectively. HPLC analysis indicated that YNVs do not contain any diosgenin and dioscin that are well‐known major components of yam to allow osteoblast activation. Additional total RNA analysis using Volcano plot and DAVID program on KEGG pathway revealed that osteogenic activity of YNVs are associated with MAPK signalling pathways. In particular, immunoblotting assay and inhibitor‐assisted Alizarin Red staining assay confirmed that YNVs activate osteoblast via BMP‐2/p‐p38‐dependent Runx2 pathway rather than other MAPK pathways for ERK1/2 and JNK. YNVs administered orally were transported through GI tract and absorbed in the small intestine, followed by bloodstream and liver. The YNVs enhanced osteogenesis in OVX‐induced postmenopausal osteoporosis mice model in vivo. Therefore, YNV is a potential therapeutic agent for the treatment of osteoporosis with excellent biocompatibility and safety.

Therapeutic Potential of Ginseng‐Derived Extracellular Vesicles in Liver Fibrosis: Modulating TIMP2/MMPs Signalling and Gut Microbiome

Presenter: Young‐Eun Cho

Andong National University, Republic of Korea

Introduction : Metabolic dysfunction‐associated fatty liver disease (MASLD) and alcohol‐associated liver disease (ALD) represent chronic liver diseases that can progress to steatohepatitis (inflammation), liver fibrosis/cirrhosis, liver failure, and death. Unfortunately, there are few medicines approved for treating liver fibrosis/cirrhosis. Therefore, in this study, we investigated the protective effects of ginseng‐derived extracellular vesicles (GDEVs) administration on liver fibrosis.

Methods : We administered GDEVs to mouse and cell models of liver fibrosis. The effects were assessed through histological analysis and biomarker measurements to evaluate fibrosis mitigation.

Results : GDEVs successfully reached the gut or liver in both in vitro and in vivo models without much degradation or cytotoxicity. GDEVs administration decreased serum endotoxin levels and restored intestinal barrier proteins with gut microbiome changes in both MASLD and ALD mouse models. Additionally, GDEVs administration reduced oxidative stress and fibrosis maker proteins to baseline levels in both models of MASLD and ALD mice. GDEVs also ameliorated liver fibrosis through regulating TIMP2/MMPs pathways in LX‐2 human stellate cells and MASLD mice.

Summary/Conclusion : These results demonstrate for the first time that GDEVs can prevent MASLD‐ and ALD‐mediated intestinal hyperpermeability and liver fibrosis through the gut‐liver axis via restoration of gut microbiome and attenuating oxidative stress and fibrosis marker proteins.

Funding : This work was supported by National Research Foundation of Korea (NRF) grant funded by Korea government (MSIT) (No.2021R1C1C1008117, 2023‐00210162, 2022R1C1C100633411).

Plant‐derived nanovesicles are a natural ally in enhancing macrophage antibacterial activity

Roberta Gasparro 1 , Christian Sanchez‐ López 2 , Vincenza Tinnirello 1 , Giulia Duca 1 , Mari Cruz Manzaneque‐López 2 , Riccardo Alessandro 1 , Antonio Marcilla 2 , Stefania Raimondo 1

1 Department of Biomedicine, Neurosciences and Advanced Diagnostics (Bi.N.D), Section of Biology, University of Palermo, Palermo, Italy. 2 Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot, Valencia, Spain.

Introduction : Plant‐derived nanovesicles (PDNVs) have recently garnered significant interest within the scientific community due to their functional properties. They carry lipids, proteins, nucleic acids, and metabolites, playing roles in cross‐kingdom communication and influencing mammalian cells. PDNVs, extensively studied by our research group, show promising potential with demonstrated anti‐inflammatory, antioxidant, and anticancer effects. Additionally, various plant's compounds, such as Citral and polysaccharides, are known to activate immune cells, potentially enhancing bacterial recognition. In this study, we aim to explore the ability of PDNVs to enhance the action of macrophages against bacteria.

Methods : Lemon (LNVs), Tangerine (TNVs) and Pomegranate (PgNVs)‐derived nanovesicles were isolated from the juice through Tangential flow filtration and Size Exclusion Chromatography and characterized by Transmission Electron Microscopy and Nanoparticle Tracking Analysis. We established a protocol for evaluating the effect of human monocytes differentiated into macrophages (THP‐1 M0) against Enterococcus faecalis or Escherichia coli. The THP‐1 M0 cells were first pre‐treated for 24 h with LNVs/TNVs/PgNVs and then grown for 3 h in the presence of bacteria. THP‐1 M0 cells and bacteria were co‐plated in LB Agar and colony‐forming units (CFU) were counted.

Results : We characterized LNVs, TNVs and PgNVs, confirming their integrity, size distribution, and positivity for HSP70 and TET8 markers. After evaluating their internalization in THP‐1 M0 cells, we demonstrated that these nanovesicles enhance macrophage activity, essential for pathogen elimination. We observed that the pre‐treatment with nanovesicles influences bacterial growth. In particular, THP‐1 M0 cells pre‐treated with LNVs/TNVs significantly reduced the bacteria's CFU compared to untreated cells. We validated the results related to Escherichia coli also on monocytes isolated from blood samples of healthy donors and differentiated into macrophages.

Summary/Conclusion : These results represent an interesting starting point for understanding how LNVs, TNVs and PgNVs can modulate the activity of macrophages and improve their response against bacteria.

Funding : This work supported by Eurostart and FFR 2024 (University of Palermo, Italy) to Stefania Raimondo, by Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Spain and Conselleria d'Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Valencia, Spain. R.G. is the recipient of Post‐doc fellowship funded by Fondazione Umberto Veronesi, C.M.S‐L of Post‐doc fellowship funded by AEI/10.13039/501100011033.

Rose petal‐derived nanovesicles: unlocking their potential role as tissue repair agents

Barone A. 1 , Ciriolo L. 2 , Pellegrini B. 3 , Fiore D. 1 , Silletta A. 3 , Tolomeo A.M. 5 , Panza S. 1˒4 , Paolino D. 1˒4

1 Department of Experimental and Clinical Medicine; 2 Department of Health Science; 3 Department of Medical and Surgical Sciences; 4 Research Center “ProHealth Translational Hub”; “Magna Graecia” University of Catanzaro, Italy; 5 Department of Cardiac, Thoracic and Vascular Science and Public Health, University of Padova, Italy Panza S. and Paolino D. are co‐senior authors .

Introduction : Plant‐derived nanovesicles (PDNVs) are emerging as promising nanocarriers for bioactive compounds. Their natural origin, biocompatibility, and potential therapeutic properties make them ideal candidates for biomedical applications. Compared to the mammalian‐derived EVs, PDNVs offer ethical sourcing and environmental benefits. The aim of this work is to investigate the physicochemical properties, stability, antioxidant activity and therapeutic potential of rose petal‐NVs (RPNVs), with a focus on their application in wound healing.

Methods : RPNVs were extracted by triturating the rose petals for 5 min, followed by sequential centrifugation, ultracentrifugation, 0.2 µm filtration and size‐exclusion chromatography, through Akta Prime system. NTA characterized the particle size and concentration, as well as TEM analysis, while Dynamic Light Scattering assessed the zeta potential. Long‐term stability was evaluated using Turbiscan Lab Expert at 25°C and 32°C. Antioxidant activity was tested using DPPH and ABTS assays, and in vitro wound healing efficacy was evaluated on BJ fibroblasts by scratch assays and RT‐PCR. Biosafety was assessed in vitro and in vivo on healthy human volunteers (Ethical Approval number: 392/2019). Informed consent was obtained from all subjects.

Results : Purification of RPNVs was optimized using Sepharose CL‐2B resin, resulting in homogeneous particles with a size range of 50‐200 nm. The RPNVs showed a negative zeta potential of ‐23 mV, ensuring stability in suspension. Long‐term stability studies confirmed no destabilization phenomena and storage after freeze drying method preserved EV integrity. Antioxidant assays showed significant radical scavenging activity. RPNVs promote an epithelial phenotype by increasing E‐Cadherin and decreasing N‐Cadherin, Alpha‐SMA and Collagenase A1 levels. Furthermore, the upregulation of MMP‐9 alongside minimal modulation of MMP‐2 suggests selective extracellular matrix remodelling. Tests on human volunteers revealed no adverse effects on skin barrier function.

Summary/Conclusion : RPNVs show potential for enhancing wound healing, demonstrated by their antioxidant activity and positive effects on key markers of tissue repair. The RPNVs remain stable under suitable storage conditions, ensuring their long‐term efficacy. In vitro and in vivo biosafety assessments confirmed their safety, supporting their potential for future dermatological applications.

Funding : Next Generation EU—Italian NRRP, (Directorial Decree n. 2021/3277)—project Tech4You—Technologies for climate change adaptation and quality of life improvement, n. ECS0000009.

Subtopic Stem Cell Derived EVs 2

Serotonin Induces miR127‐5p and IL‐10 Expression in Exosomes Secreted by Human Adipose Tissue‐derived Mesenchymal Stem Cells

Presenter: Tugba Semerci SevimLi

Eskisehir Osmangazi University, Turkey

Introduction : Exosomes are extracellular vesicles that enable communication between cells. The most valuable molecules in the cargo they carry are miRNAs. We have shown the importance of one of these miRNAs, miR‐127‐5p, in chondrogenesis and cancer in our previous studies. The central and peripheral nervous systems produce serotonin (5‐HT), a neurotransmitter, while enterochromaffin cells in the intestines produce it as a hormone. Serotonin performs its functions by interacting with a large number of receptors connected to various signalling pathways. In this study, we aimed to see how human adipose tissue‐derived mesenchymal stem cell exosomes incubated with serotonin had an effect on miR‐127 and IL‐10.

Methods : We first performed MTT analysis for serotonin. We then incubated mesenchymal stem cells with this dose. Exosome isolation and characterization were performed. We examined the expression of miR‐127‐5p and IL‐10 genes using qPCR analysis. We also performed an ELISA test for IL‐10.

Results : Serotonin increased the expression of miR‐127‐5p and IL‐10 in mesenchymal stem cell exosomes. The IL‐10 level was higher in the exosomes.

Summary/Conclusion : This study suggests that 5‐HT may be positive as it enhances the miR‐127‐5p and IL‐10 effect on mesenchymal stem cell exosomes.

Exosomes and their role in the tumour microenvironment of glioblastoma: an in vitro study

Presenter: Serena Mastantuono

University of Udine, Italy

Introduction : Glioblastoma multiforme (GBM) is the most aggressive primary brain cancer characterized by an extensive intra‐tumour and inter‐patient heterogeneity. While glioma stem cells (GSCs) are considered responsible for GBM recurrences, the crosstalk between tumour cells and tumour microenvironment (TME) strongly influences tumour behaviour. Glioma associated stem cells (GASCs) are representative of the TME since they are not tumourigenic, but, in vitro, they increase the biological aggressiveness of tumour cells through the release of exosomes. Here we aimed at evaluating the efficacy of selected drugs, acting on the TME, to blunt its tumour supporting ability, taking advantage of a patient‐derived in vitro model of TME, represented by GASCs and GASC‐derived exosomes (GDEs).

Methods : We selected 3 drugs: bacitracin, a5b1 integrin antibody and ciclopirox, known to inhibit migration and invasion of tumour cells. GASCs were treated first to select the maximum dose and the timing of treatments, and then to investigate a possible reduction in their activated phenotype evaluating proliferation, motility and anchorage‐independent growth, in vitro. GDEs were isolated from the collected supernatants of treated and not treated GASC, using ExoQuick‐TC precipitation solution and their concentration and particle size were measured by Nanosight. Moreover, U87 MG and GSCs were conditioned with GDEs, treated or not, with different drugs, to evaluate their internalization and their effects on proliferation, motility, anchorage‐independent growth, migration and invasion of tumour cells. Finally, GDEs miRNAs were extracted and sequenced to identify modifications in their contents, after drugs treatments.

Results : Drugs were able to reduce the activated phenotype of GASCs, inhibiting cell proliferation, motility and anchorage‐independent growth. GDEs were efficiently internalized into U87 and GSC being able to inhibit their proliferation, motility, migration, invasion and anchorage‐independent growth. Moreover, we found that drugs were able to modify the GDEs cargo, upregulating some oncosoppresors miRNA such as microRNA‐100‐5p, microRNA‐21‐5p and microRNA‐34a‐5p.

Summary/Conclusion : We confirmed the important supporting role, mediated by GDEs, of the TME on tumour cells behaviour. In addition, we demonstrated that the 3 selected drugs can efficiently revert its activated phenotype and change GDEs cargo reducing their tumour‐supporting function.

Funding : PRIN ARREST CUP G53D23003680006

Impact of conditioned media, proteins and EVs on inflammatory and chondrogenic markers of OA chondrocytes

Livia Kolesar Fecskeova, Jana Matejova, Lucia Slovinska, Jana Bzdilova, Denisa Harvanova

Pavol Jozef Safarik University in Kosice, Slovakia

Introduction : Current research on regenerative approaches for cartilage recovery focuses on Extracellular Vesicles (EVs) derived from various sources. EVs isolated from healthy plasma or from chorion‐ derived mesenchymal stromal cells (CHO‐MSCs) have the potential to modulate inflammation and the expression of cartilage‐related genes in Osteoarthritis (OA) chondrocytes.

Methods : This study was approved by ethics committee and patients gave their informed consents. The effect of conditioned medium (CM) and its separated protein and EVs fractions of CHO‐tissue and CHO‐MSCs on the gene expression of selected anabolic and catabolic markers of OA chondrocytes was compared. Likewise, the effect of EVs derived from various sources (CHO‐tissue, plasma and CHO‐MSCs) and isolated by various techniques (precipitation, ultracentrifugation and size‐exclusion chromatography SEC) on chondrocytes’ gene expression was analysed. EVs were characterized by particle number and size (NTA), protein content (BCA assay) and immunophenotype (flow cytometry). Changes in gene expression of chondrocytes were quantified by RT‐qPCR.

Results : Addition of CM or its separated protein fraction triggered a particularly negative reaction in the expression of catabolic and inflammatory genes (MMP1, MMP3, TIMP1, COX‐2 and TSG‐6) of chondrocytes, which we did not observe by the addition of the EVs fractions; moreover, these even decreased the expression of inflammatory genes (COX‐2, TSG‐6 and TIMP‐1). Addition of EVs of all sources and almost all extraction methods, but also CM and its protein fractions, increased the expression of chondroprotective COL2A and CHO‐MSCs‐ EVs increased the expression of COMP, irrelevant of the isolation technique. However, SEC‐enriched EVs showed superior effects in comparison with EVs isolated by other techniques. In general, plasma‐ and CHO‐tissue derived EVs were the most beneficial in terms of decreasing the expression of inflammatory genes and at the same time increasing the expression of chondrogenic genes.

Summary/Conclusion : Our results showed that treatment with CHO‐derived (tissue and MSCs) EVs and healthy plasma‐derived EVs are particularly beneficial for influencing an inflammatory process. On the other hand, increase in the expression of the chondroprotective COL2A and COMP was achieved by the addition of all components of CM, protein and EVs fractions, especially of CHO‐MSCs.

Funding : This research was supported by the projects APVV SK‐AT‐23‐0005 and VEGA 1/0103/25.

Optimizing EV yield: Continuous harvest of conditioned medium from 3D cultured Hoffa's fat pad MSCs in a vertical wheel bioreactor

Markus Rothammer 1 ; Slavomira Gulova 2 ; Alexander Otahal 1 ; Andrea De Luna 1 ; Stefan Nehrer 1

1 Center for Regenerative Medicine, Department for Health Sciences, Medicine and Research, University for Continuing Education Krems, Austria 2 Associated Tissue Bank, Faculty of Medicine, P. J. Safarik University and L. Pasteur University Hospital in Kosice, Slovakia

Introduction : Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are a subject of great interest in regenerative medicine. In this study Hoffa's fat pad (HFP)‐MSCs were 3D cultured in a vertical wheel bioreactor (VWBR) using a chemically defined and xeno‐free medium (CD‐XF medium), which allowed continuous medium collection and considerably increased EV yield compared to fetal calf serum (FCS) supplemented medium.

Methods : Primary MSCs were isolated from the HFP of five knee replacement patients after informed consent and ethical approval. First, MSCs were cultured in different media in 2D, and metabolic activity was determined using XTT assay. Tested media included the CD‐XF medium and growth media supplemented with 10% FCS, 5‐10% human platelet lysate, or 5% citrate anticoagulated human platelet‐rich plasma. CD‐XF medium and FCS‐supplemented growth medium were used to culture MSCs on microcarriers in VWBRs. The Bioreactor culture was monitored via XTT assay and cell counting. Upon reaching maximum metabolic activity, the media were harvested. To avoid contamination with bovine EVs from FCS, this medium was switched to serum‐free growth medium for a 48‐h collection phase. EVs were harvested continuously from the CD‐XF medium culture through partial media changes. EVs were isolated from the conditioned media by ultrafiltration (100 kDa cut‐off). MSC phenotype after culture was verified using flow cytometry to assay the presence of CD73, CD90 and CD105. EVs from 3D culture were characterized by nanoparticle tracking analysis (NTA) in scatter and fluorescence mode and by Western Blot.

Results : The CD‐XF medium outperformed most of the supplemented growth media tested in 2D and it allowed continuous harvesting in 3D culture, increasing the total yield of EVs. MSCs retained the positive marker expression profile. EV markers CD9 and CD73 colocalized as determined via NTA, while CD9, CD63 and Alix were successfully detected via Western Blot.

Summary/Conclusion : Culturing HFP‐MSCs in a VWBR using the investigated CD‐XF medium improves EV yield and allows continuous harvesting of conditioned media. This provides a scalable and standardizable approach for EV generation while avoiding the inherent variability of blood products.

Funding : This study is founded by the Gesellschaft für Forschungsförderung Niederösterreich, grant number FTI23‐G‐007

Improved EV‐Free Human Mesenchymal Stromal Cell Medium for Extracellular Vesicle Isolation and Production

Presenter: Mandy Chan

STEMCELL Technologies, Canada

Introduction : Extracellular vesicles (EVs) produced by mesenchymal stromal cells (MSCs) can be used in cell‐free therapies. However, supplements such as human platelet lysate and serum used in MSC culture media inherently contain EVs and introduce challenges for the consistent generation and collection of pure MSC‐derived EVs. We developed an improved EV‐free medium (MesenCult‐ACF Plus; MACFP) that can sustain MSC expansion and collection of functional EVs.

Methods : To characterize MSC‐EVs produced ex vivo, human bone marrow MSCs were cultured in animal component‐free MACFP medium for 3 days and spent medium collected to isolate EVs by ultracentrifugation (UC) or size exclusion chromatography (SEC). Analyses of EVs were performed by nanoparticle tracking analysis (NTA), western blot (WB), mass spectrometry, qPCR, and human umbilical vein endothelial cell (HUVEC) tube formation assay.

Results : MesenCult‐ACF Plus Medium was confirmed to be EV‐free by analysing ultracentrifuged samples with NTA and WB for EV markers. Direct comparison of the performance of MACFP medium to media containing non‐depleted or EV‐depleted fetal bovine serum demonstrated that only MSCs cultured in MACFP were able to expand robustly (n = 3). MSC‐EVs isolated from spent MACFP by UC ranged from 80 ‐ 150 nm in size and were positive for the EV markers CD63, CD9, and CD81 proteins. The MSC‐EVs contained the MSC‐associated microRNAs let7a, miR21, and miR26a, as per qPCR analysis. EVs from spent MACFP were biologically active, as they promoted and accelerated branch formation in HUVECs. When treated with MSC‐derived EVs, HUVECs generated tubes as early as 6 h after seeding, and the number of branch points present in tube structures were 4‐fold higher than control HUVECs (n = 5). Furthermore, EVs produced from MSC‐conditioned MACFP medium were stable when stored at ‐80°C based on NTA and WB analyses.

Summary/Conclusion : Taken together, these data demonstrate that MSC‐EVs can be produced in high yield in MACFP medium and that these possess similar physical, phenotypic, and functional characteristics as EVs in vivo.

From Variability to Consistency: A Scalable Manufacturing System for Standardized Extracellular Vesicles from telomerized human Mesenchymal Stromal Cells

Roland Prielhofer, Claudia Lindner, Melanie Reininger, Ingrid Hartl, Klaus Graumann

Phoenestra GmbH (Linz, Austria)

Introduction : Extracellular vesicles (EV) from Mesenchymal Stromal Cells (MSC) hold significant potential for regenerative medicine and other therapeutic applications. As the vesicular fraction of the secretome has been shown to achieve similar biological effects as the cells themselves, MSC‐derived EV are being extensively investigated for clinical use. However, large‐scale production of EV in consistent quality and sufficient quantities for large‐scale studies has proven challenging. To address this, innovative technologies for EV manufacturing are essential to achieve scalability and reproducibility, paving the way for standardized, GMP‐compliant production processes suitable for broader clinical and industrial applications.

Methods : Phoenestra has developed a scalable bioreactor manufacturing setup based on a proprietary basket‐impeller design, that holds macrocarriers, enabling efficient cultivation of adherent cells. It operates in perfusion mode, allowing significant volumes of conditioned media to be harvested continuously—currently tested for up to 49 days. Following harvest, conditioned media is processed by depth filtration and low‐shear ultra‐/diafiltration. The resulting EV preparations were extensively characterized through Nanoparticle Tracking Analysis for particle number, size and distribution, and nanoflow cytometry for lipid particle and EV content as well as MSC marker expression. Variability is closely monitored across and within bioreactor runs by comparing samples from different time points, ensuring consistency in EV quality and yield.

Results : Utilizing stable cell lines over extended cultivation times and the advantage of xeno‐free conditions in perfusion bioreactor cultivation, we achieved consistent EV quality across manufacturing runs. Further process optimization for key cultivation process parameters resulted in a 2‐3‐fold EV productivity increase to at least 2.5‐7.5 E+11 EV per day and yielded an improved MSC‐EV phenotype. Following MSC culture optimizations and scalable isolation and purification methods, we produced EV of consistent high quality in ample quantities already at the 250 mL bioreactor scale—sufficient to meet the demand of small clinical studies.

Summary/Conclusion : Our proprietary manufacturing system addresses the demand for consistent EV quality to meet the requirements for clinical supply. Thus, our innovations will significantly facilitate and accelerate the translation of EV products into therapeutic applications.

Proteomic profiling of extracellular vesicles from CHMP2B‐mediated frontotemporal dementia patient‐derived cells

Presenter: Cecilie Madsen

Danish Dementia Research Centre, Rigshospitalet, Denmark

Introduction : Frontotemporal Dementia (FTD) is a progressive neurodegenerative disorder and a common form of early‐onset dementia. FTD is caused by neuropathological changes in the frontal and temporal lobes and covers a spectrum of clinical manifestations including changes in behaviour, language, and executive functions. Familial forms of FTD cover 30‐40% of all cases, where a pathogenic variant in the charged multivesicular body protein 2B‐gene (CHMP2B) causes a rare form of familial FTD, also called FTD‐3. CHMP2B is part of the endosomal sorting complex required for transport (ESCRT‐III), which is important for the endo‐lysosomal system including the multivesicular bodies (MVBs). Knowing that exosomes originate from MVBs, we speculated whether the CHMP2B mutation might influence the extracellular vesicles (EVs) in FTD‐3, and if the EVs could be involved in spreading the disease between brain cells.

Methods : Using patient induced pluripotent stem cell (iPSC)‐derived microglia and neurones we investigate the protein profile of size‐exclusion‐chromatography (SEC)‐isolated EVs from FTD‐3 patient‐derived cells. By mass spectrometry, we search for proteins that are dysregulated in FTD‐3, which could potentially be driving the disease pathology, and thus could be potential therapeutic targets. This will provide insight into the potential impact of EVs in FTD‐3. By comparing results to the isogenic controls, we will investigate the influence of the CHMP2B mutation on EVs. The project is approved by the Danish scientific ethics committee.

Results : In our pilot study, we found 4407 proteins from microglial EVs. From this study we observed that the majority of proteins, 59%, were cytoplasmic and cell membrane proteins and 5% were mitochondrial proteins. However, in the group of significantly upregulated proteins, with a fold change above 1.25, the mitochondrial proteins accounted for 20% of the upregulated proteins. We are in the process of repeating the study with a larger cohort to see if we can replicate these findings and investigate the potential mitochondrial impact.

Summary/Conclusion : Results of the study will be presented.

Funding : Neuroscience Academy Denmark, Alzheimer‐forskningsfonden, Beckett fonden, Kong Christian den Tiendes Fond.

Enhancement of anti‐inflammatory properties in human mesenchymal stem cell‐derived extracellular vesicles through cytokine preconditioning

Presenter: Chadaporn Attakitbancha

Chulalongkorn University, Thailand

Introduction : Mesenchymal stem cells (MSCs) are known for their paracrine effects, particularly in immunomodulation, where extracellular vesicles (EVs) play an important role in signal transmission across cells. Recent evidence suggests that the preconditioning of MSCs with various supplements can alter EV profiles, enhancing their functional activities in response to specific environments. In this study, MSCs were preconditioned with individual and combined inflammatory cytokines, including Interleukin‐1β (IL‐1β), Interleukin‐6 (IL‐6), Tumour necrosis factor‐alpha (TNF‐α), and Interferon‐gamma (IFN‐γ) to understand MSCs responses to inflammatory conditions from an EVs perspective.

Methods : Human Umbilical cord‐derived MSCs (UC‐MSCs) were cultured in xeno‐free conditions until they reached 80% confluence. Then, they were preconditioned with specific inflammatory cytokines with specific concentrations individually and in combination. Following preconditioning, EVs were concentrated by centrifugal ultrafiltration and isolated using size‐exclusion chromatography. The total protein concentrations of EV samples were quantified using the micro‐BCA assay, and EV densities were determined by nanoparticle tracking analysis (NTA). The secretion profile of critical cytokines and growth factors was analysed via the human‐customized multiplex assay. In addition, anti‐inflammatory effects were assessed by measuring nitric oxide (NO) levels in IL‐1β induced‐fibroblasts treated with MSCs‐derived EVs.

Results : MSCs preconditioned with various inflammatory cytokines demonstrated distinct protein concentrations, EV counts, secretion profiles, and functional activities. EVs from cytokine‐preconditioned MSCs exhibited higher anti‐inflammatory cytokine levels than the negative control. In cytokine‐primed conditions, EV‐treated fibroblasts showed significantly reduced nitric oxide (NO) levels, indicating enhanced anti‐inflammatory activity.

Summary/Conclusion : Preconditioning MSCs with specific inflammatory cytokines is a promising strategy for generating EVs with tailored secretion and functional profiles.

Funding : This research was supported by the National Research Council of Thailand (NRCT)

The therapeutic effect of MSC‐EVs' cargo in acute hepatic failure

Alexandra Stamatopoulou 1 , 2 , Maria Lefkaditi 3 , Thanasis Fileles 1 , Dimitrios Bobos 1 , Adriana Psaraki 1 , Manousos Makridakis⁴, Efstathios Michalopoulos⁵, Anastasios G. Kriebardis⁶, Athanasios Stavropoulos⁷, Maria Xilouri⁷, Antonia Vlahou⁴, Aristides G. Eliopoulos 1 , Maria G. Roubelakis 1 , 2 .

1 Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens (NKUA), Athens, Greece; 2 Cell and Gene Therapy Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece; 3 Institute for Fundamental Biomedical Research, Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece; 4 Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece; 5 Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Athens, Greece; 6 Laboratory of Reliability and Quality Control in Laboratory Hematology (HemQcR), Department of Biomedical Sciences, School of Health & Caring Sciences, University of West Attica (UniWA), Egaleo, Greece; 7 Center of Clinical Research, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Attica, Greece

Introduction : Acute hepatic failure (AHF) rapidly deteriorates liver function, resulting in high mortality rates. Liver transplantation remains the gold standard for treatment with concomitant challenges such as donor shortages and lifelong immunosuppression. Our previous studies showed that factors released by fetal mesenchymal stromal cells (MSCs) and their hepatocyte progenitor‐like (HPL) cells can promote liver repair and reduce inflammation in CCL4‐mice via IL‐10 and Annexin A1. Recently, we discovered that extracellular vesicles (EVs) from fetal MSCs can serve as an alternative therapy to enhance the AHF phenotype in vivo. This study presents a novel AHF treatment approach using modified EVs from umbilical cord MSCs (UC‐MSCs) in an AHF mouse model.

Methods : We evaluated the therapeutic effects of isolated UC‐MSC‐derived EVs in ex vivo and in vivo models simulating AHF. EVs were characterized and injected intrahepatically into CCL4‐induced mice. We analysed the proteomic content of these EVs and found MFGE‐8 protein was notably enriched. Further functional analyses included administering recombinant MFGE‐8 to CCL4‐induced mice, conducting proteomic assessments of treated liver tissues, and silencing MFGE‐8 levels in EVs using a lentiviral system.

Results : UC‐MSC‐EVs significantly reduced inflammation and improved liver function by promoting oval cell proliferation in CCL4‐induced mice. LC‐MS/MS proteomic analyses revealed that MFGE‐8 in EVs aided AHF recovery by inhibiting PI3K signalling. Recombinant MFGE‐8 administration also diminished liver damage in CCL4‐mice. Proteomic analysis of MFGE‐8‐treated livers identified proteins involved in efferocytosis (e.g., Pklr, Gstt1, Gstt3) and autophagy (e.g., ATG5, ATG7, LC3B). Additionally, MFGE‐8 levels were found to be decreased in liver biopsies from AHF patients.

Summary/Conclusion : In summary, UC‐MSC‐derived EVs enhanced liver function and stimulated oval cell proliferation in CCL4‐induced mice, emphasizing the role of MFGE‐8 in liver protection. We investigated further MFGE‐8′s role in liver damage in CCL4‐induced mice. LC‐MS/MS analysis of MFGE‐8‐treated livers revealed its specific involvement in efferocytosis and autophagy. Our findings provide a proof‐of‐concept for a novel, multidisciplinary, cell‐free strategy for AHF treatment, shedding light on the reparative roles of progenitor cells in the liver.

Preconditioned HUC MSC extracellular vesicles effectively polarize intraperitoneal macrophages in mouse peritonitis model

Presenter: Polina Pikus

National Academy of Sciences of Ukraine, Ukraine

Introduction : Extracellular vesicles (EVs) are known to be promising tool in the treatment of various diseases. Of particular interest are EVs secreted from mesenchymal stem cells (MSCs) because they have a main role in modulating immune cells. Preconditioning of MSCs, which enhances their therapeutic efficacy, positively affects the immunomodulatory properties of their EVs. The aim of the study was to compare the effects of EVs secreted by native and preconditioned MSCs on intraperitoneal macrophages during resolution of inflammation in a mouse model of acute peritonitis.

Methods : MSCs were isolated from umbilical cords of both sexes and characterized according to the minimal criteria for defining MSCs (ISCT). MSCs were preconditioned with H2O2 at a dose of 30 µM. EVs were isolated by ultracentrifugation. EVs were characterized by transmission electron microscopy, ZetaView Nanoparticle Tracking Analyzer and EV markers were identified by Western blot. A model of sterile acute inflammation of the abdominal cavity in mice was induced by intraperitoneal injection of 1 mL of 3% proteose peptone solution.

Results : The maximum intensity of inflammation was 24 h after the injection of the proteose peptone solution, and at this time mice were injected with EVs in an amount equivalent to that obtained from 5 000 MSCs/mouse. 15 min after injection of MSCs at this dose led to sharp inflammation suppression, and the rate of inflammation suppression upon injection of preconditioned MSCs was even higher. Dynamics of inflammation suppression after the injection of EVs shows higher efficacy compared to MSCs. The level of IL‐10 expression in mononuclear cells obtained from the peritoneal cavity of mice after injection of EVs from native MSCs increases, suppressing inflammation compared to the positive control. But the level of IL‐10 expression is significantly higher after injection of EVs derived from H2O2‐preconditioned MSCs. Increased phagocytosis by intraperitoneal macrophages, observed during the inflammation suppression, is more intense after the injection of preconditioned MSCs.

Summary/Conclusion : The injection of EVs derived from native and H2O2‐preconditioned HUC MSCs results in potent suppression of inflammation and macrophage polarization in the mouse peritoneal cavity, with both effects being more pronounced with injection of EVs from preconditioned MSCs.

Establishment of the sensitive potency assay useful for Characterization and Quality Control of therapeutic Extracellular Vesicle product derived from human Mesenchymal Stem Cell

Tomofumi Yamamoto, Hirotaka Nishimura, Noritaka Hashii, Akiko Ishii‐Watabe

National Institute of Health Sciences, Japan

Introduction : In recent years, extracellular vesicles (EVs) have attracted attention as a new drug modality and are being developed worldwide. Mesenchymal stem cell (MSC)‐derived EVs are expected to have various effects on immune diseases and wound, and their application to humans is progressing. However, because EVs have heterogeneity, it is difficult to homogeneous EV preparations, the establishment of appropriate potency assay is the key issue for quality control of therapeutic EV products to ensure their safety and efficacy.

Methods : We used a cell‐based assay in which LPS stimulation of Raw 264.7 mouse macrophage cells induces an inflammatory response in order to establish a method for quality evaluation and control of MSC‐derived EV preparations. In this assay, the anti‐inflammatory effect of EV administration was evaluated by change in TNFα mRNA expression level. hTERT‐adipose tissue MSC was used in this study. MSC‐derived EV was isolated by ultracentrifugation method.

Results : MSC‐derived EVs showed anti‐inflammatory activity dependent on the number of particles added. Next, we checked whether the anti‐inflammatory effect was induced by components other than EVs in the supernatant of the EV samples used. No anti‐inflammatory activity was detected in the supernatant without EVs. Next, we prepared samples of degraded EVs that were subjected to repeated freeze‐thaw cycles. After several freeze‐thaw cycles, the samples showed changes in several physical properties, such as an increase in particle size and a decrease in particle concentration. However, there was no significant change in anti‐inflammatory activity, suggesting that the evaluation of biological activity using TNFα does not correlate with changes in the physical properties of EVs. Therefore, we comprehensively measured the expression levels of chemokines and cytokines after LPS stimulation to search for novel markers that correlate more sensitively with changes in physical properties. The results indicate that CXCL10 may be a more sensitive indicator of EV bioactivity than TNFα.

Summary/Conclusion : We found that CXCL10 expression level is more suitable marker in the potency assay for MSC‐derived EV preparations. It is necessary to clarify whether the change in expression level of CXCL10 reflects the mechanism of action of EVs in vivo.

Funding : AMED JP22mk0101218, Grant‐in‐Aid for Early‐Career Scientists 24K18318

Differences in the numbers of EVs released by Wharton's jelly‐derived mesenchymal stem/stromal cells obtained from different patients—potential drawback considering personalized cellular‐free therapies

Monika Sypecka 1,2 , Sylwia Katarzyna Król 3 , Anna Sarnowska 1

1 Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, PAS, Poland. 2 Doctoral School of Translational Medicine, Centre of Postgraduate Medical Education, Poland. 3 Department of Neurooncology, Mossakowski Medical Research Institute, PAS, Poland.

Introduction : Mesenchymal stem/stromal cells derived from Wharton's jelly of human umbilical cord (WJ‐MSCs) exhibit unique immunomodulative, regenerative and neuroprotective properties and are therefore considered as a potential tool in the treatment of various disorders. Their therapeutic potential mostly relies on their secretome—WJ‐MSCs secrete different factors that are either secreted into the environment as a soluble secretome or are encapsulated within extracellular vesicles (EVs). As the ability to secrete immunomodulative and neuroprotective factors usually varies significantly between donors, we hypothesize that this correlates with different numbers of EVs released by these cells. To confirm this thesis, we therefore performed a comparative study of the ability to secrete EVs by WJ‐MSCs obtained from different donors.

Methods : Umbilical cords were obtained from full‐term deliveries [according to the Ethics Committee of Warsaw Medical University guideline (KB/213/2016) and with mothers’ written consents] and MSCs were then mechanically isolated from Wharton's jelly. The cells were then cultured until 3rd passage in standard culture medium (DMEM + 5% human platelet lysate + 1% antibiotic‐antimycotic) in 5% O2, 5% CO2, 37⁰C. Next, the standard medium was removed, WJ‐MSCs were rinsed with double‐filtered PBS (0,1 µm) and a medium without platelet lysate was added for 24 h. After 24 h, cell culture medium containing EVs secreted by WJ‐MSCs was collected and EVs were isolated by ultracentrifugation (300 × g for 10 min, 4⁰C; 2000 × g for 30 min, 4⁰C; 100,000 × g for 90 min, 4⁰C). The isolated EVs were suspended in double‐filtered PBS and NTA analysis was performed.

Results : The analysis revealed that WJ‐MSCs obtained from different donors secrete significantly different numbers of EVs.

Summary/Conclusion : As significant differences in the numbers of EVs released by WJ‐MSCs obtained from different patients have been discovered, it may potentially have an impact on the efficiency of cellular‐free therapies dedicated for individual patients—in the future it may become a potential drawback considering personalized cellular‐free therapies.

Funding : This research was funded by National Science Centre grant number 2022/47/O/NZ3/01739.

GMP compliant production of EVs from UC derived MSCs

Presenter: Irena Koutna

St. Anne's University Hospital Brno, Czech Republic

Introduction : Extracellular vesicles (EVs), particularly exosomes, have garnered significant interest in recent years due to their potential as therapeutic agents. Mesenchymal stem cells (MSCs), with their regenerative and immunomodulatory properties, are a promising source of EVs for therapeutic applications. Among various sources of MSCs, the umbilical cord (UC) offers a non‐invasive and rich resource for isolation. UC‐MSCs secrete EVs that have been shown to promote tissue repair, modulate immune responses, and enhance cell survival and could be utilized for treatment of neurodegenerative disorders.

Methods : 1.Isolation of UC‐MSCs: Human UCs were obtained from healthy full‐term births with informed consent. UC‐MSCs were isolated using enzymatic digestion followed by plating in MSC growth media. Cells were expanded until reaching confluence and characterized by flow cytometry for MSC surface markers, immunosuppression properties and tri‐lineage differentiation. 2.EV Isolation: EVs were isolated from the conditioned media of UC‐MSCs which was first centrifuged at 300 × g to remove cells and debris. The supernatant was then subjected to TFF followed by SEC. 3.Characterization of EVs: The size distribution and concentration of isolated EVs were determined by nanoparticle tracking analysis (NTA). Cryo‐electron microscopy (CEM) was used to observe the morphology of the EVs. Western blot analysis was performed to detect exosome markers. 4.Functional Assays: The biological activity of UC‐MSC‐derived EVs was assessed through cell proliferation assays (MTT assay), migration assays (wound healing assay), and immunomodulatory effects on immune cells (T cell suppression assay).

Results : The UC‐MSCs cultured in a GMP EMA‐approved scheme is an excellent source for EVs because from one UC we are able to harvest up to 10 L of conditioned medium.

Summary/Conclusion : UC‐MSCs are a promising source of biologically active vesicles with significant potential for therapeutic applications for neurodegenerative disorders.

Funding : Supported by project nr. LX22NPO5107 (MEYS): Financed by European Union—Next Generation EU. Supported by the European Regional Development Fund—project CZECRIN_PHARMTECH, no. CZ.02.01.01/00/23_015/0008194. Created in collabouration with MED MUNI through the CZECRIN project (LM2023049), supported by the national budget through MEYS.

Extracellular vesicles from periodontal ligament mesenchymal stromal cells regulate macrophage polarization

Oksana Gornatiuk 1,2,3 , Oleh Andruchov 1 , Christian Behm 1

1 Competence Centre for Periodontal Research, University Clinic of Dentistry, Medical University of Vienna. 2 Institute of Genetic and Regenerative Medicine, M.D. Strazhesko National Scientific Center of Cardiology, Clinical and Regenerative Medicine, NAMS of Ukraine. 3 Institute of Molecular Biology and Genetics of NAS of Ukraine.

Introduction : Human periodontal ligament mesenchymal stromal cells (HPDLCs) are non‐hematopoietic, multipotent progenitor cells that are essential in tissue repair and immunomodulation. HPDLCs can be easily obtained by minimally invasive procedures and have been used to treat various diseases. Several studies have demonstrated that HPDLCs therapeutic effects are realized through HPDLCs ‐derived extracellular vesicles, which are involved in the regulation of macrophage polarization. However, the properties as well as roles of the hPDLCs‐EVs in M1/M2 macrophage polarization are not well known. The aim of this work was to study the influence of HPDLCs‐EVs on macrophage polarization.

Methods : Cell culture, western blotting (WB), nanoparticle tracking analysis (NTA), bead‐based flow cytometry, transmission electron microscopy (TEM), and size exclusion chromatography (SEC).

Results : HPDLCs‐EVs have been successfully isolated using ultrafiltration combined with size exclusion chromatography. The HPDLCs‐EVs size distribution was examined using NTA. It was shown their size varies from 30 nm to 300 nm. Using WB and Flow Cytometry analysis, the presence of the most common exosomes markers CD63, CD81, CD9 was confirmed. The morphology of the hPDLCs‐EVs was observed by TEM. A TEM analysis showed spherical, cup‐shaped EVs isolated by the ultrafiltration combined with SEC. Human peripheral blood mononuclear cell¬ derived macrophages were used for their polarization in vitro. M1/M2 macrophage polarization was evaluated by flow cytometry.

Summary/Conclusion : The obtained data suggest that EVs derived from human periodontal ligament cells modulate macrophage polarization.

Funding : This work was supported by Ukrainian Short‐Term Fellowships of Federation of European Biochemical Societies

Physiology and Pathology

Subtopic Organ Systems 2

Unpicking the functional components involved in bowel cancer EV/gut microbiome inter‐species cross‐talk

Rawan Maani, Melissa Lacey, Lewis Quayle, Nick Peake

Sheffield Hallam University, UK

Introduction : Bowel cancer is the 3rd most common cancer and 2nd most common cause of cancer‐related morbidity worldwide. Risk factors for developing bowel cancer include obesity and diet, with increasing evidence that disease development and progression is linked to disruption to the gut microbiome. Our previous work has shown that EVs can directly target gut‐resident bacteria such as e. coli, altering behaviours including growth and biofilm formation. The aim in this work was to better understand the molecular mechanisms linking EVs to the gut microbiome.

Methods : EVs were isolated from the isogenic bowel cancer cell lines SW480 and SW620 cultured in AD‐1000 bioreactors, which were fully characterised to the MISEV2023 guidelines using western blotting for positive (CD63, CD9) and negative (GM130) markers, DELFIA ELISA (CD9, CD63, CD81), nano‐flow cytometry and transmission electron microscopy. Enzyme targeting was performed using proteinase k, trypsin and PNGase to assess surface mediators of EV‐bacterial interaction. Metal ion content was assessed using commercial kits and depleted using chelators. Involvement of RNA and proteins in phenotypic changes assess using RNAse and proteinase k treatment respectively. E. coli strains MG1655 and 11G5 were treated with EVs before processing for RNAseq, and bioinformatic analysis was undertaken using BLAST alignment of putative bowel‐cancer associated EV‐miRNA with reference sequences for bacterial strains linked to bowel cancer.

Results : EVs from bowel cancer cells significantly impact on growth and biofilm formation in a manner dependent on disease stage and bacterial strain. Targeting EV surface proteins with proteinase k did attenuate these effects whereas PNGase did not, however EVs lysed with detergent still had a significant affect implying a role for both direct interaction and EV cargo in altering bacterial behaviour. RNAseq showed significant upregulation of genes linked to flagella function (eg FliA), and significant down‐regulation of genes associated with zinc uptake (eg zinT, znuA, znuC). EV zinc levels were observed to be disease stage‐specific, and several miRNAs were identified with potential to target across a range of disease‐linked strains.

Summary/Conclusion : EVs have potential to widely target the gut microbiome through both direct binding and indirect mechanisms, with possible involvement of both miRNA and metal ion cargo.

LIMCH1‐enriched extracellular vesicles promote vascular permeability in early‐onset preeclampsia

Seiko Matsuo 1 , Akira Yokoi 1 , Takafumi Ushida 1 , Kosuke Yoshida 1 , Hironori Suzuki 2 , Masami Kitagawa 1 , Eri Asano‐Inami 1 , Rika Miki 3 , Sho Tano 1 , Kenji Imai 1 , Yusuke Yamamoto 2 , Hiroaki Kajiyama 1 , Tomomi Kotani 1

1 Nagoya University, Japan, 2 National Cancer Center Research Institute, Japan, 3 Nozaki Tokushukai Hospital Attached Research Institute, Japan.

Introduction : Preeclampsia (PE) is a major complication of pregnancy, characterized by hypertension and various forms of end‐organ dysfunction. However, its exact pathogenesis remains incompletely understood. This study aimed to investigate a novel pathogenic mechanism related to increased vascular permeability in PE, using LC‐MS/MS analyses and detailed functional assays on serum small extracellular vesicles (sEVs) from women with PE.

Methods : We isolated sEVs from the serum of 16 women, including those with early‐onset PE and healthy pregnant controls, using serial ultracentrifugation. The isolated sEVs were then subjected to LC‐MS/MS analysis. To generate sEVs enriched with the target protein, we developed a stable overexpression cell line for the target protein using the JAR choriocarcinoma cell line. The effects of those sEVs were subsequently evaluated on human umbilical vein endothelial cells (HUVECs) and in mice. Additionally, placental immunohistochemistry for the target protein and spatial transcriptomic analysis were conducted. The study protocol was approved by the Institutional Ethics Board of our university.

Results : LC‐MS/MS analysis of serum sEVs identified LIMCH1 as an early‐onset PE‐associated EV protein. In the GEO dataset, LIMCH1 expression was significantly upregulated in PE placentas ( p < 0.01). Immunohistochemistry revealed predominant expression of LIMCH1 in syncytiotrophoblast, with a significantly higher H‐score in PE placentas compared to normal placentas ( p < 0.05). Spatial transcriptomic analysis further showed that LIMCH1 expression correlated with PLAP, a marker of placenta‐derived sEVs. Although administration of LIMCH1‐overexpressing JAR‐derived sEVs (LIMCH1‐enriched EVs) had no effect on HUVEC proliferation, the FITC‐dextran permeability assay demonstrated a significant increase in endothelial permeability with the addition of LIMCH1‐enriched EVs ( p < 0.01). Transcriptomic analysis revealed that LIMCH1‐enriched EVs impaired endothelial cell‐cell junction assembly by downregulating TJP1 (ZO‐1) expression. Furthermore, LIMCH1‐enriched EVs increased pulmonary vascular permeability in vivo. While LIMCH1‐enriched EVs promoted lung vascular permeability, they did not affect vascular permeability in the brain. Intravenous injection of sEVs into mice revealed greater uptake in the lungs than in the brain.

Summary/Conclusion : We demonstrated EV‐mediated vascular endothelial permeability, a central feature of PE pathology, and provided insights into the underlying mechanism of PE‐related pulmonary edema, which has not yet been fully elucidated.

The effect of EVs from human follicular fluid of patients with high and low response to ovarian stimulation on granulosa cell‐line KGN

Katariina J. Saretok 1 , Inge Varik 1 , Kristine Rosenberg 1,2 , Aleksander Trošin 3 , Paolo Guazzi 4 , Agne Velthut‐Meikas 1

1 Department of Chemistry and Biotechnology, Tallinn University of Technology, Estonia, 2 Nova Vita Clinic, Estonia, 3 Center for Infertility Treatment, East Tallinn Central Hospital, Estonia, 4 HansaBioMed Life Sciences, Estonia

Introduction : EVs in the ovarian follicular fluid (FF) may play a role in intercellular signalling important for female fertility. IVF, a widely used treatment option for infertile couples, is preceded by ovarian stimulation with recombinant follicle stimulating hormone (rFSH) to mature multiple oocytes. However, some women respond poorly to such stimulation jeopardizing their IVF outcome due to inability to obtain oocytes for fertilization. The role of FF EVs in case of hyporesponse to stimulation is currently unknown. The current study aims to reveal, how the FF EVs of IVF patients demonstrating normal or hyporesponse to rFSH stimulation affect the steroidogenesis and gene expression of stable granulosa cell line KGN.

Methods : The study was approved by the Research Ethics Committee of Tartu University, and a written informed consent was obtained from all study participants. Patients were segregated into normo‐ ( n = 15) or hyporesponder ( n = 16) groups according to the ovarian sensitivity index (cut‐off = 200 IU rFSH/oocyte). FF was obtained during ovarian puncture, samples with visible blood contamination were discarded. The samples were centrifuged first at 400×g and then at 2000×g for 10 min. EVs were isolated by size‐exclusion chromatography and characterized by BCA, NTA, and Western blotting. KGN cells in exosome‐depleted serum were treated with EVs in concentration of 1 × 10 9 particles/mL for 24 h. Testosterone, oestradiol and progesterone levels were measured from the culture media with ELISA. Gene expression in the treated cells was analysed by RNA sequencing.

Results : FF EVs affected collagen synthesis, cholesterol metabolism and steroidogenesis pathways in KGN cells (FDR < 0.05). EVs from hyporesponders inhibit the progesterone output of KGN cells (p < 0.05) in comparison to EVs from normoresponders and DPBS controls. Differentially expressed genes triggered by normoresponder EVs include protein C activated G protein coupled receptor signalling, while EVs from hyporesponders perturbate the gene expression of potassium channels.

Summary/Conclusion : FF EVs are needed for the regulation of gene expression in steroidogenesis, which is one of the main metabolic function of the ovaries. EVs from hyporesponders trigger minor gene expression changes in comparison to normoresponders, but diminish progesterone synthesis.

Funding : The study was funded by the Estonian Research Council (grant no PSG608).

Distinct short RNA profiles and functional impacts of small and large extracellular vesicles from human follicular fluid on KGN granulosa cells

Inge Varik 1 , Katariina Johanna Saretok 1 , Kristine Rosenberg 1,2 , Ileana Quintero 3 , Maija Puhka 3 , Nataliia Volkova 1 , Aleksander Trošin 4 , Paolo Guazzi 5 , Agne Velthut‐Meikas 1

1 Department of Chemistry and Biotechnology, Tallinn University of Technology, Estonia; 2 Nova Vita Clinic AS, Estonia; 3 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland; 4 East‐Tallinn Central Hospital, Centre for Infertility Treatment, Estonia; 5 HansaBioMed Life Sciences Ltd, Estonia.

Introduction : While several studies have isolated extracellular vesicles (EVs) from human follicular fluid (FF), research detailing the specific functions of FF EV subtypes remains limited. Understanding EV subtype‐specific roles could provide new insights into ovarian function. In this study, we isolated large (LEVs) and small EVs (SEVs) from human FF, characterized their short non‐coding RNA (ncRNA) composition, and assessed their impact on ncRNA expression in KGN cells.

Methods : Ethical approval was obtained from the University of Tartu Research Ethics Committee, and written informed consent was obtained from all study participants. FF subtypes were purified by size exclusion chromatography, followed by tangential flow filtration (pore size 200 nm), and characterized according to the MISEV2023 guidelines. KGN cells were treated with 10 8 /mL LEVs or 10 9 /mL SEVs for 24 h. Small RNA‐seq libraries were prepared for EVs and EV‐treated KGN cells and sequenced with NovaSeq X. Unique molecular counts of miRNAs and piRNAs were used for differential expression (DE) analysis with DESeq2. Over‐representation analysis (ORA) was conducted with miEAA to identify gene ontology terms related to the DE genes.

Results : Principal component analysis separated SEV and LEV samples along the PC1 score, indicating significant differences in their ncRNA content. We identified 46 DE miRNAs between SEVs and LEVs, with 43 upregulated in SEVs, and 3 in LEVs. Additionally, 45 DE piRNAs were detected, with 13 upregulated in SEVs and 32 in LEVs. ORA of DE miRNAs indicated enrichment in pathways related to cell cycle, TGF‐β signalling, DNA damage response, cellular senescence and oxidative stress. DE analysis between SEV‐ and LEV‐treated KGN cells identified 152 DE ncRNAs, including 84 DE miRNAs and 68 DE piRNAs. Similarly to DE EV miRNAs, ORA demonstrated the involvement of DE miRNAs in cell cycle, TGF‐ β signalling, and DNA damage response, but also SUMOylation and WNT signalling.

Summary/Conclusion : This study highlights differences in short ncRNA composition and function of FF EV subtypes. Differential expression of miRNAs in SEVs and LEVs, particularly in pathways like cell cycle regulation, TGF‐β signalling, and oxidative stress, suggests SEVs and LEVs may play distinct roles in maintaining follicular health.

Funding : The study was funded by the Estonian Research Council.

Endometriotic patient‐derived small extracellular vesicles carry unique signatures associated with lesion growth and disease progression

Jaelis P. Holmes, Katherine B. Zutautas, Danielle J. Sisnett, Chandrakant Tayade

Department of Biomedical and Molecular Sciences, Queen's University, Canada.

Introduction : Endometriosis (EM) is a chronic, estrogen‐dependent inflammatory disease with high heterogeneity in phenotype and symptom severity, resulting in diagnostic delays of 4–11 years. Small extracellular vesicles (sEVs), contain bioactive molecules and show distinct expression profiles between EM patients and controls, making them potential candidates for disease biomarkers. This study aims to profile sEVs in mild and severe EM cases using multi‐omics data, to unravel biomarkers associated with EM.

Methods : sEVs were isolated from ectopic lesions, matched eutopic endometrium, plasma, and peritoneal fluid samples from 24 patients (ASRM Stages I‐II, n = 8; Stages III‐IV, n = 8; controls, n = 8). Isolation involved combination of ultracentrifugation and Norgen Biotek resin technology, followed by characterization using NTA, TEM, and the MACsPlex EV kit, following MISEV2023 guidelines. sEV molecular content (miRNAs, lncRNAs, proteins, lipids) was assessed via transcriptomic, proteomic, and lipidomic analyses to identify conserved differential expression patterns relative to controls. All suppliers were ISO 0991 and ISO 13485 certified.

Results : Nanoparticle tracking analysis confirmed sEV size distribution (50‐250 nm), while TEM showed double membrane vesicles characteristic of sEVs. Preliminary MACsPlex analysis identified high expression of tetraspanins (CD9, CD63, CD83) and surface markers associated with B cells (CD24), cell adhesion/integrins (CD29, CD41b, CD42a), and platelets/endothelium (CD42a). Lipidomic analysis revealed significantly elevated saturated fatty acids and triacylglycerides in EM patient plasma and ectopic lesions compared to controls, shedding insights into EM‐associated metabolic pathways. Proteomic and transcriptomic analyses are in progress.

Summary/Conclusion : This study uniquely integrates nucleic acids, proteins, and lipids in sEVs from matched patient samples. Our results will offer a comprehensive profile of sEV signatures, and findings will aim to clarify sEV contributions to EM pathology and aid in identifying potential biomarkers for diagnostic applications.

Funding : This work is supported by the Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council (NSERC).

Small extracellular vesicles isolated from the follicular fluid of patients undergoing assisted reproductive technology: tetraspanins and PD‐L1 expression analysis

Barbara Bortot 1 , Roberta Di Florio 1 , Gabriella Zito 1 , Francesco Valle 2 , Marco Brucale 2 , Giuseppe Ricci 1,3 , Stefania Biffi 1 .

1 Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy. 2 Institute of Nanostructured Materials, Italian National Council of Research, Bologna, Italy. 3 Department of Medical, Surgical and Health Science, University of Trieste, 34129 Trieste, Italy.

Introduction : In the ovary, follicular fluid (FF) is a biological fluid that serves as the milieu for oocyte development. Extracellular vesicles (EVs) play a crucial role in coordinating the various stages of reproduction by mediating cell‐to‐cell communication within the reproductive system. PD‐1 and its ligands PD‐L1 were found in high levels in small EV fractions of human FF and had the ability to modulate T cell activation, suggesting a potential regulatory role in immune response and immunological homeostasis within the ovarian environment. CD9, CD63, and CD81 are major members of the tetraspanin family commonly used to detect sEVs.

Methods : Ten patients with a history of infertility who had undergone assisted reproductive technology treatment were included in this study. The FFs were obtained during the oocyte retrieval procedure, with each sample aspirated separately. Atomic force microscopy (AFM) was utilized to characterize FF EVs, which were first isolated through size exclusion chromatography and then pooled via ultracentrifugation. Tetraspanins and PD‐L1 expression were assessed using a highly sensitive method that integrated single‐particle antibody capture with immunofluorescence labelling on whole FF. The antibodies used included anti‐CD63, anti‐CD81, anti‐CD9, and anti‐PD‐L1.

Results : AFM and interferometric measurements of single particles showed that 70% of sEVs (small EVs) in the FF ranged between 30 and 50 nm in size. The distinctive characteristics in FF sEV subpopulations detected by single particle phenotyping were found in all cohorts of samples. FF sEVs captured with anti‐CD63 and anti‐CD81 antibodies had the same percentage (37%), whereas anti‐CD9 antibodies captured less (27%). Co‐localization studies showed that sEVs immobilized with anti‐CD63, anti‐CD81, and anti‐CD9 antibodies expressed CD81 at percentages of 42%, 46%, and 50%, respectively. CD9+ sEVs had the highest level of PD‐L1 expression, with an average of 5%, followed by the CD63+ and CD81+ sEV subpopulations, which had averages of 2% and 3% of all particles.

Summary/Conclusion : These insights show an enhanced expression of PD‐L1 in CD9+ sEVs indicating a potential regulatory function for this subgroup. These studies could help identify biomarkers for assisted reproduction and provide crucial information regarding FF sEV subpopulations.

Menstrual blood serum EVs as a source of biomarkers of unexplained infertility endotypes

Raminta Vaiciuleviciute 1 , Kieran Brennan 2 , Ilona Uzieliene 1 , Jolita Pachaleva 1 , Zaneta Kasilovskiene 3 , Lina Piesiniene 4 , Daiva Bironaite 1 , Margaret Mc Gee 2 , Eiva Bernotiene 1,5

1 Innovative Medicine Centre, Lithuania, 2 University College Dublin, Ireland, 3 Maxmeda, Lithuania, 4 Nanodiagnostika, Lithuania, 5 VilniusTech, Lithuania

Introduction : Several biomolecules have been previously associated with unexplained infertility (uIF) in blood and uterine samples, immune cells and their secreted factors, endometrial tissue, menstrual blood, serum, and stromal cells, however they do not comprehensively represent different uIF endotypes and their isolation and detection involves invasive diagnostic methods and lacks precision. This ex‐vivo study was performed to examine the potential of menstrual blood extracellular vesicles (EVs) as source of non‐invasive biomarkers of uIF.

Methods : Menstrual blood was collected on cycle day 2 from 9 fertile volunteers and 8 women with uIF. Menstrual blood serum (MBS) EVs were isolated from fertile and uIF female menstrual blood serum using Iodixanol Density Gradient Centrifugation and quantified by flow cytometry. EVs were characterized according to MISEV2023 guidelines: EV isolation was confirmed by Transmission Electron Microscopy; CD9, CD63, CD81 and CD147 EV markers were detected by flow cytometry, intracellular EV marker TSG101 and contamination markers albumin, APOA1, APOB and APOE were analysed by Western blot. Proteome analysis was performed using mass spectrometry, and data was analysed using bioinformatic tools.

Results : Comprehensive proteomic analysis of MBS EVs and EV‐depleted MBS showed significant changes in the uIF proteome, mostly affecting cell adhesion, immune response, apoptosis, response to oxidative stress and lipid metabolism. These processes were previously linked to pathologies of the female reproductive system but never investigated in uIF and were used to stratify patients into distinct molecular endotypes. AUC analysis was used to determine the optimum set of biomarkers for each of the uIF molecular endotypes.

Summary/Conclusion : This demonstrates MBS EVs carry key molecular cargo with potential to identify valuable information about alterations in the endometrium that may be associated with female infertility pathogenesis. Overall this study provides new insights into uIF that could facilitate personalised treatment approaches.

Changes in the miRNA cargo of EV‐mediated feto‐maternal communication following light treatment

Presenter: Bence P. Nagy

Semmelweis University, Hungary

Introduction : Light exposure of embryos during assisted reproduction affects embryo quality and implantation capacity in a wavelength dependent manner. We investigated the molecular mechanism of light‐induced changes through the comparative analysis of gene expression and regulatory miRNA profile of murine embryos cultured in dark environment or exposed to white / red filtered light. miRNA sequencing was used to assess the role of embryo‐derived extracellular vesicles in the endometrium‐embryo dialogue.

Methods : In vitro cultured mouse embryos (3.5 dpc) were exposed to white or red filtered light. After 24 h, the miRNA content of embryo‐derived extracellular vesicles were isolated and RNA‐sequencing was performed. Differential expression analysis and functional enrichment analysis were used for evaluating the transcriptome results.

Results : Embryo‐derived extracellular vesicles wavelength‐dependently enclosed unique miRNA cargos the target genes of which play a role in embryo implantation. We have found many significantly changed miRNA within the embryo‐derived extracellular vesicles, independent from the embryonic miRNA. Within the white light treated group, miRNA targeting genes in cell signalling and extracellular matrix reorganization were downregulated. Meanwhile following the treatment with filtered red light, there were significant elevations in levels of miRNA, targeting genes, that play a role in the implantation of embyros. Furthermore there were significant changes in miRNA targeting immunological signalling pathways, namely Fc gamma signalling, DAP12 signalling and inflammatory signallization.

Summary/Conclusion : Extracellular vesicles of light‐exposed embryos play a role in blastocyst‐decidua communication through the horizontal transfer of regulatory miRNAs. Our data prove that light exposure during in vitro fertilization modifies cell function that might affect the outcome of implantation.

Funding : This work was supported by OTKA K 147483, TKP‐EGA10 and STIA‐KF‐17.

Characterization of plasma‐derived small extracellular vesicle miRNA and protein cargo in hereditary angioedema

Linda Hofmann 1 , Robin Lochbaum 1 , Lutz Schütt 1 , Stefanie Schmitteckert 2 , Thomas K. Hoffmann 1 , Cornelia Brunner 1,3 , Jens Greve 1 , Janina Hahn 1 , Marie‐Nicole Theodoraki 1,4

1 Department of Otorhinolaryngology, Ulm University Medical Center, Germany, 2 nCounter Core Facility, University of Heidelberg, Germany, 3 Core Facility Immune Monitoring, Ulm University, Germany, 4 Department of Otorhinolaryngology, Technical University Munich, Germany

Introduction : Hereditary angioedema (HAE) is an inherited disorder causing attacks of subcutaneous tissue or mucosa swelling. The disease burden and attack frequencies vary significantly among patients. It is not fully understood why some patients are more severely affected than others and small extracellular vesicles (sEVs) may influence disease pathogenesis. So far, the sEV constellation and function in HAE are unknown. This is the first pilot study investigating sEVs as disease modulators in HAE.

Methods : Plasma was prepared from citrate blood by centrifugation at 1000 × g and 2500 × g for 10 min each. Informed consent was obtained as per ethics approval by Ulm University. sEVs were isolated by differential centrifugation, ultrafiltration, and SEC or ultracentrifugation (EV‐Track ID EV200068 , EV210344 ). Plasma‐derived sEVs from HAE patients and healthy donors (HD) were thoroughly characterized by western blot, transmission electron microscopy and nanoparticle tracking in line with the MISEV 2023 recommendations. sEV surface proteins were analysed by bead‐based flow cytometry. The miRNA cargo of sEVs was examined by nCounter technology and used to predict sEV‐based pathomechanisms in silico. All sEV readouts were analysed regarding HAE‐related changes and associations with clinical parameters and attack frequency.

Results : Total sEV protein levels were elevated in HAE patients compared to HD. In HAE patients, lower levels of sEVs carrying CD8, CD209, CD81, CD24 and CD44 were measured, indicating a redistribution of sEV subpopulations in HAE. sEV miRNA profiling revealed 84 HAE‐exclusive and 30 significantly HAE‐upregulated candidates, predicted to be involved in disease‐relevant signalling pathways. Core hubs of these interaction networks were AGO2, VEGF, RGS5, MTA1, IFG1 and BAX. A set of 12 and 36 sEV miRNAs were restricted to patients with absent swelling attacks or patients with present attacks during prophylactic therapy, respectively.

Summary/Conclusion : Our data sheds light on the sEV composition and role in HAE pathophysiology, providing a basis for developing new EV‐related diagnostic and therapeutic approaches. sEVs, via surface proteins and especially miRNAs, contribute to disease pathogenesis and a differential disease burden. They emerged as disease modulators in HAE and we are conducting further studies to reveal underlying mechanisms.

Potential of plasma‐derived small extracellular vesicles (sEVs) to differentiate between obstructive and non‐obstructive myocardial infarction (Mi versus MINOCA)

Christien M. Beez 1,2,3* , Petra Wollint 5, 6 , Felix Kneip 1, 2 , Henriette Thau 1,2 , Heike Meyborg 1,2,3 , Katrin Krüger 1,2,3 ,Jasper Iske 1,2, 3 , Miriam Weißkopf 5 , Natalia Topalovic 5 , Caroline Pietsch 5 , Maximilian Y. Emmert 1,2,3,7 , Volkmar Falk 1,2,6 , Cesarovic N. 1,2,6

1 Deutsches Herzzentrum der Charité (DHZC), Department of Cardiothoracic and Vascular Surgery, Berlin, Germany. 2 Charité—Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu Berlin, Berlin, Germany. 3 Berlin Institute of Health at Charité—Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), Berlin, Germany. 4 German Centre for Cardiovascular Research (DZHK), Partner Site Berlin, Berlin, Germany. 5 Center for Preclinical Develpoment, University Hospital Zürich, University of Zürich, Zürich, Switzerland. 6 Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland. 7 Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland.

Introduction : Significant advances have been achieved in manageing patients with obstructive myocardial infarction (MI). However, these advances are not applicable to approximately 15% of patients presenting with MI symptoms, since they suffer from myocardial infarction with non‐obstructive coronary arteries (MINOCA). Unlike patients with obstructive MI, MINOCA patients experience obstructions in smaller cardiac vessels, which can only be visualized by magnetic resonance imaging (MRI). This diagnostic process can take days, if not weeks, which is believed to contribute to an increased risk of serious complications and heightened mortality.

Methods : In order to address this challenge, the current study explores the potential of plasma‐derived small extracellular vesicles (sEVs) as diagnostic tool for the acute phase of MINOCA. Additionally, we aim to distinguish between MINOCA from obstructive MI by using our novel porcine MINOCA model alongside an obstructive MI model. Blood samples ( n = 8) were collected before (0 min) and 270 min post‐infarction induction. Plasma was isolated within 1 h of collection and stored at ‐80°C until sEVs could be isolated by ultracentrifugation. The obtained sEVs were analysed for phenotypic characteristics and transported cargo.

Results : Irrespective of infarction type, sEVs showed comparable yields in terms of protein and particle concentration, diameters smaller than 200 nm, and spheroid‐like shapes, as determined by BCA, NTA, and EM. The mass spectrometric analysis showed that at least 87% of proteins were shared across groups, with only a few distinct proteins for sEVs of MINOCA. These unique proteins are currently being assessed for their potential as diagnostic biomarkers. The study will be further complemented by the investigation of transported miRNA composition within sEVs.

Summary/Conclusion : Our preliminary findings indicate that MINOCA is distinct from MI during the acute phase, which would make the presence of a biomarker very likely. We believe that an early, non‐invasive diagnosis of MINOCA would help to significantly improve the outcomes for this unique patient population.

Funding : This work was supported by a research grant from the Swiss Heart Foundation and open access funding provided by ETH Zurich and DHZC Berlin.

MicroRNAs of blood extracellular vesicles are associated with abdominal aortic aneurysm development via the TGF‐β pathway in diabetes mellitus patients

Presenter: Kazuki Takahashi

Tokyo Medical University, Japan

Introduction : Abdominal aortic aneurysm (AAA) is a life‐threatening disease due to the risk of aortic rupture. The risk factors of AAA have been identified, including ageing, male sex, hypertension, dyslipidemia, and smoking, and it shares a risk factor with atherosclerosis. Although diabetes mellitus (DM) is one of the main risk factors for cardiovascular disease, epidemiological and basic studies have described its potential protective role on the prevalence and incidence of AAA. Multiple pathological processes and molecules contribute to AAA formation, including degradation of the extracellular matrix (ECM), inflammation, apoptosis of vascular smooth muscle cells (SMC), matrix metalloprotease 2, 9, and cytokines. On the other hand, increased ECM accumulation, enhancement of TGF‐β signalling, accumulation of collagen IV, and advanced glycation end‐products have been reported as protective mechanisms of AAA in DM. However, there is no research on the mechanism of AAA formation in DM and the microRNA (miRNA) of extracellular vesicles (EVs) for AAA formation in DM. This study focused on miRNA of the serum EVs to elucidate the mechanism of AAA formation in DM.

Methods : We compared mRNA in EVs from human serum between AAA with DM and non‐AAA with DM patients (controls). We collected serum samples from AAA with DM (N = 20) and non‐AAA with DM (N = 10). The EVs were isolated using ultracentrifugation according to MISEV 2023 guidelines. MiRNA was analysed using the next‐generation sequence. The microRNA in EVs with significant changes were subjected to pathway analysis using miRPath v.4.

Results : The significant up‐regulation is 12 miRNAs, and a down‐regulation of 12 miRNAs was detected from AAA patients compared to controls (Fold change > |1.5|, p < 0.05). The most significant microRNA was miR‐4524a‐5p. The KEGG pathway enrichment analysis of miR‐4524a‐5p indicated the TGF‐β pathway. Furthermore, eleven of the up‐regulated 12 miRNAs were also assumed to regulate this pathway.

Summary/Conclusion : The miRNA of serum EVs may regulate the TGF‐β pathway in AAA patients with DM. The activity of TGF‐β is associated with aortic aneurysm formation via SMC apoptosis, ECM degradation, and inflammatory cell infiltration.

Harnessing Artificial Circular RNA Sponges to Maintain Functionality in Senescent Cardiomyocytes

Presenter: Giorgia Senesi

Istituto Cardiocentro Ticino, Italy

Introduction : The use of lipid‐based nanocarriers for delivering biotherapeutics, such as RNA molecules, represents a promising approach in modern medicine. Extracellular vesicles (EVs) provide a natural and biocompatible means of delivering RNA‐based therapies to target cells. In our approach, we utilize EVs derived from cardiac progenitor cells (CPC) to deliver artificial circular RNA sponges (circmiRs) specifically designed to target multiple microRNAs upregulated in senescent cardiomyocytes (SenCM). This approach aims to sequester these dysregulated miRNAs, thereby mitigating their adverse effects. Our objectives include identifying upregulated miRNAs in SenCM, targeting downregulated transcripts, and engineering EV‐delivered circmiRs. We hypothesize that circmiRs can effectively modulate miRNA activity at lower doses, offering a potential advantage over senolytics, which may exacerbate cardiac dysfunction by inducing cell death and electrical uncoupling in cardiac muscle.

Methods : We conducted bulk RNA sequencing and proteomic analyses on SenCM treated with doxorubicin (Dox) compared to control cardiomyocytes (iCM). Through bioinformatic analysis, we identified target miRNAs that influence post‐transcriptional modifications in SenCM. Quantitative PCR (qPCR) was employed to validate these findings. To modulate miRNAs, we implemented a targeted sponge‐mediated RNA sequestration strategy. EVs were isolated from the conditioned CPC using size‐exclusion chromatography followed by ultracentrifugation. RNA sponges were loaded into EVs by transducing CPCs through lentiviral vector.

Results : Our transcriptomic and proteomic analyses of SenCM revealed 15 downregulated genes associated with chromatin organization, microtubule cytoskeleton dynamics, and cell cycle transitions. Bioinformatic analysis identified three miRNAs that target 11 of these 15 downregulated mRNAs and are significantly upregulated under senescent conditions. We subsequently engineered circmiRs that incorporate inhibitory sequences specific to these three miRNAs. When delivered via EVs, circmiRs were integrated into cardiomyocytes and effectively mitigated the stress conditions induced by doxorubicin.

Summary/Conclusion : Our findings indicate that the methodology of delivering circular RNA sponges via EVs can be effectively utilized to investigate the mechanisms underlying senescence activation pathways.

EVs as potential diagnostic tools in cancer‐induced cardiac atrophy

Edina Bugyik 1 , Gábor Valcz 2,3 , Júlia Opra 1 , Nóra Fekete 1 , András Försönits 1 , Krisztina V. Vukman 1 , Melinda Rezeli 4,5 , Sándor Paku 6 , Katalin Dezső 6 , Edit I Buzás 1,2,7

1 Semmelweis University, Institute of Genetics, Cell‐ and Immunobiology, Budapest, Hungary, 2 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Budapest, Hungary, 3 Image Analysis Group, 3DHISTECH Ltd., Budapest, Hungary, 4 Department of Biomedical Engineering, Lund University, Lund, Sweden, 5 Swedish National Infrastructure for Biological Mass Spectrometry (BioMS), Lund University, Lund, Sweden, 6 Semmelweis University, I st Department of Pathology and Experimental Cancer Research, Budapest, Hungary, 7 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary.

Introduction : Tumour anorexia‐cachexia syndrome is a multifaceted condition characterized by decreased skeletal muscle mass, generalized weakness, diminished quality of life, and poor prognosis. It occurs in 50‐80% of cancer patients and is estimated to be responsible for 20‐30% of all cancer‐related deaths. Although cardiac atrophy is a significant component of this syndrome, early detection of cardiac changes is still a clinical unmet need due to the focus on treating the underlying disease and limitations in diagnostic methods.

Methods : The C26 colorectal adenocarcinoma primary tumour model was used to develop tumour anorexia‐cachexia syndrome in vivo. The weight, tumour size, heart weight and tibia length of the mice were recorded upon termination. Large and small extracellular vesicles (lEVs and sEVs) were isolated from the blood samples using differential centrifugation alone or combined with size‐exclusion chromatography and were characterized using standard EV methodologies following the MISEV2023 guidelines. Wheat Germ Agglutinin (WGA‐Alexa‐488) labelling was applied for histology in order to confirm the presence of cardiac atrophy. Mass spectrometry was performed from the hearts, PFP and EV samples of healthy and diseased animals.

Results : There was a significant difference in the heart weight and body weight of control and tumour bearing groups. We confirmed a decrease in the size of cardiomyocytes in the tumour group. A total of 4993 proteins were detected during the proteomic analysis. We determined the differentially expressed proteins between the control and tumour groups. These differentially expressed proteins are mainly involved in acute phase reaction, immunity, metabolic and cell adhesion processes, apoptosis and cytoskeletal organization. The differential presence of potential heart‐derived proteins was detectable (Myh6, Myh7, Tnni3).

Summary/Conclusion : These findings provide valuable insights into the molecular changes associated with tumour‐induced cardiac atrophy. Further exploration of these proteins may lead to the development of a specific biomarker panel for early detection and intervention.

Funding : This research was funded by NVKP_16‐1‐2016‐0004, VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003, 2019‐2.1.7‐ERA‐NET‐2021‐00015. EU's Horizon 2020 No. 739593.

Ribosomal protein L36‐mediated selective loading of microRNA‐4432 into extracellular vesicles contributes to perivascular cell dysfunction in venous malformations

Jiangang Ren

Wuhan University, China

Introduction : Venous malformations (VMs), predominantly arising from activating mutations of TIE2 in endothelial cells (ECs), are characterized by dilated and tortuous vessels with a paucity of perivascular cells (PCs). The mechanisms of interaction between mutant ECs and PCs remain largely elusive. Here, we aim to investigate the characteristics of extracellular vesicles (EVs) from VM ECs, especially the microRNAs (miRNAs) carried and their roles in crosstalk between ECs and PCs in VM pathogenesis.

Methods : miRNA profiles of human umbilical vein endothelial cells overexpressing TIE2L914F (L914F cells) and TIE2WT [wildtype (WT) cells], along with their EVs, were analysed by RNA sequencing. In vitro studies using umbilical cord stem cells (UCSCs) were done to perform functional assays of VM EVs and their enriched miRNA‐4432 (miR‐4432). miRNA pulldown and RNA interference techniques were used to identify the sorting regulator of miR‐4432 into VM EVs.

Results : RNA secretion was upregulated in L914F EVs vs. WT EVs. miRNA sequencing revealed a distinct profile of L914F EVs vs. L914F cells, WT cells, and WT EVs, identifying miR‐4432 as being preferentially encapsulated in EVs from L914F cells. Functional assays demonstrated that VM EVs and EV‐carried miR‐4432 inhibited the differentiation, adhesion, and proliferation of UCSCs. Furthermore, ribosomal protein L36 (RPL36) was identified as an RNA‐binding protein and sorting regulator of miR‐4432 during the EV secretion process in L914F cells.

Summary/Conclusion : This study, for the first time, identified an interaction between VM ECs and PCs via EVs, and offers valuable data on the miRNA profiles of VM ECs and normal ECs, along with their EVs. Our findings suggest that the RPL36‐mediated selective loading of miR‐4432 into EVs may contribute to the aberrant PC coverage in VMs, providing novel insights into VM pathogenesis and potential treatment strategies.

Funding : the National Natural Science Foundation of China (81870361, 81922038, 81600385, 82101036)

The Protective Role of Exosome‐Derived miR‐30c‐1‐3p in Cardiomyocytes Against Hypoxia‐Induced Apoptosis and Cardiac Injury

Presenter: Jung Won Choi

Catholic Kwandong University

Introduction : Exosomes are small extracellular vesicles secreted by cells, playing a crucial role in intercellular communication by transporting proteins, lipids, and nucleic acids. Among their molecular cargo, microRNAs (miRNAs) are pivotal regulators of gene expression. Previous studies revealed that miR‐30c‐1‐3p levels are significantly reduced in the cardiac tissue of myocardial infarction animal models compared to controls, as confirmed by exosomal RNA sequencing. This study aimed to explore the function of exosome‐derived miR‐30c‐1‐3p.

Methods : To investigate the role of miR‐30c‐1‐3p in myocardial infarction, in vitro experiments using primary cardiomyocytes were performed.

Results : Primary cardiomyocytes were isolated from the hearts of 3‐day‐old mice and exposed to hypoxic conditions for 12, 24, and 48 h. Results indicated a marked decrease in miR‐30c‐1‐3p expression over time. Treatment with a miR‐30c‐1‐3p mimic was found to reduce hypoxia‐induced apoptosis in mouse cardiomyocytes, suggesting a protective role. Western blot analysis of conditioned media showed that cardiac injury markers such as CK‐MB, troponin‐I, and myoglobin were reduced in the mimic‐treated group under hypoxia. Proteome profiling identified changes in TIMP‐1, M‐CSF, JE, and SDF‐1 expression, further supporting the role of miR‐30c‐1‐3p in protecting cardiomyocytes against hypoxia‐induced injury and apoptosis.

Summary/Conclusion : The findings of this study highlight the protective role of exosome‐derived miR‐30c‐1‐3p in mitigating hypoxia‐induced apoptosis and cardiac injury in primary mouse cardiomyocytes. This research underscores the therapeutic potential of miRNA‐based interventions in manageing myocardial infarction and other cardiac conditions.

Funding : This work was supported by the Basic Science Research Program through the National Research Foundation of Korea NRF, funded by the Ministry of Education (NRF‐2020R1I1A2073643; funder, Sang Woo Kim) and the NRF grant funded by the Korea government (MSIT) (NRF‐2021R1A2C1005280; funder, Jung‐Won Choi).

Defective mitochondrial clearance in cardiomyocytes via impaired macrophage‐cardiomyocyte signalling contributes to adverse remodelling and left ventricular dysfunction in reperfused myocardial infarction

Bushra Saleem, Shah Bahrullah Shah, Anna Roth, Elke Winterhager, Tienush Rassaf, Ulrike Hendgen‐Cotta

University Hospital Essen, Germany

Introduction : Heart failure (HF) following reperfused myocardial infarction (repAMI) remains a major clinical challenge, with mitochondrial dysfunction playing a central role in disease progression. Although timely reperfusion is the optimal treatment for acute myocardial infarction (AMI), the risk of HF persists due to unresolved cellular damage. Mitochondria, vital for cellular energy production, are severely affected during repAMI and their impaired clearance could exacerbate cardiac injury. While cardiac tissue‐resident macrophages are essential for removing dysfunctional mitochondria via extracellular vesicles (EVs), the mechanisms behind accumulation of damaged mitochondria remain poorly understood. This study investigates how defective communication of macrophage‐cardiomyocyte together with mitochondrial debris clearence machinery contributes to mitochondrial accumulation, autophagy blockade, and adverse cardiac remodelling in repAMI.

Methods : Using electron microscopy, we identified damaged mitochondria in left ventricular biopsies from HF patients and detected defective mitochondria within EVs in the process of secretion by cardiomyocytes as well as in the cardiac interstitium.

Results : In a mouse model of repAMI, we employed immunofluorescence and confocal microscopy to track the dynamics of cardiac‐resident macrophages and their longitudinal depletion, revealing a corresponding accumulation of dysfunctional mitochondria over time. Additionally, we explored the presence of molecular pathways regulating mitochondrial secretion and degradation, uncovering disruptions in proteostasis and metabolic fitness.

Summary/Conclusion : Our findings from the HF patients biopsies and immunofluorescence in murine repAMI model suggest that impaired macrophage‐mediated clearance of damaged mitochondria via EVs leads to mitochondrial accumulation and left ventricular dysfunction. These results underscore the critical role of macrophage‐cardiomyocyte crosstalk across the basal lamina in maintaining cardiac function and suggest that targeting this signalling axis could offer novel therapeutic strategies to prevent heart failure post‐reperfusion. Future studies will focus on identifying specific molecular mediators of this mitochondrial debris removal to inform potential interventions.

MicroRNA sequence in plasma‐derived extracellular vesicles from spontaneously hypertensive rats

Presenter: Yusei Fujioka

Kitasato University, Japan

Introduction : Extracellular vesicles (EV) mediate cell‐to‐cell communication via transferring various molecules, such as microRNA (miRNA). We previously demonstrated that plasma‐derived EV from spontaneously hypertensive rats (SHR), an animal model of human essential hypertension, increased systemic blood pressure in normotensive Wistar Kyoto rats (WKY). In the present study, we performed a comprehensive analysis of miRNA contained in plasma‐derived EV from SHR and WKY to identify miRNA and their target genes that regulate the pathogenesis of hypertension.

Methods : EV were isolated from plasma of 9‐week‐old male WKY or SHR using the size‐exclusion chromatography method. After miRNA was extracted from isolated EV, miRNA‐sequence was conducted. We then conducted mRNA expression analysis in the isolated tissues of SHR and WKY by using quantitative real‐time PCR.

Results : In plasma‐derived EV from SHR, we found that three miRNAs were significantly upregulated and six miRNAs were significantly downregulated compared with those from WKY. Among the target genes of the upregulated miRNAs, the mRNA expression of several genes was downregulated in the isolated brain, heart, and kidney of SHR.

Summary/Conclusion : We revealed that plasma‐derived EV from SHR had different profiles of miRNAs compared with those of WKY. We also found that the mRNA expression of the target genes of miRNAs contained in plasma‐derived EV from SHR was downregulated in the tissues regulating systemic blood pressure. Additional research may contribute to identifying novel pathological mechanisms underlying essential hypertension.

NAT10 targeting breaks tubular epithelial cell‐exosome‐endothelial cell kill chain to suppress vascular rarefaction and AKI‐CKD transition

Presenter: Zheng Tong

The Third‐affiliated Hospital of Sun Yat‐sen University, China (People's Republic)

Introduction : Chronic kidney disease (CKD), derived from acute kidney injury (AKI), causes irreversible renal failure. Therefore, inhibiting AKI‐CKD transition is clinically vital. Vascular rarefaction, induced by apoptosis of vascular endothelial cell (VEC), is a key contributor of AKI‐CKD transition. Unfortunately, the mechanism of vascular rarefaction following AKI remains unknown.

Methods : The in vitro model of TEC(tubular epithelial cell)‐VEC communication was used to discover the role of exosome in TEC‐VEC kill chain. In vitro screen with epigenetic inhibitor library was performed to identify the decisive mediator of TEC‐exosome‐VEC kill chain. Conditional knockout mice was used to verify the in vivo role of NAT10 in vascular rarefaction and AKI‐CKD transition. acRIP‐seq and RNA‐seq were applied to investigate the mechanisms of NAT10.

Results : The injured TEC induced VEC apoptosis depending on exosomes. More importantly, targeting NAT10 significantly breaks the TEC‐exosome‐VEC kill chain. Conditional NAT10 depletion in TEC reversed vascular rarefaction and AKI‐CKD transition. Mechanistically, NAT10 constructed the TEC‐exosome‐VEC kill chain via strongly elevating the exosomal abundance of THBS1 and FBN1, the pro‐apoptotic protein of VEC. Specifically, NAT10 upregulated THBS1 and FBN1 via catalyzing mRNA ac4C. Meanwhile, NAT10 favoured package of FBN1 protein into exosomes via catalyzing protein acetylation. In addition, we found that TGF‐β and stiffening stabilized mRNA and protein of NAT10, upregulating NAT10 in succession after AKI.

Summary/Conclusion : After AKI, the injured TEC causes vascular rarefaction and AKI‐CKD transition via exosomes. NAT10, which is upregulated by TGF‐β and stiffening in injure TEC, initiates and strengthens the TEC‐exosome‐VEC kill chain via increasing exosomal abundance of THBS1 and FBN1. NAT10 orchestrates mRNA ac4C modification and protein acetylation to upregulated THBS1 and FBN1 and to favour FBN1 loading into exosomes. Therefore, targeting NAT10 with Remodelin is a promising strategy for vascular rarefaction in AKI‐CKD transition.

Funding : National Natural Science Foundation of China (No. 82170771, No. 82370756, No. 82100799)

Impact of high glucose and diabetes on extracellular vesicle‐associated mitochondrial DNA

Agafe Bless Reyes 1,2 , Fengxia Xiao 2 , Mayra Trentin‐Sonoda 2 , Marcelo Ninalaya 3,4 , Vernon Dolinsky 3,4 , Dylan Burger 1,2

1 University of Ottawa, Canada, 2 The Ottawa Hospital Research Institute, Canada, 3 The Children's Hospital Research Institute of Manitoba, Canada, 4 University of Manitoba, Canada.

Introduction : Maternal vascular dysfunction is an early feature of gestational diabetes mellitus (GDM) that persists post‐delivery and may contribute to future risk of cardiovascular disease. Our Laboratory has shown that maternal vascular injury—determined by elevated endothelial extracellular vesicles (EV)—is predictive of adverse pregnancy outcomes in type 1 diabetes. Here, we assessed the effects of the diabetic microenvironment and GDM on vascular health, focusing on the endothelial mitochondria and EV composition.

Methods : Mitochondrial proteins were assessed in high glucose (HG)‐treated human umbilical vein endothelial cells (HUVECs) and in a rat model of diet‐induced GDM (Pereira, 2015, J Physiol) by Western blot. Levels of EV‐associated mitochondrial DNA (mtDNA) from culture media and rat plasma were determined via qPCR.

Results : In vitro studies showed reduced protein levels oxidative phosphorylation Complex 2 (CII‐SDHB, p < 0.001, n = 8) in HG‐treated HUVECs. In addition, mitochondrial single‐stranded DNA binding protein (SSBP1) was increased in HG‐treated cells ( p < 0.001, n = 5). This mitochondrial injury in HG‐treated HUVECs was accompanied by approximately 2.5‐fold increases in levels of EV‐associated mtDNA NADH dehydrogenase 2 (ND2, p < 0.05, n = 8) and Cytochrome c oxidase subunit 2 (COX2, p < 0.05, n = 8) as well as increased EV‐associated SSBP1 protein levels. Decrease in CII‐SDHB levels in HG‐treated cells persisted even after a return to normal glucose conditions for two subsequent passages ( p < 0.01, n = 4). In vivo studies showed that GDM rats exhibited decreased kidney protein levels of Complex 1 (CI‐NDUFB8, p < 0.01, n = 6) and CII‐SDHB ( p < 0.01, n = 6) as well as increased protein levels of Complex 5 (CV‐ATP5A, p < 0.05, n = 6) compared with healthy rats at 3rd trimester of pregnancy. Late gestational levels of circulating EV‐associated mtDNA in GDM rats were increased compared with healthy rats ( p < 0.01, n = 10).

Summary/Conclusion : These data suggest that vascular dysfunction is present in diabetic microenvironment and in rodent GDM, which is accompanied by mitochondrial injury and increased EV‐associated mtDNA levels. Future studies may focus on mitigating endothelial mitochondrial injury in GDM to reduce GDM‐associated maternal risks.

Funding : Canadian Institutes of Health Research (CIHR PJT‐186237)

Effect of hypercholesterolemia on circulating and cardiomyocyte‐derived extracellular vesicles

Presenter: Szabolcs Hambalkó

Semmelweis University, Hungary

Introduction : Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases (CVDs) via various mechanisms that are yet not properly uncovered. Analysing how HC dysregulates extracellular vesicles (EVs) can lead to further understanding in the connection between HC and CVDs. Our aim is to assess whether and how HC affects circulating and cardiomyocyte (CM)‐secreted EVs and how can these changes affect the myocardium.

Methods : Circulating EVs were isolated with density gradient ultracentrifugation followed by size exclusion chromatography using Vezics system from male Wistar rats fed with high‐cholesterol or control chow. Plasma and EV metabolome was analysed using a Biocrates MxP Quant 500 kit. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant with ultracentrifugation. Samples were analysed with nanoparticle tracking analysis, and atomic force microscopy. Monocyte activation was measured in THP1‐ASC‐GFP cells after treatment with AC16‐EVs. AC16‐EV proteomics was measured with liquid chromatography‐tandem mass spectrometry.

Results : HC diet induced hyperlipidemia in rats and reduced the amount of certain phosphatidylcholines in circulating EVs. Furthermore, plasma EV metabolome showed only minor correlation with that of the plasma. HC treatment significantly increased EV secretion of AC16 CMs but did not affect their elasticity and total phosphatidylcholine concentration. AC16 EVs, regardless of the treatment, did not induce the activation of THP1 monocytes. HC treatment modified AC16 EV metabolome greatly, with 77 enriched proteins and 33 proteins with decreased abundance. No specific enrichment or reduction of any well‐defined molecular pathways was identified among the dysregulated proteins, however some these proteins contribute to tissue remodelling.

Summary/Conclusion : HC greatly affects metabolome of circulating EVs. Furthermore, it induces secretion and modifies proteome of CM EVs. These changes do not affect monocyte activity and may contribute to HC‐induced cardiac remodelling.

Funding : NVKP‐16‐1‐2016‐0017 National Heart Program OTKA‐139105

Subtopic Host‐pathogen & Microbiome 2

The multifaceted routes of host‐pathogen interaction mediated by Staphylococcus aureus extracellular vesicles

Julia Papail 1 , Ligia Prado 1,2 , Nathalie Daniel 1 , Danièle Vassaux 1 , Aurélie Nicolas 1 , Svetlana Chabelskaia 3 , Yann Le Gouar 1 , Nadia Berkova 1 , Julien Jardin 1 , Yves Le Loir 1 , Vasco Azevedo 4 , Éric Guédon 1

1 INRAE, Institut Agro Rennes‐Angers, STLO, France, 2 Institute of Biological and Natural Sciences, Federal University of Triângulo Mineiro, Brazil, 3 Université de Rennes, INSERM U1230, BRM, France, 4 Laboratory of Cellular and Molecular Genetics, Institute of Biological Sciences, Federal University of Minas Gerais, Brazil.

Introduction : Staphylococcus aureus is an opportunistic pathogen with a major impact on animal and human health. Its ability to colonize various hosts and cause a range of diseases is driven by its production of numerous virulence factors. Studies have shown that extracellular vesicles (EVs) produced by this bacterium carry a cargo enriched with virulence factors and participate to pathogenesis, notably by triggering inflammatory responses. Our work aims to understand how S. aureus EVs induce inflammation and influence host cell responses at the molecular and cellular levels.

Methods : EVs from Staphylococcus aureus strain N315 grown in infection‐like conditions were purified by size exclusion chromatography. They were characterized for size, concentration, morphology and content using various techniques (NTA, TEM, western blot, confocal microscopy, LC‐MS/MS, RNAseq). The effect of EVs on the expression of various immune genes in human osteoblast‐like cells (MG‐63) was assessed using RT‐qPCR. The impact of EVs was confirmed with eukaryotic reporter cell lines. EV uptake by MG‐63 was evaluated by confocal microscopy and flow cytometry in presence of various transport inhibitors. The EV content and trafficking inside the cells was followed by confocal microscopy.

Results : We showed that the pro‐inflammatory response triggered by N315 EVs is linked to their capacity to activate the NFkB signalling pathway via recognition by multiple Toll‐like receptors. Indeed, N315 EVs activate and signal through extracellular receptors such as TLR2 and TLR4, the endosomal receptor TLR7 and the cytosolic receptor NOD2. This is due to: i) EV cargo containing bacterial effectors recognized by these receptors, ii) the EV ability to be internalized, mainly by endocytosis, and iii) the endosomal and cytoplasmic release of their cargo into the infected cells. Inhibiting internalization greatly reduces EV signalling, notably on TLR7, which recognizes ssRNA. In agreement, we showed that EVs contain small RNAs with high similarity with human miRNA. Our findings suggest that EVs transport bacterial regulatory RNAs able to mimic host miRNA to modulate human genes expression.

Summary/Conclusion : S. aureus EVs use multiple routes to interact with and modulate host cells, including content delivery (eg miRNA‐like molecules) inside the cells after their internalization.

A non‐canonical activation of the ESCRT machinery is required for Leishmania donovani replication within infected cells

Presenter: Javier Rosero

University of Florida, USA

Introduction : We have previously shown that macrophages infected with L. donovani release extracellular vesicles (EVs) whose composition is modified by infection as compared to EVs from non‐infected cells. Included amongst the molecules that are significantly more abundant in EVs from infected cells, were members of the Endosomal Sorting Complex Required for Transport (ESCRT). Beyond the potential role of the ESCRT machinery in the biogenesis of EVs in Leishmania‐infected cells, we sort to explore the likelihood that the ESCRT machinery plays other roles in Leishmania‐infected cells.

Methods : Towards this end, we tracked the distribution of overexpressed tagged versions of representative members of the ESCRT machinery in Leishmania‐infected cells as compared to non‐infected cells and cells that phagocytosed Zymosan particles or latex beads. Specifically, we tracked TSG101‐GFP, CHMP2b‐mCherry, CHMP4b‐mCherry and the accessory molecule, VPS4a‐mCherry. To address the role of the ESCRT machinery in this infection, we evaluated infections in cells after knockdown of ESCRT molecules.

Results : We observed that all representative members of the ESCRT machinery were recruited to parasitophorous vacuoles that harbor Leishmania parasites (LPVs). This contrasts with limited if any recruitment of those same molecules to phagosomes that harbored either Zymosan particles or latex beads. We then determined that the expression of the phosphoinositide PI(3,4)P2 on LPVs was required for the recruitment of the ESCRT complex 3 members to the LPV. Knockdown of TSG101 did not reveal any discernable phenotype after infection with L. donovani parasites. In contrast, the knockdown of ALIX had an unexpected and significant effect on the infection. Fewer parasites were found in cells after ALIX knockdown. Moreover, in the few ALIX knockdown cells that harbored parasites, their LPVs were distended. These distended LPVs contained numerous parasites, which is dramatically different from the situation in wild type macrophages where each LPV harbors a single parasite and where LPVs divide enigmatically when parasites divide.

Summary/Conclusion : The dependency of LPV division on the availability of ALIX suggests that LPV and parasite division are dependent on the non‐canonical activation of the ESCRT machinery. This constitutes a novel function for the ESCRT machinery, which can be exploited for the control of Leishmania infections.

Funding : UF funds

Unraveling the pathogenic and proinflammatory potential of extracellular vesicles secreted by Bacillus cereus

Astrid Laimer‐Digruber, Tanja V. Edelbacher, Caroline Lassnig, Silvio Kau‐Strebinger, Belinda Maw, Karoline Kollmann, Birgit Strobl, Monika Ehling‐Schulz

University of Veterinary Medicine Vienna, Austria

Introduction : Bacillus cereus is a Gram‐positive spore‐forming bacterial pathogen that is well‐known for its ability to cause food poisoning [1]. Notably, this opportunistic pathogen is gaining increasing prominence as causing agent of non‐GI related illnesses, including systemic and local infections [2]. Recently, we showed that B. cereus secretes biologically active extracellular vesicles (EVs). In contrast to EVs of Gram‐negative bacteria, EVs in Gram‐positive bacteria are far less studied and their role in bacterial (patho)biology is largely unknown. By purifying and characterizing B. cereus EVs we could show that EVs allow to deliver its virulence factors in a protected manner to host cells [3].

Methods : Bacterial EVs were purified by employing differential centrifugation. In addition, EV‐preparations were characterized by transmission electron microscopy, nanoparticle tracking analysis and flow‐cytometry. We further analysed EV‐mediated pathogenicity using in vitro and in vivo approaches. Application of EVs on bone‐marrow derived macrophages (BMDMs) resulted in dose‐dependent release of proinflammatory cytokines and cell‐damage. By employing confocal microscopy, we were also able to closely follow the uptake of EVs with BMDMs.

Results : Systemic application of B. cereus EVs in a murine mouse model—mimicking bacterial EV release during systemic infections—led in a time and content dependent manner to a massive release of proinflammatory cytokines and inner organ damage.

Summary/Conclusion : In this study, we show for the first time that the presence of B. cereus EVs without its bacterial cells is sufficient to cause systemic inflammatory response symptoms. Therefore, it is anticipated that this research will provide new ways for understanding EV‐contribution of pathogenic Gram‐positive bacteria to systemic bacterial infections, as well as aid in the development of innovative defense mechanisms and novel therapeutic approaches.

Elucidating the mechanisms underlying the ability of pathogenic Acidovorax temperans and its derived outer membrane vesicles to promote lung cancer progression

Presenter: Haneen Abu‐Freih

Ben‐Gurion University of the Negev, Israel

Introduction : Dysbiosis is common in solid tumours, but its direct contribution to tumour development is unclear. Our previous work identified the Gram‐negative Acidovorax temperans as enriched in tumours of smokers and TP53 mutation patients, where it accelerated tumour growth through pro‐inflammatory cell infiltration in the lungs. This study investigates the role of outer membrane vesicles (OMVs) shed by A. temperans in driving inflammatory dynamics and tumourigenesis.

Methods : OMVs were isolated using serial centrifugations, filtrations, and an OptiPrep density to remove non‐vesicular elements. Purified samples were characterized using NTA, electron microscopy, and proteomics. Confocal microscopy assessed OMV cellular uptake. OMVs‐treated cells were analysed for the expression of cytokine genes using qRT‐PCR. We optimized an in vivo protocol that introduced OMVs into the mice lungs by intranasal administration, confirmed by lung ex vivo imaging to monitor OMV delivery. Immune dynamics of OMV‐treated lungs were examined through CyTOF analysis. RNA‐Seq analysis of A. temperans and OMVs was conducted to characterize differentially packaged sRNAs.

Results : A. temperans OMVs are taken up by A549 lung cancer cells and THP‐1 macrophages, inducing strong pro‐inflammatory response in both cell lines. In addition, macrophages exposed to A. temperans OMVs overexpressed SIRP‐α, linked to tumour immune escape and progression. In vivo, intranasal administration delivers the OMVs directly to the lungs where they lead to an increased secretion of pro‐inflammatory cytokines. Flow cytometry revealed selective uptake of OMVs by CD11b+ myeloid cells in mouse lungs. CyTOF analysis of OMVs‐treated lungs revealed that OMVs increased infiltration of dendritic cells, macrophages, and T helper 17 cells. OMV sRNA‐seq identified a unique RNA signature including various fragmented tRNAs that are selectively loaded in OMVs and are predicted to target host mRNAs.

Summary/Conclusion : Interactions between OMVs shed by A. temperans and lung cancer microenvironment lead to accelerated, inflammation‐based tumourigenesis.

Impact of Extracellular Vesicles from Food Preservative‐Resistant Yeast on the Host Intestinal Environment

Bao‐Hong Lee 1 , Tang‐Long Shen 2 , Yi‐Wen Wang 3 , Tzu‐Hsiang Lin 3 , Shu‐Hua Yang 3 , Wei‐Hsuan Hsu 3

1 Department of Horticulture, National Chiayi University, Chiayi, Taiwan. 2 Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan. 3 Department of Food Safety / Hygiene and Risk Management, National Cheng Kung University, Tainan, Taiwan.

Introduction : Food preservatives prevent microbial growth in products but may unintentionally promote antimicrobial‐resistant (AMR) microorganisms. Given the rising public health concerns over AMR, understanding the impact of food‐derived microbial components on gastrointestinal health and immune responses is crucial. Our previous research revealed that yeast from pickled vegetables can acquire resistance to the preservative benzoic acid (BA) through yeast‐derived extracellular vesicles (EVs). This study investigates the effect of EVs from BA‐resistant yeast on gut barrier function and inflammation.

Methods : C57BL/6 germ‐free mice were divided into three groups: a control group, a group receiving EVs from non‐resistant Pichia kudriavzevii (nPK), and a group receiving EVs from BA‐resistant P. kudriavzevii (rPK), with five mice per group. The experimental groups received 10 10 particles/mouse/day by oral gavage, while the control group received 200 µL/mouse/day of sterile water.

Results : After 12 weeks of oral administration, protein analysis of the small intestine showed significant increases in defensin, SPIB, REG‐3B, MUC5AC, MUC5B, MUC13, and toll‐like receptors (TLRs) in rPK‐treated mice compared to controls and the nPK group. In contrast, MUC2, MUC3, and ZO‐1 protein levels were significantly reduced in the rPK group, indicating compromised gut barrier function and a pro‐inflammatory state.

Summary/Conclusion : This study highlights the pivotal role of EVs as mediators of microbial influence on gut health, especially concerning AMR due to food preservatives. The observed impact of EVs from BA‐resistant yeast on gut barrier integrity and inflammation suggests potential health risks. Further research into EV‐mediated interactions between gut microbiota and the host could provide new strategies for mitigating AMR risks and protecting gastrointestinal health.

Funding : This research work and subsidiary spending were mainly supported by National Science and Technology Council (NSTC) in Taiwan under Grant No. NSTC 112‐2320‐B‐006‐057‐MY3

Subclinical mastitis induces concentration and proteomic cargo changes in CD81‐positive milk EVs

Mara D. Saenz‐de‐Juano 1 , Giulia Silvestrelli 1,5 , Vadim Krivitsky 2,4 , Adva Krivitsky 2,4 , Sibilla Sander 3 , Jean‐Christophe Leroux 2 , Jörn Dengjel 3 and Susanne E. Ulbrich 1

1 Institute of Agricultural Sciences, ETH Zurich, Switzerland. 2 Institute of Pharmaceutical Sciences, ETH Zurich, Switzerland. 3 Department of Biology, University of Fribourg, Switzerland 4 Acytronix GmbH, Switzerland. 5 Institute of Science and Technology Austria, Austria.

Introduction : Subclinical mastitis is a form of mammary gland inflammation that occurs without visible clinical symptoms, explaining its high prevalence and why it can easily spread to other animals in the herd during routine milking. Despite its hidden nature, it negatively impacts milk yield and quality, reduces reproductive performance, and compromises animal welfare. Many studies have demonstrated that mastitis induces changes in milk extracellular vesicles (EVs) cargo. However, common milk EV isolation techniques do not allow the purification of specific milk EV subpopulations. In this study, we used a novel portable microstructured electrochemical device (PMED) to isolate specific CD81‐positive EVs and to assess the differences in particle concentration and proteomic cargo in healthy quarters and quarters affected by subclinical mastitis.

Methods : Milk samples were manually obtained from 3 dairy cows with subclinical mastitis. Based on the somatic cell count (SCC), two‐quarters of each cow were selected and classified as either inflamed (high SCC) or healthy (low SCC). Milk fat, cells, and caseins were removed using differential centrifugation, acid precipitation, and filtration. Then, EVs were extracted from skimmed milk using both ultracentrifugation (UC) and the PMED modified to isolate CD81‐positive EVs. Particle size and concentration were obtained using Nanoparticle Tracking analysis (NTA), and proteomic analysis of the EV cargo was performed using bottom‐up proteomics coupled with Data‐Independent Acquisition (DIA).

Results : Subclinical mastitis increased the concentration of CD81‐positive milk EVs. When comparing high and low SCC milk EVs, CD81‐positive EVs showed a distinct cargo associated with inflammatory responses, complement cascade activation, and plasma lipoprotein regulation. Our results confirm the role of milk EVs in mastitis pathology and emphasize the importance of examining specific milk EV subpopulations.

Summary/Conclusion : The observed alterations in protein cargo between high and low SCC quarters underscore milk EVs' potential diagnostic and prognostic utility in mastitis management. The use of PMED represents a promising advancement for studying milk EV subpopulations.

Effect of lipid properties on decoy activity of bacterial vesicles against the antimicrobial peptide, LL‐37

Presenter: Mahsa Khodabakhshmajd

Lehigh University, USA

Introduction : Antimicrobial peptides are short, cationic, amphiphilic peptides that bind electrostatically to the negatively charged bacterial surface. Recently, evidence has emerged that bacterial extracellular vesicles, with their surface properties that resemble the surface of the parent bacterium, act as “decoys”, preventing the antibacterial activity of the peptides. The specific factors that regulate this decoy activity remain unclear, however.

Methods : We compared the decoy activity of vesicles produced by several Gram‐negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Aggregatibacter actinomycetemcomitans) against LL‐37. To isolate the role of specific membrane properties, including charge and saturation, in this decoy activity, we compared our results to those obtained using liposomes of controlled lipid compositions.

Results : We observed that all of the tested vesicles were protective against LL‐37 to some extent, with vesicles produced by P. aeruginosa displaying the strongest decoy activity. Liposomes with increased amounts of negatively charged phosphatidylglycerol (PG) were more protective than those composed of neutrally charged lipids; however, charge alone did not fully explain the results obtained with the bacterial vesicles, as P. aeruginosa were not the most highly charged vesicles used. In addition to charge, we have observed that lipid saturation and the resulting membrane fluidity impact the binding of LL‐37 and resulting decoy activity.

Summary/Conclusion : Together, these results demonstrate that both lipid charge and saturation influence the ability of LL‐37 to bind to vesicles and therefore regulate the decoy activity of the vesicles.

Funding : This work was supported by the United States National Institutes of Health and the Pennsylvania Department of Health.

Nanoplastics Indirectly Compromise Lettuce Growth in Hydroponic Systems via Microbial Extracellular Vesicles Derived from Curvibacter Fontanus

Wei‐Hsuan Hsu 1 , Tang‐Long Shen 2 , Bao‐Hong Lee 3

1 Department of Food Safety / Hygiene and Risk Management, National Cheng Kung University, Tainan, Taiwan. 2 Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan. 3 Department of Horticulture, National Chiayi University, Chiayi, Taiwan.

Introduction : Recent studies confirm that nanoplastics (NP) cause severe microbial imbalances in various ecosystems, significantly affecting microbial diversity and abundance. Hydroponic systems vital for lettuce production are increasingly threatened by NP contamination in irrigation water and this issue is gaining global attention. This study investigates microbial species in hydroponic irrigation water altered by NP exposure and their impact on lettuce growth.

Methods : Lettuce (Lactuca sativa L.) was cultivated in two hydroponic models to assess the effects of NP treatments and Curvibacter fontanus‐derived extracellular vesicles (EVs). Model‐1 involved continuous water conditions, while Model‐2 incorporated water changes every four days, both using a nutrient solution with specific concentrations of minerals and NP. Over ten days, growth metrics such as leaf dimensions and root length were recorded. Microbial assays involved centrifuging water samples for DNA extraction and sequencing of the 16S rRNA gene. The uptake of NP by C. fontanus was visualized using confocal microscopy. EVs were isolated from cultured C. fontanus, characterized by size and morphology, and applied to hydroponically grown lettuce to evaluate their effects on oxidative stress, measured by enzyme activity and malondialdehyde levels.

Results : While NP did not directly harm or accumulate in lettuce, significant changes in water parameters and microbial communities were observed, particularly an increase in Curvibacter fontanus abundance. Inoculation of sterile irrigation water with NP and C. fontanus led to lettuce mortality, suggesting C. fontanus as a critical mediator. Furthermore, extracellular vesicles (EVs) isolated from C. fontanus, treated with NP, were shown to suppress leaf development, growth, antioxidant defenses, and lettuce survival.

Summary/Conclusion : This study concludes that NP‐induced microbial shifts, particularly involving C. fontanus EVs, indirectly harm hydroponic lettuce production.

Funding : This research work and subsidiary spending were mainly supported by the National Science and Technology Council (NSTC) in Taiwan under Grant No. NSTC 112‐2628‐B‐415‐001‐MY3 (Young Scholar Fellowship Program).

Systemic transmission of CEVd (citrus exocortis viroid) via host plant derived extracellular vesicles

Presenter: Tang‐Long Shen

National Taiwan University, Taiwan (Republic of China)

Introduction : Viroids are a type of plant pathogens solely consisted of naked, circular single‐stranded RNAs that do not encode any protein. Citrus exocortis viroid (CEVd) enables infecting not only citrus but also many Solanaceae spp. Neverthelss, the pathogenesis and the systemic symptomatic mechanistic nature of CEVd are largely unknown.

Methods : To clarify whether CEVd enables transmission through tomato‐derived extracellular vesicles (tEVs), we isolated tEVs from stems or leaves of tomato plants at six wpi or mock.

Results : After measurement of the isolated tEV particle concentration, protein, and RNA amounts, as well as examination by transmission electron microscopy (TEM), the CEVd RNA genome was detected in the CEVd‐infected tEVs but not in the mock, implicating that CEVd might be able to transmit in the host plant via tEVs. Additionally, tEVs derived from CEVd‐infected leaves bear several catabolic and defense pathway‐associated proteins, including catalase, subtilisin‐like protease, and pathogenesis‐related (PR) proteins at a relatively high expression level compared to the mock counterpart. Furthermore, to speculate the pathogenicity of the CEVd‐contained tEVs, we re‐inoculated the CEVd‐infected tEVs on tomatoes and subsequently found significant decreases in the biomass and the leaf area at 4 wpi compared to the mock control.

Summary/Conclusion : Our study provides first and novel insight for EV‐mediated transmission of CEVd within infected tomatoes attributed to the systemic symptom development during CEVd pathogenesis.

Funding : NSTC 112‐2313‐B‐002‐024‐MY3

Influence of Protein Corona Removal on Extracellular Vesicles from Aspergillus fumigatus: Implications for Immune Evasion and Fungal Pathogenesis

Presenter: Lucas F. B. Nogueira

University of São Paulo, Brazil

Introduction : Extracellular vesicles (EVs) produced by Aspergillus fumigatus play a pivotal role in fungal communication and host‐pathogen interactions, especially during pulmonary infections. These vesicles acquire a protein corona when exposed to biological fluids, which significantly alters their surface properties and biological behaviour. Understanding the impact of this protein corona on EV functions, particularly in interactions with immune and fungal cells, is critical for elucidating their role in pathogenesis.

Methods : EVs were isolated from Aspergillus fumigatus cultures and subjected to a high ionic strength solution followed by ultracentrifugation to remove the protein corona. Nanoparticle tracking analysis (NTA) was used to assess the size distribution of the EVs before and after protein corona removal. Scanning electron microscopy (SEM) was employed to observe changes in morphology, and zeta potential measurements were taken to evaluate surface charge alterations. Protein content and composition were analysed through BCA assay and SDS‐PAGE. Functional assays were conducted to examine the uptake of EVs by macrophages and their interaction with fungal cells.

Results : NTA revealed that the hydrodynamic diameter of EVs was initially around 130 nm, but after removal of the protein corona, a new distribution appeared, with vesicles measuring greater than 200 nm. SEM images confirmed that EV morphology was largely maintained, though subtle shape changes were observed. The zeta potential shifted from ‐12.4 mV to ‐2.3 mV after corona removal, indicating substantial changes in surface protein composition. Protein quantification and SDS‐PAGE analysis showed significant differences between native and corona‐free EVs. Functionally, EVs without their protein corona exhibited reduced uptake by macrophages, suggesting the corona's importance in immune recognition. In contrast, corona‐free EVs displayed altered interactions with fungal cells, potentially influencing biofilm formation and fungal virulence.

Summary/Conclusion : The protein corona significantly modulates the biological activity of Aspergillus fumigatus EVs, influencing both immune recognition and fungal interactions. Its removal resulted in altered EV size, surface charge, and reduced macrophage uptake, while enhancing interactions with fungal cells. These findings highlight the role of the protein corona in immune evasion and fungal pathogenesis, offering potential avenues for developing therapeutic strategies targeting these processes in Aspergillus fumigatus infections.

Funding : This work was supported by FAPESP (2024/00403‐2).

Eimeria falciformis proteins from host plasma‐derived extracellular vesicles

Joshua Seun Olajide 1,2 , Olusanya Olasehinde 3 , Cai Jianping 1

1 Chinese Academy of Agricultural Sciences, China, 2 Obafemi Awolowo University, Nigeria, 3 University of Benin, Nigeria Cai Jianping is senior co‐author

Introduction : During host‐parasite interactions, extracellular vesicles (EVs) are secreted by intracellular protozoan parasites and parasite‐infected host cells. Secreted EVs usually contain proteins with important roles in disease outcome. Eimeria falciformis clinical manifestation is prominent on the 7th day post infection, coinciding with oocyst shedding and completion of parasite life cycle. This study examined plasma‐derived EVs with parasite‐proteins in E. falciformis‐infected mice.

Methods : Blood samples were collected from E. falciformis‐infected mice (7 days post‐infection) and from uninfected host. EVs were isolated from the host plasma using serial centrifugation. 5 mL of the supernatant was equilibrated with PBS (40%) and EDTA. Centrifugations were followed by ultrafiltration(0.45 µm) and ultracentrifuged at 120,000 × g for 18 h at 4°C. The pellet was resolved on iodixanol gradient solutions. Seven gradient layers were pooled, diluted with PBS and washed for 4 h at 120,000 × g . EVs were characterized by TEM and NTA, and the protein composition were profiled by LC–MS/MS.

Results : Identified EVs were spherical to circular in shapes. 53 and 154 proteins were identified from the infected and uninfected hosts respectively. Out of these, 31 host‐derived proteins were common to both groups. Common EVs markers were HSP70, 14‐3‐3 protein and histones. E. falciformis‐infected host plasm‐derived EVs had ras‐related protein and MHC class II as specific EVs markers. Identified parasite proteins were 69. Including synthesis, regulation of eukaryotic cellular processes, chromatin binding and stress‐response proteins. Enrichment analysis indicated that these proteins were from extracellular exosomes, vesicles, region and organelles, while response to endogenous stimulus, toxic substance and chemicals were significant biological processes of EV proteins isolated from infected host. Major molecular function were protein and enzyme binding. Antigen processing and presentation and protein processing were significant annotated genes. Protein‐protein interaction analysis showed that parasite proteins are in complex formation with overlapping genes.

Summary/Conclusion : Identified parasite EV proteins are involved in parasite survival and pathogenesis while EV proteins from the host indicated oxidative stress. Nonetheless, identified proteins are important diagnostic and pathogenic molecules during coccidiosis.

Funding : Key Technologies Research and Development Program (Key Technologies R&D Program) 2017YFD050040320, Innovative Special Project of Agricultural Science and Technology (Grant No. CAAS‐ASTIP2014‐LVRI‐09)

Investigating amyloid‐β effects on extracellular vesicle secretion and characteristics using nanofluidic channel microscopy

Presenter: Vesa Halipi

Chalmers University of Technology, Sweden

Introduction : Aggregation of the amyloid‐β (Aβ) peptide into amyloid fibrils is a central pathogenic feature in Alzheimer's disease. This process is intricately connected to the endolysosomal system, where defects can lead to accumulation of Aβ and other disease‐related proteins. Endolysosomal dysfunction in neurodegenerative disorders has been shown to involve exosomes, a type of extracellular vesicle (EV) released via endosomal trafficking, which may modulate amyloid formation and facilitate cell‐to‐cell propagation. We aim to leverage nanofluidic channel microscopy to investigate how Aβ(1‐42) exposure affects EV secretion and characteristics in vitro to elucidate EV roles in AD progression.

Methods : We have investigated how Aβ(1‐42) uptake is affected by human neuroblastoma SH‐SY5Y EVs using confocal microscopy and flow cytometry. Furthermore, we utilize nanofluidic channel microscopy to monitor EV secretion dynamics and alterations in EV characteristics in response to Aβ exposure, using chemically engineered EVs expressing a fluorescent CD63 EV marker. This approach allows for direct investigation of Aβ(1‐42)‐EV interactions at the single‐particle level, requiring low sample volumes and minimal to no EV isolation.

Results : EVs enhance uptake of the aggregation‐prone Aβ(1‐42) peptide in human neuroblastoma SH‐SY5Y cells, with an even greater effect on uptake observed for EVs from SH‐SY5Y cells previously exposed to Aβ(1‐42). Initial findings with nanoparticle tracking analysis and flow cytometry also suggest an increase in EV secretion in Aβ‐exposed cells, supporting the hypothesis that EV release is modulated in AD contexts, potentially enhancing pathogenic Aβ spread. We have set up a pipeline utilizing nanofluidic channel microscopy to achieve high‐resolution single‐particle profiling of EVs, enabling precise characterization of EV size, concentration, and populations possibly containing Aβ(1‐42).

Summary/Conclusion : This study shows how EVs influence Aβ(1‐42) uptake and introduces nanofluidic channel microscopy as an innovative approach for characterizing EV release and morphology in response to Aβ, as well as detailing Aβ(1‐42)‐EV interactions. These findings deepen our understanding of EV roles in AD pathology and provide potential therapeutic insights into targeting EV‐related Aβ propagation.

Bacteria‐derived extracellular vesicles: new players in host‐pathogen interaction

Presenter: Marco Catania

University of Siena / University of Catania, Italy

Introduction : Bacterial Extracellular Vesicles (bEVs) are membranous structures released by both gram‐negative and gram‐positive bacteria. bEVs serve as an additional means of communication with host cells and other bacteria. They play a critical role in host‐pathogen interactions, transferring resistance and virulence factors and directly inducing host immune responses. This is relevant given the global health challenge posed by infections from antibiotic‐resistant bacteria, with resistant Enterococcus strains greatly contributing to mortality rates. Gaining insight into these novel mechanisms of interaction is thus essential for developing new therapeutic strategies and enhancing infection management.

Methods : bEVs were isolated from three Enterococcus strains: E. faecalis vancomycin‐susceptible (VSE), E. faecalis vancomycin‐resistant (VRE), and E. faecium from clinical samples. Strains were inoculated in DMEM, grown overnight at 37°C and harvested upon reaching the early exponential phase, at approximately 18 h. Cultures were centrifuged at 8000 × g for 10 min, filtered (0.22 µm), and ultracentrifuged at 100,000 × g for 75 min. The pellets were washed in PBS and ultracentrifuged again at the same speed. Next, bEVs were analysed for size and concentration via Dynamic Light Scattering and Nanoparticle Tracking Analysis. DNA from bEVs was then isolated, quantified, and sequenced using Illumina MiSeq.

Results : The analysis of size and concentration indicated significant quantitative differences in bEVs among the strains. Clinical samples and VSE produced more bEVs than VRE, and these vesicles were significantly larger than VRE‐bEVs. Notably, vesicles from clinical sample contained the highest DNA levels, while VRE‐bEVs, despite their lower abundance, held more DNA than VSE‐bEVs. Interestingly, DNA sequencing results revealed a concordance between the resistome and virulome of clinical strain and VRE cells with their respective vesicles.

Summary/Conclusion : The differences among Enterococci in terms of size and concentration suggest that bEVs production is influenced by their strain of origin. Differences in DNA quantification and the concordance between cells and vesicles resistome and virulome suggest a possible involvement of bEVs in infection and the existence of specific cargo loading mechanisms. The potential role of bEVs in host‐pathogen interactions will be further investigated by functional assays in co‐culture models integrated with omics analysis.

Proteomic alterations are associated with bacterial ribosomal protein expression in human vesicles under various antibiotic treatments in Staphylococcus aureus‐spiked blood

Dapi Menglin Chiang 1,2,7 , Michael W. Pfaffl 1 , Gustav Schelling 3 , Agnes Meidert 3 , Florian Brandes 3 , Christina Ludwig 4 , Susanne I. Wudy 4 , Benedikt Kirchner 1 , Mia S.C. Yu 1 , Christian Zenner 5 , Laurent Muller 6,7 and Marlene Reithmair 2

1 Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich (TUM), 85354 Freising, Germany 2 Institute of Human Genetics, University Hospital, LMU Munich, 80336 Munich, Germany 3 Department of Anesthesiology, University Hospital, LMU Munich, 81377 Munich, Germany 4 Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich (TUM), Freising, Germany 5 Intestinal Microbiome, ZIEL—Institute for Food & Health, School of Life Sciences, Technical University of Munich (TUM), Freising, Germany 6 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Basel, Switzerland 7 Department of Biomedicine, University of Basel, Switzerland

Introduction : Sepsis is a critical global health issue, causing 20% of annual deaths, with 71% linked to antibiotic‐resistant infections. Timely antibiotic treatment and bacterial detection in blood are essential for patient outcomes. Bacterial membrane vesicles (bMVs), ranging from 20 to 400 nm, carry proteins and virulence factors that modulate immune responses and may contribute to organ damage. However, proteomic differences between human extracellular vesicles (EVs) and circulating bMVs under simulated Staphylococcus aureus (SA) infection and antibiotic treatment remain unclear.

Methods : In this study, 5 mL of EDTA‐blood from healthy donors was spiked with SA (MOI: 0.0001), either alone or together with commonly applied antibiotics in various concentrations: Piperacillin‐Tazobactam (Pip‐Tazo: 25, 50, and 100 µg/mL), Vancomycin (6.25, 12.5, and 25 µg/mL), and Moxifloxacin (0.18, 0.73, and 2.9 µg/mL). EVs were isolated using the Miltenyi Biotec Pan EV Isolation Kit (human EVs), followed by incubation of unbound plasma with in‐house coupled 1 µm magnetic beads with anti‐OmpA/GroEL antibodies (bEVs). Staining of human EVs and bMV‐BacBead complexes with CellMask Deep Red (CMD), anti‐PanEV, and anti‐SA antibodies enabled analysis via bead‐based flow cytometry. High‐resolution liquid chromatography‐mass spectrometry (HRLC‐MS/MS) was applied to identify proteins from plasma, human EV, and bMV fractions in antibiotic groups, SA‐only, and MOCK controls.

Results : Flow cytometry showed elevated CMD+ CD45+ PanEV+ SA+ levels on human EVs at the lowest Pip‐Tazo and highest Vancomycin concentrations compared to the SA‐only group, although no significant changes were observed with Moxifloxacin. HRLC‐MS/MS analysis of the human EV fraction identified a cluster of five stable human proteins and two SA ribosomal proteins associated with bacterial infection across all bacterial treatment groups, compared to the MOCK control. Additionally, two SA ribosomal proteins were significantly elevated in the Vancomycin group compared to the SA‐only group. However, only a few proteins were detectable in the bMV fraction.

Summary/Conclusion : In conclusion, bMV‐associated proteins were detected in human EVs. During Vancomycin and Pip‐Tazo treatment, bacterial‐associated EVs were more abundant in circulation than in SA‐only conditions. These findings suggest that bacterial‐associated EVs may contribute to either host defense or aid SA proliferation. Furthermore, the found SA ribosomal proteins could serve as future early sepsis biomarkers.

Bacterial extracellular vesicle production by caries‐relevant Streptococcus mutans biofilms is modulated by collagen glycation

Camila Leiva‐Sabadini 1 , Paula Saavedra 1 , Pablo Berrios 1 , Estefanía Tarifeño‐Saldivia 2 , Christina M.A.P. Schuh 3 , Sebastian Aguayo 1

1 Pontificia Universidad Católica de Chile, 2 University of Concepción, Chile, 3 Universidad del Desarrollo, Chile.

Introduction : Streptococcus mutans is the main bacteria in the development and progression of dental caries, a disease that impacts billions of people globally. Recent research has highlighted important mechanobiological changes in tooth collagen as a result of glycation, yet no studies have researched whether these alterations influence the production of bacterial extracellular vesicles (bEVs) by S. mutans biofilms and promote disease. Thus, this study aimed to conduct a multimodal analysis of oral biofilm‐derived S. mutans bEVs formed on both native and glycated type‐I collagen substrates.

Methods : Coverslips were coated with type‐I collagen and experimental glycation was obtained through incubation with methylglyoxal (MGO) and monitored via autofluorescence quantification for 96 h. Subsequently, S. mutans UA 159 biofilms were grown for 24 h under aerobic conditions and at 37 C on collagen‐coated substrates and bEVs from the biofilms were isolated with a combination of ultrafiltration (Amicon) and ultracentrifugation. All bEVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy, fluorescence microscopy with lypophilic dye staining, atomic force microscopy, and mass‐spectrometry‐based proteomics. Protein enrichment, abundance, and differential expression were analysed across the different bEV groups.

Results : NTA and microscopy analysis demonstrated that bEVs isolated from biofilms growing on both types of collagen surfaces were smaller in size than their counterparts isolated previously from planktonic cultures. No differences in particle yield and protein concentration were found between bEVs from the native collagen and the glycated‐collagen adhered biofilms. However, proteomic analysis demonstrated different cargo signatures among groups and a decreased protein diversity for bEVs isolated from glycated collagen‐bound biofilms, as well as the differential expression of several virulence‐associated proteins in the bEV cargo.

Summary/Conclusion : Collagen‐glycation by MGO induces relevant morphological and protein cargo alterations in S. mutans bEVs that have potential biological and clinical impact on cariogenic biofilm promotion, and may be an important driver for disease development in the oral cavity.

Funding : ANID FONDECYT 1220804

Subtopic Immunology & immunity 2

Small extracellular vesicles protein cargo and their role in micro‐clotting in long COVID

Shadi Hambo 1 , Xuejun Cai 1 , Trishla Adhikari 2 , Marc Gentzel 3 , Kristin Tausche 4,5 , Manuel Gado 2 , Kaladhar Kamalasanan 6 , Bilal Alashkar Alhamwe 7 , Elke Pogge von Strandmann 7 ,

Nikolaos Perakakis 2,8‐9 , Hani Harb 1 , 1 Institute for Medical Microbiology and Virology, University Hospital Dresden, Technical University Dresden, Germany, 2 Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany, 3 Center for Molecular and Cellular Bioengineering (CMCB), Core Facility Mass Spectrometry & Proteomics, Technische Universität Dresden, Germany, 4 Department of Medicine I, University Hospital Dresden, Technische Universität Dresden, Germany, 5 East German Lung Center / Ostdeutsches Lungenzentrum Dresden‐Coswig, Germany, 6 Department of Pharmaceutics, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, India, 7 Institute for Tumor Immunology, Philipps‐University Marburg, Germany, 8 Paul Langerhans Institute Dresden (PLID), Helmholtz Center Munich, University Hospital and Faculty of Medicine, TU Dresden, Germany, 9 German Center for Diabetes Research (DZD e.V.), Germany

Introduction : It has been demonstrated that epithelial cells‐derived extracellular vesicles (EVs) protein content play a significant role in the high coagulation rate in patients with COVID19, leading to thrombo‐inflammation. furthermore, the development of microclots is considered a signature of long COVID sequela. In this study, we aimed to investigate the role of EVs in the pathogenesis of microclots in long COVID.

Methods : We isolated small EVs (sEVs) (< 200 nm) from plasma of either healthy (HC) probands or long COVID (LC) patients using size exclusion chromatography. sEVs were characterized using tunable recessive pulse sensing, electron microscopy and western blot. To investigate their role in microclotting, we ran a label‐free quantitative proteomics of these sEVs. Functionally, flow cytometric analysis and Raman spectrometry were used to measure platelets activation and function upon treatment with sEVs.

Results : Our proteomics analysis of sEVs from both HC probands and LC patients has revealed 665 differentially expressed proteins. Gene set enrichment analysis of these proteins showed that different pathways related to clotting are activated in LC patients including heparin, glycosaminoglycan, and cofactor binding pathways. Furthermore, multiple key thrombosis and clotting proteins were significantly up‐ or downregulated including fibronectin (FN1), and Histidine rich glycoprotein (HRG). Functionally, flow cytometry analysis showed a significant increase in platelet activation markers (P‐selectin and CD63) in platelets treated with LC sEVs. Moreover, four h of stimulation of platelets with LC‐sEVs isolated resulted in different mean Raman spectra compared to platelets stimulated with HC‐sEVs. In particular, there were differences regions corresponding to the protein (amide I and III) and lipid (cholesterol, phospholipid) structures, indicating the activation of platelets by changing their shape due to restructuring of membrane components and release of granules.

Summary/Conclusion : Plasma LC‐sEVs showed the ability to activate platelets mediated by its protein content specially FN1 and HRG. This activation caused platelets degranulation, which can trigger the distinct coagulation and thrombosis pathways. This could allow for thrombo‐inflammation and micro‐clotting formation in long COVID patients.

T cell extracellular vesicles promote eosinophil survival through the cytokine cargo IL‐3

Presenter: Kaitlyn E. Bunn

Vanderbilt University, USA

Introduction : T cells orchestrate diverse immune responses, from infections to autoimmunity and allergy. To effectively coordinate tissue inflammation, T cells communicate with other cells in the local microenvironment through cell contact mediated and secreted signals, including extracellular vesicles (EVs). EVs have emerged as mediators of communication between immune cells, but the functions of T cell EVs in communicating signals in tissue inflammation are incompletely understood. In this study, we investigate how T cell EVs contribute to inflammation through their interactions with eosinophils, critical effectors of allergic inflammation, in the pathologic context of allergic asthma.

Methods : We leveraged T cell‐specific membrane tagging and single vesicle flow cytometry to quantify T cell EVs in the airways during induced allergic inflammation in mice. We further treated in vitro differentiated eosinophils with EVs isolated from primary T cell culture media and performed cell death assays and RNA sequencing. Additionally, we utilized small molecule inhibitor, blocking antibody, and enzyme immunoassay studies to identify the active cargo carried by T cell EVs. Finally, we administered T cell EVs into the airways of mice with allergic inflammation and assessed lung eosinophilia.

Results : T cell EVs are increased >10‐fold in lung lining fluid during allergic inflammation in mouse models of asthma. In vitro, T cell EVs promote eosinophil survival and induce pro‐survival, activation, and metabolic gene programs in eosinophils. T cell EVs carry the surface cargo IL‐3, which inhibits apoptosis in eosinophils through the activation of Jak/Stat signalling. The effect of EV‐associated IL‐3 is more potent at lower doses and longer lasting than that of soluble IL‐3. This effect depends on T cell activation state, as only T cells activated through the T cell receptor secrete EVs carrying IL‐3 that support eosinophil survival. In vivo, instillation of activated T cell EVs into the airways is sufficient to prolong eosinophilia during an allergic reaction.

Summary/Conclusion : This study supports a role for T cell EVs and their cytokine cargoes as a critical signalling axis in allergic lung inflammation. As cytokine‐based communication is ubiquitous during immune responses, the capacity of EVs to serve as vehicles for cytokines may be a fundamental mechanism for inflammation‐associated pathology.

Neutrophilic granulocytes regulate the activity of other inflammatory cells through pro‐and anti‐inflammatory EV production

Ákos M. Lőrincz 1,2 , Mátka Nagy 1 , Viktória Szeifert 1 , Ferenc Kolonics 1 , Nóra Borsos 1 , Roland Csépányi‐Kömi 1 , Erzsébet Ligeti 1

1 Semmelweis University, Hungary, 2 Szent György Hospital, Hungary

Introduction : Neutrophils (PMNs) produce extracellular vesicles (EVs) predominantly ranging in diameter from 100 to 600 nm. We previously characterized three distinct EV populations: spEVs produced by resting PMNs, apoEVs generated during apoptosis, and ozEVs induced by opsonized zymosan particles. Only ozEVs exhibited antibacterial effects, and we have explored the specific signalling pathway leading to their production. This study investigates the roles of these EV populations in intercellular communication with other myeloid and endothelial cells.

Methods : PMNs and monocytes were isolated from healthy adults in accordance with the Declaration of Helsinki and with ethics approval from ETT TUKEB (31937‐7/2020/EÜIG). Human PMNs were either stimulated with opsonized zymosan or left unstimulated. The EV fractions were isolated by differential centrifugation and filtration. These distinct EV fractions were then co‐incubated with resting neutrophils, monocytes, monocyte‐derived macrophages, and HUVEC cells. Phagocytosis was assessed by flow cytometry, ROS production via chemiluminescence, and IL‐8 and TGF‐beta production by ELISA. Cell surface markers were quantified by flow cytometry, and macrophage migration was monitored by microscopy.

Results : PMN‐derived EVs did not influence phagocytosis in PMNs and only apoEV in monocytes. However, spEVs inhibited ROS production in both cell types, while ozEVs elevated ROS production in PMNs. SpEVs significantly increased anti‐inflammatory TGF‐beta production, whereas ozEVs increased pro‐inflammatory IL‐8 secretion in PMNs, monocytes, and HUVEC cells. SpEV‐treatment lowered CD11b amount on the monocyte cell surface. OzEV increased CD11b expression on monocytes and E‐selectin and VCAM‐1 on HUVEC cells. Additionally, apoEVs enhanced macrophage migration.

Summary/Conclusion : According to these data, we hypothesized that the PMN EVs do not simply reflect the state of the mother cell but also deliver addressed information to the different partner cells. This selective EV‐mediated communication underscores the role of neutrophils in dynamic modulation of immune responses and highlights their possible therapeutic roles in immunomodulation.

Funding : EFOP‐3.6.3‐VEKOP‐16‐2017‐00009, NKFIH FK 137770, TKP2021‐EGA‐24, János Bolyai Research Scholarship of the Hungarian Academy of Sciences

Neutrophil‐derived extracellular vesicles improve the viability of leukocytes

Mátka Nagy 1 , Kristóf Molnár 1 , Vanda Vikman 1 , Katalin Kohán 1 , Viktória Szeifert 1 , Delaram Khamari 1 , Ágnes Kittel 2 , Erzsébet Ligeti 1 , Roland Csépányi‐Kömi 1 , Ákos M. Lőrincz 1,3

1 Semmelweis University, Hungary, 2 HUN‐REN, Hungary, 3 Szent György Hospital, Hungary

Introduction : Neutrophils (PMNs) are the predominant leukocyte population in human blood, they facilitate intercellular communication through the release of extracellular vesicles (EVs). Our Laboratory focuses on EVs generated in three biologically characteristic states of neutrophils: spontaneously released spEVs from resting neutrophils, apoEVs produced by neutrophil undergoing apoptosis and ozEVs formed during complex biological activation with opsonized zymosan. The aim of our current work is to investigate the PMN EVs effect on other immune cells viability.

Methods : Neutrophils, lymphocytes and monocytes were isolated from human blood, macrophages were differentiated from monocytes using M‐CSF. PMNs were incubated with or without activating agent for 20 min or left in a pro‐apoptotic environment for 24 h. After cellular elimination, PMN EVs were isolated by two‐step centrifugation and gravitational filtration. Right after isolation, leukocytes were treated with EVs for different incubation periods. Cell count and viability was assessed by measuring annexin V and propidium iodide positivity using FC, LDH release was measured from cell culture supernatants. Caspase‐3 activity of EV treated cells was also assessed. During the macrophage differentiation, cell culture supernatants was measured by FC and LDH assay, fluorescence microscopy images were taken of the fully differentiated macrophages.

Results : All three PMN EVs improved the viabilityof lymphocytes, while they had no effect on neutrophils. Monocytes cell viability was slightly enhanced by apoEVs and spEVs. In the case of the monocyte‐derived macrophages, spEVs and apoEVs significantly increased the differentiation success. Based on the supernatant measurements, spEVs and apoEVs improved the viability of the cells from the beginning of the differentation, resulted in three times more cells in the spEV and apoEV‐treated samples compared to the untreated samples. However, there was no difference after EV treatment in the viability of the differentiated macrophages.

Summary/Conclusion : Depending on the prevailing circumstances, neutrophils produce extracellular vesicles that can selectively enhance the viability of partner immune cells. This selectivity underscores the complex ways in which neutrophils dynamically modulate inflammation through EV production. By exploiting this property, neutrophil EVs can be utilized for therapeutic applications.

Funding : EFOP‐3.6.3‐VEKOP‐16‐2017‐00009; NKFIH FK137770 ; TKP2021‐EGA‐24; János Bolyai Research Scholarship of the Hungarian Academy of Sciences

The role of mast cell‐derived extracellular vesicles in T‐cell activation and the adaptive immune response

Kelsey Fletcher 1 , Tamás Visnovitz 1 , Dorina Lenzinger 1 , Andrea Ferencz 2 , Edit I.Buzás 1,3,4 , Krisztina V. Vukman 1

1 Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, 2 Department of Surgical Research and Techniques Semmelweis University Budapest, 3 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, 4 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary.

Introduction : Mast cells (MCs) play a critical role in T‐cell activation, particularly in facilitating Th1 adaptive immune responses. Extracellular vesicles (EVs) produced by MCs have emerged as significant mediators in this process.

Methods : MCs were activated with bacterial and viral antigens, followed by isolation of MC‐derived large and small EVs using differential centrifugation, density gradient centrifugation and size exclusion chromatography. These EVs were characterised by flow cytometry, electron microscopy, TRSP, SPV and protein assays to confirm their EV nature. Their uptake and effects on the viability, proliferation, cytokine secretion of spleen cells and isolated CD4+T‐cells were assessed in vitro using flow cytometry, resazurin assay, qPCR and ELISA. Additionally, in vivo experiments were conducted by injecting EVs into mice and analysing their impact on CD4+ T‐cells.

Results : Our findings reveal that T‐cells internalised the vesicles both in vitro and in vivo. EVs derived from MCs activated with bacterial antigens stimulated T‐cells, leading to cytokine production (IFN‐gamma, IL‐5, IL‐2). Interestingly, only EVs from unstimulated MCs promoted T‐cell proliferation, whereas bacterial antigen‐activated EVs reduced this activity without affecting T‐cell viability. Notably, larger EVs appeared to exert a more potent effect on T‐cell activation; when T‐cells were treated solely with soluble mediators (without large EVs), activation did not occur. Furthermore, specific proteins within these larger EVs were identified as potential contributors to T‐cell activation, with ICAM1 on T‐cells emerging as a key player in this response.

Summary/Conclusion : In conclusion, our study highlights the essential role of MC‐derived EVs in T‐cell activation, emphasising the often‐overlooked importance of larger EVs in adaptive immune responses. Although exosomes have been widely studied in this context, larger EVs have recieved limited attention. Our findings suggest that these larger EVs, alongside their smaller counterparts, may play an equally—if not more— significant role in immunomodulation, offering novel insights into immune cell communication and adaptive immunity.

Funding : NVKP_16‐1‐2016‐0004 grant of the Hungarian National Research, Development and Innovation Office (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, the Therapeutic Thematic Program TKP2021‐EGA‐23. RRF‐2.3.121‐2022‐00003 (National Cardiovascular Laboratory Program), 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 Research and Innovation Program (No. 739593) and János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

5′‐tRF‐Gly‐GCC in EVs confer platinum resistance in ovarian cancer

Presenter: Yuya Monoe

Osaka University, Japan

Introduction : Tumour‐infiltrating T cells have been reported to be important in the acquisition of platinum drug resistance in ovarian cancer, but the underlying mechanisms are unknown. Previously, we have found that colon cancer EVs harbor large amounts of 5′tRF‐GlyGCC, a known T cell inhibitor (ISEV2023). Therefore, we aimed to determine the expression of tRFs in ovarian cancer EVs and its effect on T cells and platinum drug resistance.

Methods : Clinical specimens: The ovarian cancer specimens were obtained from patients undergone primary resection at the Osaka Medical and Pharmaceutical University, Japan. Written informed consent was obtained from each patient, and the study was approved by the ethics review board of the Osaka Medical and Pharmaceutical University. Tumour tissue‐derived EVs: Tissue‐immersed medium (normal ovaries, platinum‐resistant and sensitive ovarian cancer tissues) were centrifuged at 2000 × g , for 30 min, and the collected supernatants were subjected to the ultracentrifuge method (100,000 × g × 2) for recovery of tissue‐exudative EVs (Te‐EVs). The size and concentration of EVs were determined using qNano. RNA‐seq of Te‐EVs: RNA were isolated from Te‐EVs by using miRNeasy kit. RNA libraries were constructed using the NEBNext Small RNA Library Prep Set and sequenced on a HiSeq 2500 platform.

Results : Compared to normal ovarian Te‐EVs and platinum‐sensitive ovarian cancer Te‐EVs, platinum‐resistant Te‐EVs were significantly taken up by CD4 T cells and their activity was suppressed. RNAseq analysis showed that ovarian cancer Te‐EVs harbored more 5′tRF‐GlyGCC than that of normal ovarian Te‐EVs. Overexpression of 5′tRF‐GlyGCC in normal ovarian Te‐EVs suppressed T cell activity. Furthermore, ovarian cancer EVs contribute to the acquisition of platinum drug resistance in a 5′tRF‐GlyGCC‐dependent manner in a mouse ovarian cancer model.

Summary/Conclusion : 5′tRF‐GlyGCC in ovarian cancer EVs suppress T cells and contribute to the acquisition of platinum drug resistance.

Collagen Hydrogel Encapsulating Calcium Silicate Stimulated‐Adipose Mesenchymal Stem Cells‐Derived Extracellular Vesicle for Immunomodulation and Accelerating Diabetic Wound Healing

Presenter: Ming‐You Shie

China Medical University, Taiwan (Republic of China)

Introduction : Diabetic wounds are characterized by impaired healing due to chronic inflammation, reduced angiogenesis, and insufficient collagen deposition. Extracellular vesicles (EVs), derived from adipose‐derived mesenchymal stem cells (ADSC), have shown potential in promoting wound healing through immunomodulation and tissue regeneration. This study explores the therapeutic effects of calcium silicate (CS)‐stimulated ADSC‐derived EVs (CSEV) incorporated into collagen hydrogel for diabetic wound healing.

Methods : The in vitro and in vivo diabetic wound models were utilized, including a diabetic human dermal fibroblast (HDF) model and a diabetic rabbit wound model. CSEV were characterized for their physical and biological properties, and their therapeutic efficacy was assessed, particularly in improving wound healing and regulating oxidative stress. Molecular analysis revealed that CSEV contains distinct miRNA and proteins linked to angiogenesis, wound healing, and anti‐inflammatory effects, highlighting their potential for diabetic wound treatment.

Results : CSEVs were characterized by their enhanced protein content, surface marker expression, and bioactive cargo, including pro‐angiogenic and anti‐inflammatory factors. In vitro studies demonstrated that CSEV‐loaded collagen significantly reduced reactive oxygen species (ROS) production and enhanced cell proliferation and migration compared to standard EV‐loaded collagen. Cytokine profiling further revealed the upregulation of anti‐inflammatory cytokines (IL‐10) and extracellular matrix components (Col I) in the CSEV‐treated groups. In vivo, histological evaluation of diabetic rabbit models treated with CSEV‐loaded collagen exhibited superior re‐epithelialization and organized collagen deposition, indicating accelerated wound closure.

Summary/Conclusion : The findings of this study demonstrate that CS‐stimulated EVs (CSEV), incorporated into collagen hydrogel dressings, significantly enhance the healing of diabetic wounds. CSEV exhibit superior bioactivity, as evidenced by increased protein content, enhanced cellular uptake, and modulation of inflammatory and regenerative pathways. The sustained release of CSEVs from the collagen matrix supports long‐term therapeutic efficacy, promoting angiogenesis, reducing oxidative stress, and accelerating re‐epithelialization and collagen deposition in vivo. These results highlight the potential of CSEV‐loaded collagen dressings as a novel therapeutic strategy for improving outcomes in diabetic wound care, particularly in cases where traditional treatments are insufficient due to impaired tissue regeneration.

Immunomodulatory effect of mesenchymal stromal cell‐derived extracellular vesicles on dendritic cells

Kimingi W. Hannah, Buckley Fiona, Contereras Kallens Pamina, Shen Yeyu, Aris M. Hannah, Treacy Oliver, Brennan Á. Meadhbh

University of Galway, Ireland

Introduction : Dendritic cells (DCs) are professional antigen‐presenting cells (APCs) that link the innate and adaptive immune systems. Overactive DCs are implicated in inflammation‐related bone loss by activating T‐cells as reported in rheumatoid arthritis (RA) and chronic periodontitis. Therefore, there is a need for therapies that can modulate DCs towards tissue‐repair effectors. It has been shown that mesenchymal stromal cells (MSCs), and more recently their secreted extracellular vesicles (EVs), can influence the immune system. This study investigates the influence of MSC‐EVs' potential to modulate dendritic cell differentiation and activation.

Methods : Murine MSCs were cultured on tissue culture plastic in EV‐depleted media for 48 h. EVs released by the MSCs were isolated by using differential centrifugation, ultrafiltration, and size exclusion chromatography (SEC). EVs were characterized by particle size and concentration using nanoparticle tracking analysis (NTA), by protein content using bicinchoninic acid (BCA), for morphology, as well as surface marker expression. MSC‐EVs were treated on monocyte‐derived dendritic cells isolated from C57BL/6 mice. EVs were treated at increasing dosages (0, 4e+009, 8e+009, and 2.25e+010 EV particles per mL) on days 0, 3, and 6 for differentiation assay and one day after activation for activation assay. The uptake of the EVs by DCs was monitored by tracking palmitoylated‐tandem dimer Tomato (PalmtdTomato) EVs using confocal microscopy. DC surface markers analysed using flow cytometry, and cytokine secretion, were employed to assess the effect of MSC‐EVs on differentiation, activation, and intracellular cytokine release by dendritic cells.

Results : The size of the isolated MSC‐EVs ranged between 30 and 380 nm with the majority of EVs having the size of 130 ± 40 nm. MSC‐derived EVs caused a significant dose‐dependent reduction in the expression of MHC‐II, CD11c, CD40, and CD86 surface markers and increased expression of CD11b surface marker and PD‐L1 immune checkpoint marker.

Summary/Conclusion : MSC‐EVs inhibited the differentiation and activation of monocyte‐derived dendritic cells. To understand the underlying mechanisms behind these findings we are currently investigating the role of EV‐treated dendritic cells on the activation and proliferation of T‐cells. Further, we will investigate the transcriptome, miRNA, and protein cargo profiles of the murine MSC‐EVs.

Funding : Science Foundation Ireland (SFI).

Type 2 inflammation tunes extracellular vesicle dynamics in fibroblasts

Aiden Blumer, Julian Krause, Silvio Kau Strebinger

University of Veterinary Medicine Vienna, Austria

Introduction : Type 2 inflammation, driven by Th2 cytokines such as interleukin‐4 (IL‐4) and IL‐13, is implicated in chronic inflammatory diseases affecting respiratory and cutaneous tissues. This immune pathway, notably driven by IL‐4 and IL‐13 via JAK/STAT signalling, has recently been linked to skin fibrosis in secondary lymphedema—focus area of our investigation. Given that IL‐4 and IL‐13 can alter endosomal‐lysosomal traits, we hypothesize that genes regulated by STAT proteins, or their physical interactions influence extracellular vesicle (EV) dynamics in lymphedema, affecting both local and systemic disease processes. We regard fibroblasts as an underestimated cell type in this context, which may reveal novel insights into tissue remodelling and immune modulation.

Methods : We performed a comprehensive database search to compile the STAT1/3/6 interactome and identify genes regulated by these proteins. Data were cross‐referenced with Gene Ontoloy (GO) annotations to pinpoint connections of IL‐4/IL‐13‐STAT signalling with EV biogenesis. An extensive literature review assessed previously identified direct STAT interactions to uncover associations with EV biognesis. To pre‐investigate EV dynamics in fibroblasts, NIH‐3T3 cells were treated with IL‐4, IL‐13, and their combination at various time points up to 24 hs. Cleared culture supernatant (500 and 1500 g) was analysed using NTA, flow cytometry, and TEM. Particle counts were normalized to cell numbers.

Results : Preliminary data reveal many STAT interactions impacting EV‐related processes in human and murine cells. These interactions extend beyond transcriptional targets to include direct protein‐protein associations, suggesting a multi‐faceted role of STATs in modulating EV biogenesis within inflamed tissues. Type 2 inflammation was found to tune EV release in murine fibroblasts, which reflects in silico results.

Summary/Conclusion : This study highlights a potential mechanistic link between dysregulated Type 2 immune responses and EV dynamics in fibroblasts, which may underlie local progression of lymphedema and its systemic manifestation. Further experiments are necessary to explore the precise molecular underpinnings of this association.

Macrophage‐derived chimeric extracellular vesicles mediating bacterial‐induced immune dysregulation in sepsis: mechanisms and clinical implications as biomarkers for immune assessment and therapy

Presenter: Bingbing Ru

Southern Medical University, China (People's Republic)

Introduction : Sepsis, a lethal organ dysfunction caused by dysregulated immunity, claims millions of lives annually. Macrophages (Mφ) drive sepsis pathogenesis, and their extracellular vesicles (EVs) may propagate systemic immune dysregulation. We identified macrophage‐derived EVs carrying bacterial components (chimeric EVs, CEVs) in sepsis patients, hypothesizing CEVs mediate remote monocyte activation and serve as biomarkers.

Methods : To elucidate the role of macrophage‐derived extracellular vesicles (Mφ‐EVs) in immune regulation during sepsis, we isolated Mφ‐EVs from serum EVs of sepsis patients and healthy controls using magnetic bead‐based specific isolation and performed proteomic analysis. Bacterial components in Mφ‐EVs were characterized via immunogold labelling, EV‐specific ELISA, and nano‐flow cytometry. CEVs were extracted from E. coli‐phagocytosing THP‐1 cells using magnetic beads and intraperitoneally injected into sepsis mouse models. Systemic immune effects of CEVs were assessed by flow cytometry analysis of peripheral blood and peritoneal lavage cells (monocytes, macrophages, neutrophils) and ELISA quantification of serum TNF‐α and IL‐6. CellMask‐labelled CEVs and M1 Mφ‐EVs were intravenously injected into mice, and their cellular uptake was analysed via flow cytometry. Primary murine monocytes and sepsis mice were used to evaluate CEV‐induced monocyte recruitment and inflammatory differentiation. Transcriptomic and enrichment analyses of CEV‐treated THP‐1 cells were conducted to identify regulatory mechanisms. To assess whether CEV levels reflect macrophage functional states, macrophages from sepsis mice at different disease stages were isolated for functional evaluation.

Results : Sepsis patient Mφ‐EVs showed elevated immune proteins involved in pathogen recognition, inflammation, chemotaxis, complement activation, and antimicrobial defense. Bacterial proteins, including LPS colocalized with the EV marker CD63, were uniquely detected in sepsis patient Mφ‐EVs. In murine models, pre‐injection of CEVs significantly increased circulating monocyte percentages, reduced bone marrow and peritoneal monocytes, and elevated CD80, CD86, CCR2 pro‐inflammatory markers and IL‐6, TNF‐α. CEVs activated TLR/NF‐κB pathways in monocytes, driving inflammatory differentiation. Circulating CEV levels positively correlated with macrophage phagocytosis, inflammatory polarization, and cytokine secretion (r = 0.75–0.89, p < 0.001).

Summary/Conclusion : CEVs drive sepsis‐associated immune dysregulation by shuttling bacterial components to monocytes, triggering pro‐inflammatory differentiation via NF‐κB/IRF3 pathways. The novel CEV liquid biopsy technique offers a robust tool for real‐time immune monitoring and immunotherapy evaluation in sepsis.

Impact of tumour microenvironment on small Extracellular Vesicle interactions with antigen presenting cells and immune modulation

Presenter: Maria Luisa L. Fiani

Istituto Superiore di Sanità, Italy

Introduction : Tumour microenvironment (TME) immnosuppression is a major obstacle to overcome for effective therapies against solid tumours, with small extracellular vesicles (sEVs) playing a central role in intercellular communication. A hallmark of TME is hypoxia that alter sEVs secretion and composition, including glycosylation and cargo content. The key players in the TME are immature dendritic cells (iDCs) and tumour‐associated macrophages (TAMs), both characterized for the expression on their surface of the mannose receptor (MR), a potential pathway for sEVs uptake. The aim of this study is to investigate if sEVs, released under normoxic or hypoxic conditions, once internalized via MR, influence TME potentially promoting mechanisms of immune suppression and tumour progression.

Methods : Fluorescent sEVs (Bodipy sEVs) were obtained by pulsing melanoma cells under normoxic (N‐sEVs) or hypoxic (H‐sEVs) conditions with BODIPY FL C16, isolated through differential ultracentrifugation and quantified by Flow Cytometry (FC). Monocyte derived immature Dendritic Cells (iDCs) and M2‐macrophages were used to study sEVs role in phenotypic and functional changes on antigen presenting cells. Uptake and induction of phenotypic changes and proliferation in mature DCs and CD4+ T‐cells was evaluated by FC. Functional assays, such as ELISA, were performed to observe changes in cytokine levels in cells supernatant after treatment with sEVs.

Results : Bodipy N‐ and H‐ sEVs were specifically taken up by iDCs. N‐sEVs induced the expression of CD80 and CD86, markers involved in the activation of T lymphocytes, as well as CD83 in the same manner as LPS. No effects observed with H‐sEVs. MR expression is increased following internalization of H‐sEVs compared to N‐sEVs internalized via the MR. This result suggested that H‐sEVs appear to maintain DCs in an immature state. MLR data showed that N‐sEVs induce T‐cells proliferation more than H‐sEVs.

Summary/Conclusion : This work addresses a critical gap in understanding the role of sEVs in tumour progression and immune system modulation. By elucidating the mechanisms of N‐ and H‐sEVs uptake and their capacity to reprogram immune cells, this approach could pave the way for the development of targeted immunotherapies to overcome TME‐mediated immunosuppression, offering new therapeutic avenues for metastatic melanoma and other tumours.

Funding : Italian Ministry of Health (grant RF‐2019‐12369719)

TGFβ+ small extracellular vesicles as potential contributors to the pathogenesis of chronic rhinosinusitis

Katarzyna Piszczatowska 1 , Katarzyna Czerwaty 2 , Karolina Dżaman 2 , Nils Ludwig 1 , Mirosław J. Szczepański 1,2

1 Department of Biochemistry, Medical University of Warsaw, Poland, 2 Department of Otolaryngology, The Medical Centre of Postgraduate Education, Poland

Introduction : Chronic rhinosinusitis (CRS) is an inflammatory disease affecting almost 5‐12% of the worldwide population and is categorized into two main subtypes: CRS with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP). One of the key aspects of CRS pathogenesis is tissue remodelling (TR). TGFβ–related pathways are widely known for their participation in TR not only in CRS, but also in various other diseases. In our recent study, we detected increased TGFβ expression levels in the upper respiratory epithelium of CRSwNP patients. The aim of this study was to characterize the quantity, quality and TGFβ cargo content in plasma–derived small extracellular vesicles (sEVs) obtained from patients diagnosed with CRSsNP or CRSwNP qualified for Functional Endoscopic Sinus Surgery (FESS).

Methods : sEVs were isolated from plasma using pre‐clearing with differential centrifugation and Size Exclusion Chromatography (SEC). Subsequent characterization was performed by Nanoparticle Tracking Analysis (NTA), Cryo‐Electron Microscopy (Cryo‐EM), and western blotting (WB).

Results : sEV sizes ranged from 83 to 109 nm and concentrations were determined as 1,90E+09 to 2,6E+11 particles/mL. We observed differences in the concentration value between patients. Cryo‐EM imaging revealed the characteristic shape of sEVs with bilayer membranes and sizes comparable to the NTA findings. WB demonstrated the presence of CD9 and CD63, TGFβ as well as the negative marker Grp94.

Summary/Conclusion : In our study, we demonstrated the presence of TGFβ+ sEVs in the plasma obtained from CRS patients. Those sEVs might be involved in the CRS pathogenesis, however, further investigation with the use of further methodologies as well as wider patient cohorts are urgently needed.

Funding : Young Investigator Grant from Medical University of Warsaw: 1WK/1/M/MB/N/23

Extracellular vesicles as carriers of mitochondrial damage‐associated molecular patterns

Kristína Lichá 1 , Jakub Janko 1 , Tomáš Dallos 2 , Barbora Balážiová 2 , Peter Celec 1,3 , Barbora Tamášová 1 , Michal Pastorek 1

1 Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia, 2 Comenius University Medical Faculty in Bratislava and National Institute of Children's Diseases Bratislava, Slovakia, 3 Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia

Introduction : Extracellular vesicles (EVs) can act as carriers of extracellular DNA protecting it from degradation in circulation by nucleases. EV‐DNA play roles in different physiological and pathological processes. We have previously investigated that healthy plasma‐derived small EVs (sEVs) contain nuclear (ncDNA) and mitochondrial DNA (mtDNA) both inside and outside vesicles, with prevalence of mtDNA. EV‐mtDNA and proteins can act as damage‐associated molecular patterns (DAMPs). MtDNA inside vesicles is protected from pattern‐recognition receptors, preventing it from being recognized and triggering inflammatory reaction. During trauma injury, cells release sEVs and mitochondrial DAMPs into the circulation. The aim of this study was to investigate EV‐mtDNA‐DAMPs in plasma from patients after traumatic joint injury.

Methods : sEVs were isolated from healthy human plasma by iodixanol‐density gradient ultracentrifugation or using qEVoriginal 35 nm columns (Izon Science) and characterized by nanoparticle tracking analysis, transmission electron microscopy and western blot. DNA was extracted using a commercial kit (Qiagen) and further analysed. Plasma was drawn from healthy controls and patients after traumatic joint injury (n = 7) into K3EDTA vacutainer tubes. Diluted plasma was stained with MitoTracker Green for mitochondria labelling and To‐Pro‐3 for DNA detection in microparticles (EV‐mtDNA‐DAMPs). Markers of sEVs—CD63 and CD81 was detected in microparticles.

Results : Our recent results show healthy plasma‐derived EVs contain approximately 60‐75 % of DNA associated with surface and about 25 – 40% is protected inside the sEVs. MtDNA was prevalent in sEVs compared to ncDNA and protected against nucleases. In this study, EV‐mt‐DAMPs were confirmed in patients with traumatic joint injury using flow cytometry staining. The number of mitochondria particles and DNA‐associated particles was higher compared to controls, which suggests an increased presence of EV‐mtDNA‐DAMPs in the plasma of posttraumatic patients. Detection of EV markers using flow cytometry staining was problematic and must be optimised.

Summary/Conclusion : This study demonstrates a protected presence of mtDNA in healthy plasma‐derived EVs. MtDNA can act as EV‐mt‐DAMPs in disease states and offers a simple method for their detection using flow cytometry to predict inflammatory processes caused by traumatic joint injury.

Funding : Slovak Research and Development Agency APVV‐21‐0370 and Slovak Research and Development Agency APVV‐21‐0378 The role of infectious and sterile inducers of neutrophil extracellular trap formation in sepsis

The role of extracellular vesicles in allergic sensitization

Presenter: Davis Tucis

University of Nottingham, UK

Introduction : Type I allergy is increasing worldwide, causing pressure on healthcare infrastructures and lowering the life quality of affected individuals. Allergic sensitization is the phase encompassing the first encounter with an allergen, involving the priming of T cells by dendritic cells (DCs) to generate a Th2 phenotype. However, the mechanisms driving a Th2 response are still much debated. In recent years, extracellular vesicles (EVs) have gained popularity due to their ability to influence the immune system. Thus, the focus of this study was to examine how EVs could drive allergic sensitization, utilising an in vitro, human DC and naïve T cell assay.

Methods : Primary human DCs were derived from freshly isolated monocytes (immunomagnetic separation) from PBMCs, cultured with GM‐CSF and IL‐4 for 5 days. DCs were stimulated with 100 ng/mL of LPS or 10 µg/mL of OVA for 24 h. Subsequently, the EVs generated were isolated using SEC with IZON columns, and fractions from 1 to 7 were concentrated using 10 kDa Amicron columns. EVs were then added to freshly isolated autologous naïve T cells (immunomagnetic separation) at different time points ranging from 44 to 92 h. The T cells were collected and phenotyped by Spectral flow cytometry.

Results : Following Kaluza analysis on naïve T cells, it was shown that OVA‐stimulated DC‐derived EVs from allergic individuals drive these naïve T cells towards a Th2 phenotype when co‐stimulated with CD3 and CD28 by increasing IL‐4 production and suppressing IFN‐γ production. While, in healthy individuals LPS‐stimulated DC‐derived EVs suppress IFN‐γ production. Interestingly, EVs without co‐stimulation of CD3 and CD28 did not show the same results.

Summary/Conclusion : By generating EVs from OVA‐stimulated DCs it was shown that EVs shift naïve T cells towards Th2 phenotype by increasing IL‐4 and decreasing IFN‐γ production in egg allergic individuals. It also shows that EVs act as the 3rd signal for the naïve T cells, as EVs without co‐stimulation with CD3 and CD28 did not affect the response.

Funding : No external funding.

Metabolite cargo of bacterial extracellular vesicles is altered in a mouse model of Alzheimer's disease

Samuel Wachamo, Alisha Thakur, Mallarie Broadway, Brett Moreau, Alban Gaultier

University of Virginia, USA

Introduction : Alzheimer's disease (AD) is the most common form of dementia affecting 50 million people worldwide, with estimated global costs of $1 trillion annually. AD slowly destroys memory and thinking skills, and subsequently, the ability to perform activities of daily living. The pathological hallmarks of AD include deposition of amyloid beta, formation of neurofibrillary tangles composed of hyperphosphorylated Tau, and neuroinflammation, which result in loss of synapses, neuronal dysfunction, and eventually, neuronal death. However, what triggers the pathological hallmarks of AD remains elusive. Emerging evidence implicates that the alteration of the microbiota and its associated microbial metabolites precedes overt signs and symptoms of AD, but the precise mechanisms by which this contributes to AD pathology are poorly understood. We hypothesized that altered microbiota in AD contributes to the etiology and progression of the disease by releasing bacterial extracellular vesicles (BEVs) that contain bioactive microbial metabolites.

Methods : BEVs from the whole intestinal contents of WT (C57BL/6J) and 5XFAD mice (an animal model of amyloid beta plaque deposition) were isolated by ultracentrifugation followed by size exclusion chromatography. BEVs were characterized by BCA assay (total protein), ZetaView nanoparticle tracking analysis (size and concentration), transmission electron microscopy (size and morphology), western blot (BEV specific markers), and metabolomics (cargo).

Results : Our preliminary results revealed that the metabolic profiles cluster separately between BEVs from WT and 5XFAD. BEVs also contain metabolites that have been associated with AD. These results will be further validated using plasma samples from mice and AD patients and healthy controls. Furthermore, our studies demonstrated that BEVs can traffic to the brain.

Summary/Conclusion : We discovered that BEVs contain bioactive metabolites, which can potentially regulate local and systemic immune response in AD. Ongoing and future studies will leverage Cre‐LoxP system and determine the functional roles of BEVs in the context of AD. These studies will provide valuable insights into the role BEVs in AD pathogenesis and can potentially identify biomarkers and therapeutic targets for AD.

Funding : National Institutes of Health (T32 GM007267 and 5T32AI007496‐29), Owens Family Foundation, and the Miller Family.

Endothelin receptor A antagonists impair macrophages’ vesicular trafficking breaking down Syndecan‐Syntenin‐Alix sorting complex

Presenter: Vanessa Biemmi

Università della Svizzera Italiana Lugano, Switzerland

Introduction : Extracellular vesicles play a pivotal role in intercellular communication, governing diverse physiological and pathological processes. Owing to their stability in various body fluids, these small vesicles establish connections between distant cells, mediating the transfer of small molecules and modulating inflammatory responses. Inflammatory EVs derived from macrophages (M1 EVs) notably exacerbate post‐myocardial infarction (MI) inflammation and increase cardiomyocyte cytotoxicity. The study explores mechanisms behind small EVs release by M1 inflammatory macrophages (M1 sEVs) and evaluates the potential of endothelin receptor A antagonists (ETRA‐a) to impairs macrophage EV sorting.

Methods : Buffy‐coat‐derived M0 macrophages were differentiated into the M1 inflammatory phenotype with cytokine stimulation. Through serial centrifugation, large vesicles (lEV) were isolated from the 20K pellet, while small vesicles (sEV) were collected from the 100K ultracentrifugation pellet and characterized per MISEV guidelines.

Results : Following polarization, M1 macrophages showed increased sEV secretion, with consistent morphology and size verified by Particle Metrix, TEM, and FC analyses of tetraspanin markers. Polarization did not impact lEV concentration or profile. In vitro, ETRA‐a significantly decreased sEV release in M1 macrophages without affecting lEV release, as shown by Particle‐Metrix and FC. TEM confirmed that EV morphology and size remained unchanged after treatment. ETRA‐a disrupts SRC kinase activation, impairing Syndecan‐Syntenin‐Alix alternative sorting complex formation, as shown by proximity ligation assay. The inability of endosomal vesicles to be committed to the sorting route drives both early endosomes Rab5+ and RAb7+ late endosomes to fuse with lysosomes driving vesicular cargoes to the degradative pathway. This mechanism is not Cacl2 dependent. Furthermore, ETRA‐a ability to impair M1 sEVs offers cardioprotective effects by minimising cytotoxicity on IPS‐derived cardiomyocytes.

Summary/Conclusion : The study highlights the role of ETRA in regulating M1 EVs release through Syndecan‐Syntenin‐Alix sorting complex. ETRA‐a impairs complex formation, disrupting the release of pro‐inflammatory EVs and altering endosomal maturation. Our findings connect immune cell‐derived EVs directly to cardiomyocyte health. By clarifying the role of M1 sEVs in myocardial health, this study supports ETRA‐a as a potential therapeutic strategy to protect heart function after MI and mitigate inflammation in various pathological conditions.

CD169 and HLA‐DR in blood cells and circulating extracellular vesicles as a new markers for monitoring SARS‐CoV‐2 infection and Long COVID associated sequelae

Marialaura Fanelli 1 , Vita Petrone 1 , Rossella Chirico 1 , Luigi Coppola 2 , Chiara Sorace 2 , Chiara Cipriani 1 , Martina Giudice 1 , Claudia M. Radu 3 , Elisabetta Teti 2 , Vincenzo Malagnino 2,4 , Marco Iannetta 2,4 , Sergio Bernardini 1 , Emanuela Balestrieri 1 , Loredana Sarmati 2,4 , Sandro Grelli 1,5 , Antonella Minutolo 1* , Claudia Matteucci 1*

1 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, 00133, Italy; 2 Infectious Diseases Clinic, Policlinic of Tor Vergata, Rome, 00133, Italy; 3 Thrombotic and Hemorrhagic Diseases Unit Department of Medicine—DIMED University of Padua, Italy; 4 Department of Systems Medicine, University of Rome Tor Vergata, Rome, 00133, Italy; 5 Virology Unit, Policlinic of Tor Vergata, Rome, 00133, Italy.

Introduction : Elevated inflammation and immune dysregulation are main consequences of SARS‐CoV‐2 infection. The dysregulated inflammatory state persists after COVID‐19, generating the post‐acute sequelae of SARS‐CoV‐2 infection in Long COVID individuals (LC). The role of CD169 as a marker in the early diagnosis of SARS‐CoV‐2 infection and its association with severity and clinical outcome was demonstrated in COVID‐19 patients (COV). We evaluated the expression of CD169 and HLA‐DR on leukocytes subpopulations and on circulating extracellular vesicles (EVs) from COV and LC to better elucidate their involvement in immunological dysfunction.

Methods : Blood samples from COV, LC, and Healthy Donors (HDs) were collected at Tor Vergata University Hospital of Rome. Leukocytes subpopulations and circulating EVs were characterized for HLA‐DR and CD169 expression in COV, LC and HD through flow cytometry (CytoFLEX). The different markers analysed were associated with clinical and biochemical parameters in COV and LC.

Results : CD169 RMFI was found significantly higher in COV than in HDs and LC, resulting in a good marker of viral infection. Whereas the percentage of CD169+ monocytes was high in COV and LC, and the percentage of HLA‐DR+ monocytes was low with respect to HD. Notably, the percentage of activated monocytes CD169+HLA‐DR+ were high in COV and persisted in LC. The percentage of HLA‐DR+CD169+ monocytes correlated inversely with a‐PTT ratio in COV, and directly with the numbers of platelets in LC. The percentage and number of HLA‐DR+CD169+ EVs were significantly elevated in COV and, although lower, persist in LC compared to HD. The analyses of different size EVs in COV, LC and HD showed a different expression of HLA‐DR and CD169 markers.

Summary/Conclusion : CD169 RMFI and myeloid activation markers were confirmed as predictive markers of COVID‐19, and these markers persisted in LC. This data opens a new possibilities for studying the use of CD169 and HLA‐DR at cellular and EVs level as markers during acute infection or in post‐infection sequelae.

Funding : Funded by HERVCOV project (GA101057302) HORIZON‐HLTH‐2021‐DISEASE‐04 Personalised medicine and infectious diseases: understanding the individual host response to viruses.

Pseudomonas aeruginosa microvesicles modulate macrophage activation

Presenter: Giulia Bernabè

University of Padua, Italy

Introduction : Pseudomonas aeruginosa is a Gram‐negative opportunistic pathogen producing several virulent factors in response to quorum sensing (QS) system activation. Indeed, P. aeruginosa uses QS‐related autoinducing molecules such as homoserine lactones (i.e., 3OC12‐HSL) or 2‐heptyl‐3‐hydroxy‐4 (1H)‐quinolone (PQS) also to modulate host immunity. Bacterial membrane vesicles (BMVs) are reported to carry factors involved in host‐pathogen interactions, thus modulating infection outcomes. However, protocols for isolating and characterizing BMVs and insights into the BMVs‐related mechanisms are still lacking.

Methods : BMVs were isolated through sequential centrifugation and ultracentrifugation from cultures of P. aeruginosa standard strain (PAO1) and QS‐deficient mutant (DQSPAO1). Bacteria were growing up in the presence or not of sub‐MIC concentration of ciprofloxacin, as a stressor agent, or 3OC12‐HSL, to induce the QS system. BMVs quantity and integrity were assessed via Nanoparticle Tracking Analysis (NTA) and flow cytometry. The presence of 3OC12‐HSL and PQS in BMVs was evaluated using biosensors. To investigate immune‐modulatory effects, BMVs were incubated with human peripheral blood monocyte‐derived macrophages polarized to pro‐inflammatory (MqM1) or anti‐inflammatory (MqM2) phenotype; cytokines were quantified in the conditioned media.

Results : NTA characterized the purified BMVs yielding a concentration of 1.0×10 1 ⁰ particles/mL with an average diameter of 163±1.2 nm. Ciprofloxacin treatment significantly enhanced the production of BMVs enriched in the QS molecules 3OC12‐HSL and PQS, as revealed by biosensor‐based assays. Using lipid‐specific fluorescent probes and flow cytometry analysis, we revealed that the antibiotic treatment triggered the formation of structurally different BMVs sub‐populations. BMVs obtained from unstimulated PAO1 cultures induced the release of inflammatory cytokines in MqM1, whereas ciprofloxacin triggered in PAO‐1 the release of BMVs that inhibited LPS‐induced MqM1 activation. Moreover, activation of QS induced in PAO1 by 3OC12‐HSL supported the production of BMVs endowed with anti‐inflammatory effects in MqM2. BMVs obtained from ΔQSPAO1 showed no significant immune‐regulatory effects, regardless of bacterial stimulation.

Summary/Conclusion : This study reveals the critical role of P. aeruginosa derived BMVs in mediating host‐pathogen interaction, depending on the environmental stressors. The enhanced presence of QS molecules following ciprofloxacin treatment suggests that BMVs drive peculiar host immune activity during antibiotic therapeutic interventions.

Pseudomonas Quinolone Signal trafficked by Bacterial Membrane Vesicles (BMVs) impair monocyte recruitment and differentiation in Alveolar Epithelium

Priyakshi Gogoi, Ambalika Roy, Dipankar Ghosh

Special Centre for Molecular Medicine, Jawaharlal University, New Delhi, India

Introduction : The cross‐talk between alveolar macrophages and pneumocytes is essential to deploy circulating monocytes to infection sites. This study reveals that PQS trafficked by BMVs significantly impairs monocyte recruitment and activation into M1 macrophages, disrupting immune homeostasis.

Methods : BMVs were purified using density gradient from clinical strains of P. aeruginosa biofilms and characterized using NTA and TEM and Q‐TOF LCMS. PQS trafficked within BMVs were quantitated using Triple Quad LC‐MS/MS. AT2 ‐ Macrophage ALI co‐culture model was developed using A549 pneumocytes and THP‐1 derived macrophages. PQS extracted from predetermined BMV numbers were exposed to the apical surface of the ALI co‐cultures with LPS. The cells and culture supernatants were quantitated for chemokines CCL2, CCL4, CXCL8, CXCL10 and CXCL11 using qRT‐PCR and CBA Array respectively. Parallel sets were used to study chemotaxis of THP‐1 monocytes and CD14+ sorted cells from human blood using a Combined Boyden‐Flow Cytometry Assay. Monocytes were exposed to PQS under M1‐polarizing conditions and assessed for differentiation through surface marker expression, cytokine profiling, and functional assays.

Results : Biofilm‐associated BMVs from clinical P. aeruginosa strains contained elevated PQS levels than the type strain PA‐O1. In the AT2 ‐ Macrophage co‐culture model under ALI conditions, LPS activation induced significantly higher chemokines production (CCL2, CCL4, CXCL8, CXCL10, CXCL11) compared to a 2‐D model, with CCL2 and CXCL8 being most expressed. Notably, PQS suppressed the expression of the chemokines at both mRNA and protein levels by inhibiting the nuclear translocation of NF‐ĸB p65. Suppression of chemokines led to impaired chemotaxis of THP‐1 and CD14+ monocytes. Furthermore, PQS exposure under antigenic stimulation attenuated monocyte activation into M1 macrophages by inhibiting surface markers expression, cytokines and dysregulating energy metabolism. This significantly reduced the overall antimicrobial potential in these cells.

Summary/Conclusion : Pulmonary macrophages and pneumocytes work in tandem with circulating monocytes for effective host defense against P. aeruginosa biofilm infections. P. aeruginosa BMVs present unique pathogenic mechanisms that can remotely compromise this first line of defense and exacerbate infections in the lung.

Funding : This research is part of Priyakshi Gogoi's and Ambalika Roy's PhD projects at Jawaharlal Nehru University, funded by the Department of Biotechnology, Government of India.

Urinary EVs as biomarker for renal dysfunction and signs of premature ageing in young women suffering of anorexia nervosa

Diego Prudente 1 , Enrica Favaro 1 , Valentina Ponzo 1 , Ilaria Goitre 1 , Giovanni Abbate Daga 2 , Simona Bo 1 , Benedetta Bussolati 1 and Cristina Grange 1

1 Department of Medical Sciences, University of Turin, 10126 Turin, Italy. 2 Department of Neurosciences, Eating Disorders Unit, University of Turin, 10126 Turin, Italy

Introduction : Anorexia nervosa (AN) is a severe psychiatric disorder with high mortality rate. There are currently no validated biomarkers for AN severity. Moreover, patients with AN experience physical and psychological stress that may impact on a premature ageing. Data on accelerated cellular senescence and inflammation are still controversial. In this context, extracellular vesicles present in urine may be a source of valuable information to better understand the complexity of this pathology. Beyond kidney status, uEVs may reveal signs of inflammation, ageing and, in general, multi‐organ dysfunction.

Methods : In this study, adult female patients ( n = 20), diagnosed with AN according to DSM‐5 criteria, were recruited from the Psychiatry Department and the Outpatient Clinics of the Dietetics and Clinical Nutrition Unit at the Città della Scienza e della Salute of Torino. Healthy women, matched by age and sex were enrolled as control. Urine were collected from spontaneous clean catch voids. uEV were characterized combining the cytofluorimetric analysis of uEV surface markers, obtained with MACSplex Exosome Kit performed on pre‐cleared urine, with the quantification of uEVs‐Klotho measured by ELISA performed on ultracentrifugation‐isolated EVs.

Results : We isolated a comparable number of uEVs from AN patients and healthy subjects. We observed the presence of renal progenitor markers (CD133, CD24, SSEA4) and of the epithelial marker CD326 in both experimental conditions. However, the expression of renal uEVs markers CD24, CD133 and the epithelial marker CD326 was increased in AN patients. Of interest, the levels of inflammatory markers CD3 and HLA‐DR appeared reduced in AN patients in comparison of healthy subjects All the results will be correlated with the anthropometric measurements (height, weight, and BMI) and Laboratory test results (including serum creatinine and AST/ALT levels).

Summary/Conclusion : The results presented here suggested that uEV may provide crucial insights into the pathophysiology of AN, thereby opening possible future therapeutic strategies. Urinary EV markers (CD133 and CD24) could be informative of renal regenerative reserve and the levels resulted increased in AN patients. Inflammatory marker levels may reflect systemic inflammation.

Funding : PONV_RILO_24_01, Ricerca Locale 2024 to Dr. Grange Cristina

Metabolic Imaging of Plasma‐Derived Small Extracellular Vesicles (sEV) Interaction with Head‐Neck Cancer and Endothelial Cells

C. Guder 1 , L. Englert 2,3 , F. Gasparin 2,3 , E. Dorado 4 , M. A. Pleitez, B. Wollenberg 1 , Z. Takats 3,5 , V. Ntziachristos 2,3 , M. N. Theodoraki 1,6

V. Ntziachristos and M. N. Theodoraki are co‐senior authors.

1 Department of ENT, TUM University Clinic, Germany, 2 Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Germany, 3 Chair of Biological Imaging at the Central Institute for Translational Cancer Research (TranslaTUM), School of Medicine and Health, Technical University of Munich, Germany, 4 Department of Metabolism, Digestion and Reproduction, Imperial College London, United Kingdom, 5 Department of Immunomedicine, University of Regensburg, Germany, 6 Department of ENT, head and neck, University of Ulm, Germany

Introduction : Small extracellular vesicles (sEV) are emitted by all cells into the surrounding extracellular environment, facilitating communication between cells. Tumour‐derived‐EVs (TEX) are frequently found in the plasma and saliva of cancer patients. There are already known mechanisms of TEX weakening the anti‐tumour immune response mediated by proteins (e.g. PDL‐1 and CD44v3) and by RNA (e.g. miRNA). Studies additionally indicate a connection between TEX and changes in metabolism. Nevertheless, the exact impact of TEX on the tumour microenvironment remains unknown. This study investigates the interaction between TEX and protein, carbohydrate, and lipid metabolism of recipient cells.

Methods : sEVs were isolated from the plasma of head and neck squamous cell carcinoma (HNSCC) patients by size exclusion chromatography. The isolated TEX were characterized by nanoparticle‐tracking‐analysis, western blot (CD63, CD9, TSG101 and CD81) and transmission electron microscopy. To investigate the interaction between sEV and protein, carbohydrate, and lipid metabolism, different cell types (e.g. UD‐SCC‐5, HUVEC) were treated with TEX over different time periods. Label‐free mid‐infrared optoacoustic microscopy (MiROM) and multiphoton microscopy were used to detect changes in metabolism. Additionally, metabolic changes were validated by immune assays and ‐omics analyses.

Results : Label‐free metabolic imaging indicates early changes in lipid metabolism, but not in metabolism related to proteins and carbohydrates, after treatment of UD‐SSC‐5 with TEX. For HUVEC cells no significant differences in the metabolism after co‐incubation with TEX of HNSCC patients were detected. Further immune assays and ‐omics results support these observations. Changes in proteins were visible at later time points.

Summary/Conclusion : The results of this study support the hypothesis of the connection between TEX and changes in metabolism. While the metabolites of nonmalignant HUVEC cells remained unaltered after treatment with TEX of HNSCC patients, the lipid metabolism of HNSCC cells was altered.

During skin sensitization keratinocytes release small extracellular vesicles containing hapten‐modified proteins necessary for the development of allergic contact dermatitis

Adriana T. Larregina 1,2 , Juliana S. Powell 3 , Olga Tkacheva 1 , William J. Shufesky 3 , Beer Stolz D 4,5 , Sullivan M 4,5 , Simon C. Watkins 4,5 , and Adrian E. Morelli 2,3 .

Departments of Dermatology 1 Immunology 2 Surgery 3 and Cell Biology and Physiology and Center for Biologic Imaging University of Pittsburgh. Pittsburgh, PA, USA.

Introduction : Allergic contact dermatitis (ACD) is a severe inflammatory skin disease that affects up to 18% of the population in USA and Europe. The onset of ACD requires the activation of the inflammasome in keratinocytes resulting in the synthesis and secretion of IL‐1β. These mechanisms are potentiated by substance P (SP) a pro‐inflammatory neuropeptide that signals with high affinity the neurokinin 1 receptor (NK1R). The events by which keratinocytes that are targets of neurogenic inflammation promote ACD remain poorly understood. Because IL‐1β is a leaderless protein released by non‐conventional secretory pathways, we hypothesized that contact sensitizers activate the inflammasome in keratinocytes, which release IL‐1β in small extracellular vesicles (sEVs) and that these effects require SP.

Methods : In vitro studies utilized mouse or human immortalized keratinocytes treated with the contact sensitizer 2,4‐dinitrochlorobenzene (DNCB) in the presence or not of SP. Increase of multivesicular bodies (MVB) was quantified by transmission electron microscopy and analysis of MVB fusion with the cell membrane was assessed by Total Internal Reflection Fluorescence Microscopy in keratinocytes stable transfected with pLenti‐Hluo_M153R‐CD63mCherry. Quantification and size of secreted EVs was evaluated by nanotracking particle analysis. In vivo studies induced ACD in K14‐NK1RKO or in K14‐Rab27aKO mice and their littermate controls.

Results : Signalling mouse and human keratinocytes with SP stimulated the formation of MVBs that fused to cell membrane and increased the secretion of sEVs. These effects required the expression of NK1R and Rab27a since they were abrogated in keratinocytes from K14‐NK1Rfl/fl or K‐14‐Rab27aKO mice. sEVs secreted by DNCB and SP‐signaled keratinocytes expressed CD63, CD81 and TSG101 and contained IL‐1β. Molecular studies showed that keratinocytes treated with SP have increased transcripts of proteins involved in the activation of signals 1 and 2 of the inflammasome and augmented amount of IL‐1β. Induction of ACD in K14‐NK1Rfl/fl or in K14‐Rab27aKO mice was impared compared to their littermate controls.

Summary/Conclusion : Our data demonstrating that SP signalling of keratinocytes exposed to allergen potentiates the secretion of sEVs that are necessary for ACD support the therapeutic potential of blocking the SP‐NK1R axis for the treatment of the disease.

Funding : NIH R01 AI168142 and NIH R01 AI174273

Circulating extracellular vesicles containing miRNAs alleviate sociability deficits in mice

Eisuke Dohi 1,2,4 * , Ken Matoba 1 * , Phoebe A. Garcia 1 * , Jose Francis‐Oliveira 1 * , Frida Anguiano 1 , Takahiro Kochi 1 , Jana Badrani 1 , Julia See 2 , Norimichi Ito 1 , Rei Mitani 2 , Indigo V.L. Rose 2 , Eric Y. Choi 2 , Takashi Imai 2 , David K. Crossma 1 , Mikhail V. Pletnikov 2,3 , Kenneth W. Witwer 2 , Minae Niwa 1,2 and Shin‐ichi Kano 1,2,†

1 University of Alabama at Birmingham, USA, 2 Johns Hopkins University School of Medicine, USA, 3 Present address: Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, SUNY University at Buffalo, Buffalo, NY, USA, 4 Present address: National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan * These authors equally contribute to this work .

Introduction : Extracellular vesicles (EVs) circulating in the bloodstream have attracted attention as key mediators of intercellular communication and potential regulators of brain function and behaviour. In this study, we investigated the therapeutic potential of EVs and their associated microRNAs (miRNAs) from wild‐type (WT) mice to address sociability deficits in immunodeficient Rag1‐/‐ mice, aiming to clarify the underlying molecular mechanisms.

Methods : EVs were isolated with ultracentrifugation from the sera of WT mice or from Rag1‐/‐ mice and injected intravenously into Rag1‐/‐ mice. Behavioural tests were evaluated using a three‐chamber social interaction test. Additionally, immunohistochemistry and electrophysiology were employed to evaluate neuronal signalling within the medial prefrontal cortex (mPFC). We conducted simultaneous RNAseq of the brain and miRNAseq of EVs to identify brain molecules involved in sociability and their regulatory miRNAs.

Results : WT‐derived EVs attenuated sociability deficits in Rag1‐/‐ mice without impacting social novelty preference. Notably, the injected EVs were detected in the mPFC, co‐localizing with neurones. Electrophysiological analysis revealed that postsynaptic inhibitory currents were reduced in Rag1‐/‐ mice, but this deficit was ameliorated following EV exposure. Mechanistically, miR‐23a‐3p and miR‐103‐3p within the EVs decreased the expression of PKCε, enhancing postsynaptic GABAA receptor presence and restoring inhibitory signalling in mPFC pyramidal neurones, thereby ameliorating sociability deficits.

Summary/Conclusion : Our findings demonstrate that circulating blood EVs and their associated miRNAs can positively influence sociability by modifying neuronal signalling in the mPFC, offering potential therapeutic strategies for social impairments.

Funding : This research was funded by grants from the NIH. KM is supported by UFPF and TK is supported by JSPS.

High‐fat diet‐induced alterations in adipose tissue extracellular vesicle miRNA content may drive NF‐κB activation and microglial inflammatory response in the CNS

Adam M. Allouch 1,2 , Sarah E. Elzinga 1,2 , Bhumsoo Kim 1,2 , Rosemary E. Henn 1,2 , Mohamed H. Noureldein 1,2 , Faye E. Mendelson 1,2 , John M. Hayes 1,2 , Diana M. Rigan 1,2 , Masha G. Savelieff 1,4 , Junguk Hur 1,4, Kai Guo 1,4 , Yin Zou 3 , Sunitha Nagrath 3 , Eva L. Feldman 1,2

1 University of Michigan, Department of Neurology, Ann Arbor, Michigan, USA; 2 Neuroneetwork for Emerging Therapies, USA; 3 University of Michigan Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA; 4 University of North Dakota Department of Biomedical Sciences, Grand Forks, North Dakota, USA

Introduction : Obesity, prediabetes, and diabetes induce adipose tissue inflammation, which contributes to many diabetic complications, including nervous system complications. Adipose tissue inflammation is associated with increased production and release of adipose tissue‐derived extracellular vesicles (ATEVs), which can cross the blood‐brain barrier and promote central nervous system (CNS) inflammation, possibly by activating microglia. As miRNAs are suggested contributors to peripheral and CNS inflammation, we aimed to assess the effect of obesity on ATEV miRNA content and inflammatory gene expression profiles of microglia, the primary immune cells in the CNS.

Methods : We induced obesity/prediabetes by feeding 5‐week‐old male C57BL/6 mice standard diet (SD) or high‐fat diet (HFD) for 1 or 3 months, then isolated ATEVs from white epididymal adipose tissue. ATEV size and concentration was confirmed via nanoparticle tracking analyses and miRNA content analysed via NanoString nCounter. Single‐cell RNA sequencing data from hippocampal microglia was used to understand the potential effects of ATEV miRNAs on microglial inflammatory gene expression in vivo. ATEVs were also used to treat a human microglial cell line to assess microglia uptake of ATEVs and inflammatory gene expression after treatment. miRNA mimics and inhibitors of differentially expressed ATEV miRNAs were used to recapitulate ATEV‐mediated neuroinflammation in vitro.

Results : Obesity reduced expression of ATEV miRNAs let‐7f, miR‐141, and miR‐504. Enrichment analysis showed that let‐7f and miR‐141 regulate genes in the NF‐κB pathway, and their reduction is associated with NF‐κB activation. In HFD mice, ATEV production increased in white adipose tissue, and microglia uptake of these ATEVs led to elevated pro‐inflammatory gene expression, indicating NF‐κB activation.

Summary/Conclusion : These data suggest that HFD alters ATEV miRNA content, disrupting microglial inflammatory responses over time, implicating adipose‐microglia crosstalk as a mechanism for peripheral inflammation spreading to the CNS.

Funding : NIH Grant U01AG057562. NIH Grant U24DK115255. Michigan Alzheimer's Disease Research Center P30AG072931. NIDDK Grant T32DK007245. JDRF Grant 5COE‐2019‐861‐S‐B. Edith S. Briskin/SKS Foundation Neuroneetwork Emerging Scholar Fund. Robert E. Nederlander Sr. Program for Alzheimer's Research. Andrea and Lawrence A. Wolfe Brain Health Initiative Fund. A. Alfred Taubman Medical Research Institut. Neuroneetwork for Emerging Therapies. Neuroneetwork for Emerging Therapies Tauber Family Student Internship

Extracellular particle bound YRNA levels increase during airway inflammation

Cherie Saffold, Antiana Richardson, Quanhu Sheng, Marisol Ramirez‐Solano, Kasey Vickers, Heather Pua

Vanderbilt University Medical Center, USA

Introduction : Extracellular YRNAs (exYRNAs) are abundant in biofluids, with levels that fluctuate during disease. exYNRAs levels increase in response to inflammatory stimuli. In addition, exYRNAs packaged in extracellular particles (EPs) can trigger innate immune responses in recipient cells. Together, these observations implicate exYRNAs in immunologic communication. Despite observations linking exYRNAs with inflammatory processes, little is known about how they regulate inflammatory responses. Therefore, we investigated how exYRNA dynamics coordinate with immune processes in murine airway inflammation models.

Methods : To assess exYRNA levels during airway inflammation, we used three models: OVA, house dust mite, and papain. To quantify full length exYRNAs (RNY1 and RNY3) and their fragments in bronchoalveolar lavage fluid (BALF), we designed qPCR primers to detect 5’ and 3’ ends. To elucidate how exYRNAs are packaged during airway inflammation, we performed RNAse sensitivity assays on whole BALF, SEC‐enriched EVs, and sub‐100 kDa ultrafiltrates. To determine BALF exYRNA length distribution, we performed RNA sequencing using a template switching reaction capable of processing highly structured RNAs and eliminating size bias between full length and fragment YRNAs. Reads were trimmed (cutadapt), aligned (hisat2), and counted (featurecounts). Unique exYRNA sequences were identified using linux.

Results : BALF exRNY1 5’ end levels are increased in all three airway inflammation models one day after airway inflammatory challenge. Levels then decrease rapidly, despite persistent tissue inflammation. Approximately 50% of exRNY1 5’ ends are contained within detergent‐sensitive SEC‐enriched EVs. The other 50% of exRNY1 5’ ends are contained in proteinase‐sensitive sub‐100 kDa EPs. Notably, exRNY1 3’ ends and exRNY3 are only present in EVs. Both EV and EP exRNY1 5’ ends increase during airway inflammation. Pilot RNA seq experiments show that BALF contains YRNA fragments but minimal full length YRNA.

Summary/Conclusion : exRNY1 5’ ends, which are predominantly fragments, increase during the acute phase of airway inflammation. These YRNAs are partitioned into EV and EP compartments, which supports their potential role in intercellular communication. These data lay the foundation for elucidating exYRNA's role in inflammatory disease.

Role of lemur tail kinase 2 in remodelling of cystic fibrosis lung

Presenter: Agnieszka Swiatecka‐Urban

University of Virginia, USA

Introduction : Cystic fibrosis (CF) is a muco‐obstructive lung disease characterized by chronic dysregulated inflammation, infection, and tissue repair, leading to lung remodelling characterized by structural damage and respiratory failure. Neither the dysregulated inflammation nor chronic infection is entirely reversible with the novel high‐efficiency CFTR modulator therapy. The transmembrane lemur tail kinase (LMTK)2 is a TGF‐β1 mediator and specific activator of pro‐fibrotic Smad3. It is abundant in the apical membrane domain of differentiated bronchial epithelial cells. We examined how LMTK2 mediates the profibrotic program in the airway.

Methods : Cell models were primary differentiated human bronchial epithelial (HBE) cells from CF, and controls; immortalized human bronchial epithelial cells (16HBE) cells from CF and controls. Imaging was done by immunostaining followed by confocal fluorescence microscopy, immunogold electron microscopy (EM), and Cryo‐EM. Nanoparticle tracking analysis (NTA). Western blotting for tetraspanin CD9, a marker of EVs and LMTK2. 3‐6 experiments per group.

Results : Confocal microscopy studies showed exclusively apical localization of LMTKs in HBE cells from CF patients, compared to controls. The distribution of LMTK2 was patchy, corresponding with the distribution of cilia. EM images confirmed the presence of LMTK2 in the cilia and enrichment in periciliary vesicles. Next, we examined LMTK2 abundance in EVs secreted into the medium of CF 16HBE cells and controls in the presence of TGF‐β1 or vehicle control. We confirmed the isolation of EVs using cryoEM. NTA analysis showed a similar concentration of EVs secreted by CF and control cells. The size distribution was also similar. Neither size nor concentration of EVs was affected by TGF‐β1. LMTK2 was significantly enriched in EVs secreted by CF cells in the presence of vehicle control. TGF‐β1 increased the LMTK2 abundance in EVs secreted by CF cells.

Summary/Conclusion : Our data demonstrated that TGF‐β1 increases the secretion of LMTK2 by CF airway epithelial cells into EVs. LMTK2 may mediate the paracrine TGF‐β signalling in the CF airway. Our data warrant further studies of LMTK2 as a potential biomarker for monitoring disease progression and/or therapeutic target in CF.

Funding : NIH R01144539 to A.S‐U.; R01DK133598 to U.E. Cystic Fibrosis Foundation SWIATE24A0‐MPI. American Diabetes Association, Grant/Award 7‐22‐ICTSPM‐19 to U.E.

Subtopic Host‐pathogen & Microbiome 2

Extracellular vesicles derived from vaginal bacteria initiate the progression of early‐phase ovarian carcinoma

Presenter: Eri Asano‐Inami

Nagoya University, Japan

Introduction : Microbiota contributes to the development of tumours and other diseases, but the relationship vaginal bacteria and ovarian cancer are entirely unclear. Extracellular vesicles (EVs) play a crucial role in cancer biology, and bacteria secrete EVs, named bacterial EVs (BEV). Ovarian carcinoma was thought to arise from ovarian surface epithelial cells. Recent understanding of high‐grade serous carcinoma HGSC has revealed the tubal epithelial hypothesis, which arises when fallopian tube epithelial cells (FT cells). However, the detailed mechanism by which FT cells that have acquired oncogenic potential develop as ovarian cancer is not known, and this study aimed to elucidate the mechanism of tumourigenesis of HGSC promoted by vaginal BEV.

Methods : Four vaginal commensal bacteria were selected using 16s sequencing data of the vaginal microflora, and the selected bacteria were cultured with monitoring OD values and BEVs were isolated using an optimal continuous centrifugation method. Patient BEV was isolated using cellulose nanofibre (CNF)‐based EV sheets In vitro and in vivo functional analysis was performed to determine the effect of BEV on FT cells.

Results : Cell viability analysis showed that none of the BEVs affected FT cell proliferation. On the other hand, migration ability showed that specific Lactobacillus species promoted cell motility (p < 0.05). Furthermore, the addition of BEV to FT cells seeded in sheets on dishes coated with basement membrane components increased the number of cells that were shed (p < 0.05). In vivo experiments showed that vaginally administered BEV was taken up by FT cells, which was confirmed by in vivo and in vitro imaging. We collected BEVs on FT from 15 ovarian cancer patients using CNF‐based EV sheets, and small RNA sequencing for BEV‐RNA revealed that the profiles between HGSC and benign patients were different, and specific sequences of Lactobacillus species were detected. mRNA sequencing for FT cells treated with BEV from specific Lactobacillus species showed that gene expression was significantly altered, and IPA/GSEA analyses identified several cancer‐related pathways.

Summary/Conclusion : These results demonstrate an unknown early mechanism of ovarian cancer development from pathogenic BEV exposure and identify a novel target gene in FT cells as a new therapeutic target.

Lactobacillus extracellular vesicles suppress the systemic inflammation and provide the therapeutic effects in a sepsis mouse model

Soyeon Lee 1 , Haekang Yang 1 , Eunbi Jo 1 , Chul Won Seo 1 , Shinwon Chae 1 , Woocheol Jung 1 , Yoon‐Jin Lee 1,2 , Eun‐Young Lee 1,2 , and Dongsic Choi 1,2

1 Department of Biochemistry, 2 Research Center for Extracellular Particles, College of Medicine, Soonchunhyang University, Cheonan, Chungcheongnam‐do, Republic of Korea

Introduction : Bacteria secrete the small extracellular vesicles (EVs) known as outer membrane vesicles from Gram negative bacteria and membrane vesicles from Gram positive bacteria. Theses bacterial EVs have emerged as key regulators in host immune system. Especially, Lactobacillus probiotics, widely used in dietary supplements, have showed the beneficial properties related in their anti‐inflammatory effect through the bacteria–host interactions possibly via EVs. To address the differential therapeutic effect of Lactobacillus species‐derived EVs, we investigated their anti‐inflammatory effect in a sepsis mouse model induced by cecal ligation and puncture (CLP).

Methods : Bacterial culture supernatant from Limosilactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus crispatus, Limosilactobacillus fermentum, Lactiplantibacillus paraplantarum, and Lactobacillus acidophilus were concentrated with a 100‐kDa cut‐off. The concentrated supernatant was ultracentrifuged at 150,000 × g for EV sedimentation. Their size and concentration were measured by nanoparticle tracking analyses. Sepsis was induced in C57BL/6 mice through CLP, where the cecum was ligated and punctured with a 21‐gauge needle. EVs were injected by intravenous into mouse 1‐h after CLP operation. Therapeutic effects were measured by survival, cell counting by Diff‐Quick staining in bronchoalveolar lavage fluid (BALF), and expression of cytokines by PCR. Animal protocols were approved by the IACUCs (SCH24‐0053).

Results : Isolated Lactobacillus EVs showed the spherical round shape under 100 nm size by electron microscope. In a sepsis model, most of Lactobacillus EVs by intravenous administration represented the increased survival with mild morbidity. Especially, Limosilactobacillus reuteri EVs showed significant therapeutic effect in about 80% survival. Also, these mice exhibited the decreased neutrophil infiltration in BALF, accompanying with increased anti‐inflammatory IL‐10 but decreased TNF‐α and IL‐6. Hematoxylin and eosin staining of lung tissue showed the reduced tissue damage, representing the suppression of inflammation by Limosilactobacillus reuteri EVs.

Summary/Conclusion : In this study, we found the Lactobacillus EVs reduce the mortality and morbidity in a sepsis mouse model. These findings suggest that Lactobacillus EVs could be applicable in therapeutics for modulating excessive immune responses and improving survival in diverse inflammatory diseases.

Funding : This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS‐2024‐00348103 and RS‐2023‐00219563).

Proteomic analyses of extracellular vesicles derived from Lactobacillus reuteri and Lactobacillus rhamnosus

Eunbi Jo 1 , Haekang Yang 1 , Soyeon Lee 1 , Shinwon Chae 1 , Woocheol Jung 1 , Chul Won Seo 1 , Yoon‐Jin Lee 1,2 , Eun‐Young Lee 1,2 , and Dongsic Choi 1,2

1 Department of Biochemistry, 2 Research Center for Extracellular Particles, College of Medicine, Soonchunhyang University, Cheonan, Chungcheongnam‐do, Republic of Korea

Introduction : Bacteria release the extracellular vesicles (EVs) ranged from 20 nm to 100 nm, which play diverse pathophysiological roles such as antibiotics resistance, transfer of materials, and quorum sensing. Especially, Lactobacillus are widely used as probiotics in dietary supplements with the diverse beneficial properties including immune‐modulatory activity. To address the reliable protein content of Lactobacillus EVs, we established the confident procedure of EV isolation by optimized size exclusion chromatography (SEC) and their proteomic contents were identified by mass spectrometry.

Methods : Lactobacillus reuteri and rhamnosus were obtained from the Korean Collection for Type Cultures. Bacteria cultured in MRS broth under anaerobic conditions at 37°C. Supernatant was concentrated using a 100 kDa cut‐off filter and loaded onto a SEC column. EV‐enriched fractions were collected and analysed by nanoparticle tracking analyses (NTA) and micro BCA assay. Quantitative proteomics were conducted to reveal unique the proteome of Lactobacillus reuteri‐ and rhamnosus‐derived EVs. To address the anti‐inflammatory effect, LPS‐activated RAW264.7 cells were treated with EVs and cytokine expressions were quantified by PCR. EVs were labelled by lipophilic fluorescent DiR and treated to the RAW264.7 cells. Their uptake efficiency and trafficking were quantified by Lionheart FX automated microscope.

Results : Silver staining, electron microscope, and NTA showed the enriched bacterial EVs in SEC fractions. To compare the purity, particles per protein amount was applied. The SEC method was 1.76E9 particles/µg, while the ultracentrifugation method had a purity of 7.35E7 particles/µg, implying better purity in SEC method. Proteomics identified the EV proteins enriched with membrane and cytosol, implying selective sorting into EVs form parental bacteria. These proteins are closely related in facilitating protein transfer to the other cells, biogenesis, modulating immune system, and host‐pathogen interaction. Especially, enzymes including glucosamidase, amidase, and dextransucrase were highly enriched in both EVs.

Summary/Conclusion : We established the effective isolation method of bacterial EVs by SEC with confident identification of EV proteomes. This procedure would provide the information of more reliable EV cargos with immune modulation in host‐pathogen interaction.

Funding : This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS‐2024‐00348103 and RS‐2023‐00219563)

Investigating the temporal stability of gut microbiota‐derived extracellular vesicle release in healthy adults

Presenter: Suzette Soerensen,

Aalborg University, Denmark

Introduction : Gut microbiota‐derived extracellular vesicles (GM‐EVs) are believed to play an important role in microbiome‐host communication in health and disease. The growing number of studies investigating GM‐EVs from clinical samples, often only rely on a single time point collection. The gut microbiota composition is known to be stable over time, it has, however, not been investigated if GM‐EV secretion is likewise stable. More knowledge on possible temporal fluctuations in GM‐EV secretion is therefore highly warranted.

Methods : Fecal samples were collected from five healthy donors once every week for a period of five weeks. GM‐EVs were isolated by centrifugation followed by filtration and size exclusion chromatography. GM‐EV secretion was evaluated by particle number, particle size, and gram‐positive to gram‐negative EV ratio. Additionally, we investigated the bacterial origin of GM‐EVs by Oxford Nanopore sequencing of near full‐length 16S genes. Finally, the analysis of bacterial protein cargo of secreted GM‐EVs will be performed by mass spectrometry to assess for any changes in functional capacity. All participants gave informed consent.

Results : Our 16S sequencing data revealed a clear distinction between the gut microbiota composition and the GM‐EV composition for all five donors. This includes a higher alpha‐diversity within the gut microbiota compared to the GM‐EVs. As for the beta‐diversity, the GM‐EV samples form a distinct cluster, which is separated from the gut microbiota samples. Notably, samples from the same donor cluster together within the GM‐EV group. Temporal fluctuations were smaller than inter‐donor differences, suggesting a stable GM‐EV composition over time.

Summary/Conclusion : The GM‐EV composition appears to be stable over time, justifying single time point collections in gut microbiota EV studies.

Funding : The Lundbeck Foundation

Subtopic Tissue Stress and Remodelling 2

Highly multiplexed biochip to decipher potential modifications of EVs phenotype and size under cytotoxic conditions

Geetika Raizada 1 , Joan Guillouzouic 3 , Alain Rouleau 1 , Eric Lesniewska 2 , Eric Le Ferrec 3 , Céline Elie‐Caille 1 , Wilfrid Boireau 1

1 Université Marie et Louis Pasteur, CNRS, Institut FEMTO‐ST, 25030 Besançon, France 2 ICB UMR 6303 CNRS, F‐21078 Dijon, France 3 Univ Rennes, Inserm, EHESP, IRSET (Institut de Recherche en Santé Environnement et Travail), UMR_S 1085, F‐35000, Rennes, France

Introduction : The project consists in obtaining the on‐chip phenotype profile of EVs originating from control HMEC‐1 cell culture and B[a]P‐treated HMEC‐1 cell culture conditions. The motivation was to observe if there is differences in the expression of certain surface proteins due to the cytotoxic conditions.

Methods : The NBA platform, developed earlier by our group (1,2) was provided the basis for this purpose. It employs a multimodal approach by combining techniques such as SPRi and AFM, with SPRi identifying EV phenotypes based on specific ligands grafted onto a biochip and enabling immunocapture. AFM provides precise metrological data on EVs selectively captured on ligand arrays, enabling us to correlate specific surface protein expression with the morphological parameters of the EVs. To offer a more thorough screening approach, we broadened the range of ligands used for selective EV capture, through miniaturizing the ligand spots. That transition enabled to go from manual spotting (with 16 spots of 1.3mm 2 ) to automatic nano spotting (with 100 spots of 0.125 mm 2 ).

Results : We successfully simultaneously monitored the EVs capture on 11 ligands, on the same biochip. SPRi experiments revealed significant differences in responses for Anti‐CD36, Anti‐CD81, and Anti‐ApoA between the two conditions, likely due to B[a]P exposure. To complement this, AFM scanning and thorough statistical analysis further highlighted size‐related differences between EVs from the treated and control conditions, with Anti‐CD81, Anti‐ApoA, Annexin‐V, Anti‐NOX2, and Anti‐VEGFR1 emerging as potential key biomarkers for the cytotoxic condition.

Summary/Conclusion : These findings suggest that B[a]P exposure may drive not only an increase in EV size but also selective expression of these markers, indicating shifts in the phenotypic profile of large EVs (lEVs) specifically. Those results complete the characterization of these same objects we already engaged through Raman analysis (3,4). This focused investigation of lEVs, through both SPRi and AFM, underscores the potential of lEV‐specific markers to reveal phenotypic changes linked to cytotoxic responses, offering valuable insights into EV heterogeneity under cell stress conditions.

Funding : References 1.Obeid et al., 2017, 10.1016/j.bios.2016.08.100 2.Obeid et al., 2019, 10.1016/j.nano.2019.02.026 3.Raizada et al., 2023, 10.3791/64210 4.Raizada et al., 2024, 10.1007/s00216‐024‐05567‐4 Funding : Region Bourgogne Franche‐Comté, Graduate school

Raman characterization of EVs to study the reversal of arsenic impairment by rehabilitative or pharmacological treatment

Silvia Picciolini 1 , Alice Gualerzi 1 , Martina Gerli 1 , Francesca Rodà 1,2 , Aurora Mangolini 1 , Zachary Clemens 3 , Aaron Barchowsky 4 , Amrita Sahu 3,4,5 , Marzia Bedoni 1

1 IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy; 2 Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy; 3 Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 4 Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 5 McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Introduction : Arsenic (As) exposure impairs tissue function resulting in reduced mobility and overall quality of life. Although there still exists a dearth of interventions aimed to restore As‐impairment, preliminary findings from the Sahu and Barchowsky labouratories have demonstrated that neuromuscular electrical stimulation (E‐stim) and pharmacological intervention with mitochondrially targetted peptide‐SS‐31 improves As‐impaired functional regeneration of skeletal muscle. It has also been demonstrated that As disrupts extracellular vesicle (EV) mediated skeletal muscle signalling in regenerating muscle fibres. However, the regulatory cargoes within the EVs driving As‐effects are yet to be investigated. Herein, we propose to determine the effect of E‐stim and SS‐31 intervention on circulating biochemical signatures of EVs in As‐exposed animals by using Raman Spectroscopy.

Methods : Blood EVs were isolated by size‐exclusion chromatography and ultracentrifugation. Size distribution and concentration have been assessed by nanoparticle tracking analysis. EV samples were analysed by means of a Raman microspectroscope equipped with 532 laser line and multivariate statistical analysis were performed in order to compare the Raman fingerprint of EVs from As‐exposed mice with and without E‐Stim or SS‐31 treatment.

Results : We found that EVs size and concentrations are not affected by As exposure or the interventions. Raman data showed that EV fingerprints are significantly different between As and treated mice with the main modifications in spectral regions related to lipids and proteins. We also observed some spectra differences that could be related to changes in the presence and conformations of specific molecules involved in oxidative stress condition (cytochrome, glutathione and nucleic acids).

Summary/Conclusion : Our findings demonstrated that both rehabilitative and pharmacological treatments alter the biochemical composition of circulating EVs of As‐exposed mice, suggesting that they could improve As‐impaired autocrine, paracrine, and endocrine signalling capabilities mediated through EVs. Moreover, our results showed that Raman EV characterization can represent a valuable method to identify potential involvement of molecules in oxidative stress.

Funding : This research was funded by the Italian Ministry of Health, The Pittsburgh Foundation, USA and National Institute of Environmental Health, USA.

Loss of ovarian hormones and acute exercise affect specifically the nucleic acid cargo of circulating extracellular vesicles in female rats

Presenter: Sira M. Karvinen

University of Jyväskylä, Finland

Introduction : Loss of ovarian hormones (i.e. hormonal ageing) has negative health effects that are not fully mitigated by exercise. Ageing and exercise have shown to affect specifically the nucleic acid cargo of circulating extracellular vesicles (EVs). Whether loss of ovarian hormones affects specifically the nucleic acid cargo of circulating EVs is not known. This study investigated the effects of loss of ovarian hormones and acute exercise on the molecular cargo of circulating EVs in female rats.

Methods : Female Wistar rats ( n = 40) were matched for body mass and maximal running capacity, and subjected either to ovariectomy (OVX), to induce loss of ovarian hormones, or sham surgery ( n = 20/group). There groups were further divided into control or acute exercise group, that performed maximal running test prior to sample collection (sham/control, sham/max, OVX/control and OVX/max, n = 10/group). Plasma EVs were isolated by density‐gradient ultracentrifugation followed by size‐exclusion chromatography. The EV isolation was validated by dot blot, electron microscopy and nanoparticle tracking analysis. The EV cargo was studied via Raman spectroscopy from pooled samples and nucleic acid to lipid (NA/L) and protein to lipid (P/L) ratios were calculated. The EV small‐RNA cargo of individual samples was sequenced by outsourced service and analysed with Tools for Integrative Genome analysis of Extracellular sRNAs (TIGER) pipeline.

Results : We observed a significant effect of acute exercise and interaction of OVX and acute exercise in NA/L ratio ( p < 0.050). Sham group had higher NA/L ratio than OVX group when examining the control groups ( p = 0.019). OVX/max had higher NA/L ratio compared with OVX/control group ( p = 0.001). There were no significant findings from P/L ratio ( p ≥ 0.106). When exploring the small‐RNA content of the EVs in each study group, 10‐13% were mapped to host and 26‐36% to nonhost origin. Of the host sequences, most abundant were ribosomal RNA‐derived sRNA (rDR) (50%–55%), microRNA (19%–30%) and transfer RNA‐derived sRNA (tDR) (9%–14%) sequences. Of the nonhost sequences, most abundant ones originated from rDR (46%–48%) and algae (8%).

Summary/Conclusion : Our results highlight that loss of ovarian hormones and acute exercise affect specifically the nucleic acid cargo of circulating EVs.

Funding : Research Council of Finland (grants to 332946 and 354603 to SK).

Acute mental stress affects the kinetics of circulating extracellular vesicles

Judith Eberz 1 , Elsa Kratz 1 , Lea Walther 1 , Alexandra Brahmer 1 , Carsten Geiß 1 , Tim Ebert 2 , Nils Gassen 2 , Marianne Müller 3 , Eva‐Maria Krämer‐Albers 1

1 Johannes Gutenberg University Mainz, Germany, 2 University Hospital Bonn, Germany, 3 University Medical Center Mainz, Germany

Introduction : Extracellular vesicles (EVs) circulating in the bloodstream potentially cross the blood‐brain barrier (BBB), facilitating interaction between the brain and periphery. The composition and kinetics of circulating EVs can correlate with acute brain states and provide insight into the dynamics of neurological disorders, including stress‐related diseases. Since physiological stress in form of acute exercise is known to trigger the release of EVs into the circulation, we asked whether acute mental stress elicits a similar EV response. To this end, we assessed the dynamics of circulating EVs in a cohort of first‐time bungee jumpers and performed multiparametric profiling of EVs in plasma samples.

Methods : Blood plasma was collected longitudinally at two time points prior and four time points after bungee jump (−1 week, ‐20 mins, +6 mins, +1 h, +2 h, +4 h, +1 week) and analysed for platelet background and hemolysis according to the MIBlood recommendations. CD63+ EVs were separated from plasma by immuno‐isolation. Phenotyping of CD63+ EVs was performed by multiplex bead‐based flow cytometry using the MACS‐Plex EV‐Immuno and EV‐Neuro assay (Miltenyi Biotec). Data were expressed as fold‐change post versus prior to the jump.

Results : Bungee jumping was accompanied by an increase of different EV populations up to 4 h after the jump, followed by a decline to baseline after 1 week. EVs following these kinetics were carrying integrins (CD29, CD49e) and other surface markers characteristic of lymphocytes (CD3, CD45), platelets (CD41b, CD42a) or endothelial cells (CD31, CD105). Brain‐specific markers were not detected among increasing EVs. To reveal the physiological background and trigger of EV release during acute mental stress, we are currently investigating EV kinetics in another cohort subjected to dexamethasone injection and sleep deprivation.

Summary/Conclusion : Acute mental stress, as occurring during bungee‐jumping, leads to a transient increase of circulating EVs originating from peripheral cell sources. Monitoring circulating EV profiles could thus inform about distinct stress states. Current and future studies include different stress models to explore relevant EV populations and their functional role in stress signalling and adaptation.

Funding : This research was funded by the Leibniz‐Wissenschaftscampus NanoBrain. We thank Miltenyi Biotec for providing us with a prototype of the Neuro‐EV platform.

Loss of ovarian hormones diminishes acute exercise response of circulating microRNAs carried via extracellular vesicles in female rats—effects on target tissue protein expression

Veera Puumalainen 1,2 , Anni Maja 3 , Tia‐Marje Korhonen 1 , Tuuli A. Nissinen 1 , Maarit Lehti 1 , Sira Karvinen 1,2

1 Faculty of Sport and Health Sciences, University of Jyväskylä, Finland, 2 Gerontology Research Center, University of Jyväskylä, Finland, 3 Department of Biological and Environmental Science, University of Jyväskylä, Finland

Introduction : The role of exercise in health is well‐established, but the mechanisms are unclear. Loss of ovarian hormones (i.e., menopause) results in negative health effects, which are not completely mitigated by exercise. The profile of circulating microRNAs (miRs) changes in response to exercise and menopause. We have previously shown that menopause affects the exercise‐responsive miR cargo of extracellular vesicles (EVs). However, it is not known how these changes impact the expression of miR target proteins at tissue level.

Methods : Female Wistar rats were divided to two groups: ovariectomy (OVX), to induce loss of ovarian hormones ( n = 20), and sham, surgery without the removal of ovaries ( n = 20). Half of each group performed maximal running test to induce an acute exercise stimulus, resulting in four groups: sham/control, sham/max, OVX/control and OVX/max ( n = 10/group). Plasma EVs were isolated by density‐gradient ultracentrifugation followed by size‐exclusion chromatography. EV isolation was validated by dot blot, electron microscopy and nanoparticle tracking analysis. To study the miR cargo of EVs, total RNA was extracted and sequenced by an outsourced service. Differential expression of miRs was performed with DeSeq2. Target proteins of miRs were analysed with miRPath v3.0 and quantified by Western blot from gastrocnemius muscle and liver.

Results : The levels of nine miRs were significantly different between sham/control and OVX/control. Of these, miR‐1b, miR‐133a‐3p, miR‐378a‐3p, miR‐145‐5p, miR‐146a‐5p, let‐7g‐5p, and miR‐30c‐5p were downregulated, whereas miR‐100‐5p and miR‐206‐3p showed upregulation ( p < 0.05). When examining the effect of acute exercise in sham groups, miR‐100‐5p, miR‐221‐3p, and miR‐127‐3p exhibited upregulation ( p < 0.05). Contrarily, no differences between the OVX groups were observed ( p > 0.39). At protein level, muscle mTOR and liver p38 MAPK expressions were lower in OVX than in sham ( p < 0.05). There was an interaction of OVX and acute exercise in muscle PKLR expression ( p < 0.01).

Summary/Conclusion : Our results confirm that the loss of ovarian hormones blunts the exercise‐induced miR response of EVs in female rats, which we have previously reported in women. Furthermore, the changed miR cargo of EVs may affect the protein expression of the EV target tissues, highlighting the functional relevance of EV‐carried miRs.

Funding : Research Council of Finland (grants 332946 and 354603 to SK).

Protein cargo of extracellular vesicles from cerebrospinal fluid and serum in humans is modulated by intense aerobic exercise

Presenter: Nikoleta Alchus Laiferová

Slovak Academy of Sciences, Slovakia

Introduction : The health benefits of regular physical exercise are driven by a range of mechanisms acting in synergy. Extracellular vesicles (EVs) released into circulation during exercise are promising mediators of interorgan crosstalk, potentially involved in exercise‐induced adaptations and modulation of brain plasticity. The aim of this study is to identify the changes in protein content of EVs, isolated from cerebrospinal fluid (CSF) and serum, induced by acute intense aerobic exercise.

Methods : EVs were isolated from CSF and serum of young healthy active adults (M/F 13/6; median age 25(IQR 22‐31yrs), BMI 23.2(IQR 21.7‐24.5 kg/m2) & VO2max (spiroergometry) 47(IQR 38.1‐51.2 mL/kg/min) by ExoQuick (System‐Biosciences, US). Sampling of CSF was performed (i) before and (ii) 60 min after a monitored outdoor 90 min run by atraumatic lumbar puncture. Serum was obtained at baseline (R1), immediately after (R2), and 60 min after (R3) the run (90 min, 80%HRmax). EVs were characterized by Nanopartical Tracking Analysis (Nanosight‐NS500, Malvern, UK), TEM and immunoblotting & Exo‐Check Array (CD63, CD81, GAPDH, ApoE, Albumin, GM130). Cytokine content of EVs isolated from CSF and serum was determined (RayBio array C‐2000, US). Running‐induced changes cognitive performance (MemTrax, Cogstate) were assessed.

Results : Ten out of 174 cytokines were significantly downregulated in EVs from CSF after exercise (fold change range ‐0,42 to ‐1,81). Acute exercise reduced levels of two and upregulated levels of six cytokines in serum EVs (fold change R2/R1 ‐0, 40 to 1,22). Compared to baseline, two cytokines were upregulated 1 h postexercise (fold change R3/R1 0,39–0,74), while 1 h post‐exercise recovery decreased levels of four and increased levels of one cytokine (fold change R3/R2, ‐0, 95 to 0,34) in serum EVs.

Summary/Conclusion : The regulated cytokines participate in the regulation of essential metabolic pathways, immune functions, vascular integrity and angiogenesis, as well as in extracellular matrix remodelling. Our results support the role of EVs as mediators of adaptive responses to exercise.

Funding : APVV 20‐0466, ADDIT‐CE Horizon Europe, VEGA 2‐0076‐22, FWF‐KLI‐1122, EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project No. 09I03‐03‐V04.

Association of Physical Activity and Neighborhood Deprivation with Extracellular Vesicle Characteristics in African American Women with Obesity

Presenter: Anca D. Dobrian,

Eastern Virginia Medical School, USA

Introduction : African American (AA) women have disproportionate cardiovascular disease (CVD) risk due to increased exposure to adverse social determinants of health (SDoH), including lower individual‐ and neighborhood‐level socioeconomic status (SES). Physical activity (PA) and plasma extracellular vesicles (EVs) are known to modulate CVD risk, but their relationship with SDoH remains unclear. This study investigates associations between SDoH, PA, and plasma EVs in a pilot cohort of at‐risk AA women.

Methods : Participants joined the Step It Up pilot study, utilizing a place‐tailored digital app to increase PA. Objective PA was assessed through daily step counts via Fitbit. Neighborhood socioeconomic deprivation (NSD) was determined from U.S. Census data. Individual household SES was self‐reported. EVs were isolated using size‐exclusion chromatography from fasting EDTA plasma samples and characterized using nanoparticle‐tracking analysis. EV miRNA cargo was determined using the NanoString platform. Functional effects of EVs on endothelial barrier function and cell migration were measured using electric cell‐substrate impedance sensing methodology. Multivariable regression analysis was conducted, adjusted for BMI and ASCVD 10‐year risk score.

Results : In AA women ( N = 24, age: 57 ± 12, BMI: 35 ± 6, ASCVD: 9 ± 5), NSD associated with decreased EV size ( β = −0.49, p = 0.01). Higher daily step counts associated with increasing EV size ( β = 0.48, p = 0.02). EV miR‐1246, miR‐28‐5p, and miR‐765 exhibited distinct expression patterns associated with EV size, NSD, or daily step count. In vitro, endothelial cell (EC) barrier integrity associated with EV miR‐28‐5‐p ( r = 0.73, p = 0.007) and miR‐765 contents ( r = −0.58, p = 0.049) while EC migration potential after wounding were associated with miR‐28‐5‐p content ( r = −0.67, p = 0.02).

Summary/Conclusion : Our findings show that smaller EV size associates with high NSD and low PA. Increased miR‐28‐5p cargo in EVs may support EC function, influencing CVD pathogenesis. By examining the relationships between NSD, PA, and EV characteristics in this high‐risk population, we aimed to uncover novel biological mechanisms underlying health disparities. Our comprehensive approach, combining community‐based sampling with advanced EV and miRNA profiling, provides unique insights into how SDoH may influence CV risk at the molecular level. Furthermore, through in vitro experiments, we determined the impact of the isolated EVs and their cargo on endothelial function, a hallmark of CVD development and progression.

Modulation of Osteoclastogenesis by Mesenchymal Stem Cell‐Derived Apoptotic Bodies: A RANKL Dependent Role in Monocyte

Presenter: Yeyu Shen

University of Galway, Ireland

Introduction : Human mesenchymal stromal cells (MSCs) die by apoptosis when transplanted with biomaterials in preclinical and clinical studies and initiate a bone regeneration cascade. As a pivotal role in fracture healing, osteoclasts initiate bone healing by clearing necrotic tissue, promoting new bone formation, and facilitating bone remodelling, while excessively active osteoclasts can also impact the bone quality following fracture healing. Osteoclast presence has been shown to be a prerequisite for bone healing facilitated by transplanted MSCs. The aim of this study was to investigate if apoptotic bodies derived from MSCs (MSC‐ABs) impact bone regeneration by regulating monocyte‐to‐osteoclast differentiation.

Methods : Apoptosis was induced in human bone marrow‐derived MSCs by ultraviolet (UV) irradiation in EV‐free media and verified by flow cytometry of apoptotic MSCs, scanning electron microscopy, and caspase 3 quantification. MSCs‐ABs were isolated from conditioned media via differentiatial centrifugation and characterized by total protein and RNA content, morphology, and surface marker expression by flow cytometry. MSC‐ABs were treated onto human peripheral blood‐derived CD14+ monocytes in an in vitro osteoclast differentiation protocol.

Results : Results suggested that the MSCs‐ABs have a pro‐osteoclastic effect, assessed by tartrate‐resistant acid phosphatase (TRAP, predominantly found in osteoclasts) staining and also by measuring the phosphatase concentration assay in the culture supernatant. Conversely, MSCs‐ABs had an inhibitory effect on osteoclast formation in the presence of the receptor activator of nuclear factor kappa‐B ligand (RANK‐L). Additionally, the enzyme‐linked immunosorbent assay revealed that AB‐treated cells secreted significantly higher levels of IL‐6 and IL‐10, which are known to have a dual effect on osteoclastogenesis.

Summary/Conclusion : To understand these findings, we are currently sequencing the RNA of the MSC‐ABs, then polymerase chain reaction and western blotting could be used for verifying underlying mechanisms. This study may contribute to clarifying the currently ambiguous mechanisms by which MSC‐ABs regulate osteoclast differentiation and promote bone regeneration, which may provide a promising therapeutic way for clinical bone regeneration.

Funding : This research is founded by the European Council Starting Grant, project number 852152. This research is also funded by the Chinese Scholarship Council (CSC).

Loss of MMP9 causes cranium deformities via disturbing cranial suture fusion in mice

Presenter: Jingwen Yang

Wuhan University, China (People's Republic)

Introduction : Cranial sutures function as growth centres for calvarial bones. Abnormal suture closure will cause permanent cranium deformities. MMP9 is a member of the gelatinases that degrades components of the extracellular matrix. MMP9 has been reported to regulate bone development and remodelling. However, the function of MMP9 in cranial suture and cranium development is still unknown.

Methods : We used Mmp9 global knockout mice and analysed the cranial suture and cranium via micro‐CT and histological staining. The underlying mechanisms were determined using unbiased RNA‐Seq analysis and confirmed via ex vivo 3D culture and in vivo inhibitor application.

Results : We identified that the expression of Mmp9 was specifically elevated during fusion of posterior frontal (PF) suture compared with other patent sutures in mice. Interestingly, inhibition of MMP9 ex vivo or knockout of Mmp9 in mice (Mmp9‐/‐) disturbed the fusion of PF suture. Histological analysis showed that knockout of Mmp9 resulted in wider distance between osteogenic fronts, suppressed cell condensation and endocranial bone formation in PF suture. Proliferation, chondrogenesis and osteogenesis of suture cells were decreased in Mmp9‐/‐ mice, leading to the PF suture defects. Moreover, transcriptome analysis of PF suture revealed upregulated ribosome biogenesis and downregulated IGF signalling associated with abnormal closure of PF suture in Mmp9‐/‐ mice. Inhibition of the ribosome biogenesis partially rescued PF suture defects caused by Mmp9 knockout.

Summary/Conclusion : These results indicate that MMP9 is critical for the fusion of cranial sutures, thus suggesting MMP9 as a potential therapeutic target for cranial suture diseases.

Funding : This work was supported by the National Natural Science Foundation of China (No. 82170915 and No. 31500788).

Calcific aortic valve disease augments vesicular microRNA‐145‐5p to regulate the calcification of valvular interstitial cells via cellular crosstalk

Presenter: Mohammed Rabiul Hosen

University of Bonn, Germany

Introduction : Calcific aortic valve disease (CAVD) is one of the leading causes of cardiovascular death in the elderly population worldwide. MicroRNAs are highly dysregulated in patients with CAVD undergoing surgical aortic valve replacement (SAVR). However, the miRNA‐dependent mechanisms regulating inflammation and calcification or miRNA‐mediated cell‐cell crosstalk during the pathogenesis of CAVD remain poorly understood. We investigated the role of extracellular vesicle (EV)‐associated miR‐145‐5p, which we showed to be strongly upregulated in CAVD in mice and humans during valve calcification.

Methods : Human miRNA arrays identified dysregulated miRNAs in CAVD tissue explants compared to non‐calcified HV tissue explants from patients undergoing SAVR. Echocardiographic parameters were measured in conjunction with the quantification of dysregulated miRNAs in a murine CAVD model. Integrated OMICS analyses were performed to analyse molecular miRNA signatures and their effects on signalling pathways. RNA‐seq, high‐throughput TF assays, osteogenesis, and proteomic arrays revealed that several genes, miRNAs, TFs, and proteins are critical for calcification and apoptosis involved in the pathogenesis of CAVD.

Results : Among several miRNAs dysregulated in valve explants from CAVD patients, miR‐145‐5p was the most highly sex‐independently dysregulated miRNA (AUC, 0.780, p value, 0.01). MiRNA arrays utilizing CAVD samples from patients and mice showed that the expression of miR‐145‐5p is significantly upregulated and positively correlated with cardiac function based on echocardiography. In vitro experiments confirmed that miR‐145‐5p is encapsulated in EVs and transported into VIC of the AV. The results of integrated OMICs show that miR‐145‐5p is related to markers of inflammation, calcification, and apoptosis. The calcification experiments demonstrated that miR‐145‐5p regulates the ALPL gene, a hallmark of calcification in heart valve cells. EV‐mediated shuttling of miR‐145‐5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3′ untranslated region to inhibit its translation, thereby diminishing the calcification of target VIC.

Summary/Conclusion : Elevated levels of pro‐calcific and pro‐apoptotic EV‐associated miR‐145‐5p contribute to the progression of CAVD via the ZEB2‐ALPL axis, which could potentially be therapeutically targeted to minimize the burden of CAVD.

Funding : German Cardiac Society (DGK), Ernst und Grimmke Foundation, and Corona Foundation Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Grant No. 397484323 ‐ TRR259 ‐ Project B04

Elucidating mechanism behind the impact of regulatory T cell‐derived extracellular vesicles on osteogenesis

Eylem Baysal 1 , Niyaz Al‐Sharabi 1 , Neha Rana 1 , Daniela E. Costea 1,2 , Meadhbh Brennan 3 and Salwa Suliman 1

1 University of Bergen, Norway. 2 Haukeland University Hospital, Norway. 3 University of Galway, Ireland.

Introduction : Regulatory T cells (Tregs), a specialized subset of CD4+ T cells, play a crucial role in immune regulation and have also been implicated in promoting bone repair. Emerging evidence highlights the immunomodulatory potential of extracellular vesicles derived from Tregs (Treg‐EVs), however, their precise mechanism of impact on osteogenesis remains understudied. Therefore, our study aimed to assess the role of Treg‐EVs and the phosphatidylinositol 3‑kinase (PI3K)/AKT, a crucial pathway in osteogenesis, in modulating the osteogenic potential of bone marrow‐derived mesenchymal stromal/stem cells (BMSCs).

Methods : Tregs were isolated from the peripheral blood of healthy donors ( n = 6) following informed consent. Tregs were expanded for 13 days, followed by 24 h of serum starvation. Thereafter, conditioned media were collected, pooled and concentrated. Treg‐EVs were isolated using size‐exclusion chromatography and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. In the presence or absence of PI3K/AKT pathway inhibitor, BMSCs were treated with Treg‐EVs (80 µg/mL), and morphology, metabolic activity, migration, and osteogenic potential of BMSCs were evaluated at mRNA and protein levels.

Results : Treg‐EVs were shown to express CD25, CD73, HLA‐DR, CD63, and CD81 and uptake of Treg‐EV by BMSCs was confirmed by confocal imaging after 24 h. Treg‐EVs significantly enhanced BMSCs metabolic activity after 4 and 7 days, albeit insignificant when PI3K/AKT pathway was inhibited. Treg‐EVs reduced BMSCs migration after 48 and 72 h, also during PI3K/AKT pathway inhibition. During osteogenic differentiation, PI3K/AKT pathway was highly activated in BMSCs from 4 days when treated with Treg‐EVs compared to BMSCs not treated with Treg‐EVs. Moreover, Treg‐EVs significantly enhanced alkaline phosphatase enzyme activity and osteogenic markers at mRNA and protein level in BMSCs after 7 days with and without PI3K/AKT pathway inhibition. Mineralization after 10 days was enhanced, even with pathway inhibition.

Summary/Conclusion : Treg‐EVs promote BMSCs viability and osteogenic differentiation while reducing migration. This positive impact of Treg‐EVs on osteogenesis was sustained despite PI3K/AKT pathway inhibition, suggesting a potential PI3K/AKT independent pathway for enhancing osteogenesis. These findings provide insights into Treg‐EVs mechanisms for enhancing osteogenesis and highlight their therapeutic potential in bone regeneration.

Funding : Funded by Trond Mohn Foundation (TMS2021STG03) and Norwegian Research Council (314473).

Hematopoietic stem cell extracellular vesicles condition the bone marrow endothelium

Presenter: Danielle Kobulsky

Children's Hospital of Philadelphia, USA

Introduction : Crosstalk between hematopoietic stem and progenitor cells (HSPCs) and specialized bone marrow (BM) niche cells is critical for engraftment after therapeutic HSPC transplantation. Emerging evidence supports a dynamic role for HSPCs in shaping their niche to promote BM occupancy. However, key signalling events originating from HSPCs that govern these processes, including through secreted extracellular vesicles (EVs), are not well understood.

Methods : To better understand the composition and functions of secreted HSPC vesicles, small EVs were isolated from brief ex vivo cultures of human CD34+ progenitors and donor‐matched plasma from mobilized leukapheresis products. Further density‐gradient enrichment was performed for quantitative proteomic analysis, and characterization of vesicles was performed according to MISEV2023 guidelines. Key differentially expressed proteins were validated in murine HSPC EVs for downstream functional studies.

Results : HSPC EVs are enriched in integrin α4β7, a receptor for vascular cell adhesion molecule‐1 (VCAM‐1), compared to plasma EVs in both murine and human model systems (6.5‐fold, p < 0.05). Using a well‐established model of primary murine BM endothelial cells (ECs), we demonstrate robust uptake of HSPC EVs into ECs that is reduced after pharmacologic and genetic disruption of α4β7/VCAM‐1 interactions. Upon uptake into ECs, HSPC EVs induce canonical NF‐κB activation and downstream transcriptional upregulation of CC motif chemokine receptor 2 ligands (CCR2L; including Ccl2, Ccl7, and Ccl12) (> 8‐fold, p < 0.05). Small RNA sequencing studies of HSPC EVs revealed KLF4, a negative regulator of NF‐κB, to be an enriched cellular target of EV miRNA, and transcriptionally suppressed in ECs after EV uptake (0.7‐fold, p < 0.01). Functionally, recapitulation of vesicle‐mediated HSPC:EC signalling by intraosseus injection of EVs or CCR2L improved donor cell homing and engraftment in murine bone marrows after transplantation.

Summary/Conclusion : Overall these findings support a novel, integrin‐dependent role for HSPC EVs in shaping the EC secretome to promote BM homing. Importantly, our study identifies a therapeutically accessible axis of HSPC:EC vesicle crosstalk to inform potential targeted conditioning strategies to improve HSPC transplantation outcomes.

Funding : This work was supported by the National Institutes of Health grants R01HL164633 (P.K.), 4‐R38‐ HL143613 ‐04 (S.N.H.), and T32 HL‐07439 (S.N.H.).

The role of circulating endothelial cells and endothelial extracellular vesicles as new biomarkers in colorectal cancer

Francesca D'Ascanio 1,2,3 , Davide Brocco 4 , Domenico De Bellis 1,2 , Giulia Colasante 1,2 , Arianna Aquilini‐Mummolo 1,2 ,Francesco Dotta 1,6 , Piero Chiacchiaretta 1,6 , Nicola Tinari 1,5 , Alessandro Cama 4 , Pasquale Simeone 1,2 , Paola Lanuti 1,2 .

1 Center for Advanced Studies and Technology (CAST), Italy, 2 Department of Medicine and Ageing Sciences, Italy, 3 Department of Humanities, Law and Economics, “Leonardo da Vinci” University, Italy, 4 Department of Pharmacy,Italy, 5 Department of Medical, Oral and Biotechnological Sciences, Italy, 6 Department of Innovative Technologies in Medicine and Dentistry, Italy

Introduction : The balance between circulating endothelial progenitor cells (CEPC) and circulating endothelial cells (CEC) reflects the endothelial homeostatic processes involved in cancer neo‐angiogenesis. Hematopoietic stem cells (HSCs) and endothelial‐derived extracellular vesicles (EVs) also play a role in this process. We undertook the task to describe CEC, CEPC, HSC and endothelial EVs in colorectal cancer (CRC) patients, with the aim to investigate their role as circulating biomarkers in CRC diagnosis, prognosis and response to treatment.

Methods : We recruited 131 cancer patients and 41 healthy volunteers. Blood samples were collected at the start of the study and during follow‐up. Different circulating endothelial cell population, as well as HSC were analysed using a polychromatic flow cytometry approach. Furthermore, a patented innovative flow cytometry method was used to identify, subtype, and count endothelial extracellular vesicles. The 1‐H MULTIOMICS dimension reduction algorithm was also optimized and applied to flow cytometry data; UMAP and t‐SNE were used to evaluate the impact of the therapy on the circulating subsets of CRC circulating cells.

Results : We identified several types of circulating cells and EVs. We found that HSC levels were significantly lower in CRC patients compared to healthy individuals. This decrease was also seen in other types of cancer. We also discovered a specific population of cells (CD45dim/CD34+/CD117+/CD146+) that was associated with better treatment response and overall survival in CRC patients. Higher levels of these cells were linked to longer survival. On the other hand, lower levels of circulating endothelial EVs were associated with better survival.

Summary/Conclusion : Our findings suggest that circulating cells and EVs involved in blood vessel remodelling play a crucial role in CRC. These elements could be useful for diagnosing and monitoring CRC. Further studies with larger patient groups are needed to validate these results and potentially improve personalized treatment strategies. Flow cytometry is a promising tool for liquid biopsy in cancer assessment.

M2 macrophage‐derived exosome‐functionalized janus fibrous scaffold regulate foreign body response to accelerate bone and muscle remodelling

Presenter: Shue Jin

West China Hospital of Sichuan University, China (People's Republic)

Introduction : During biomaterials‐mediated foreign body response (FBR), an abnormality in any of the interaction between implant materials and host tissue cells, such as a disturbance in the temporal or spatial of the cells involved in FBR, can delay or aggravate the FBR, and ultimately cause implant failure. Fortunately, manipulating exosomes secreted by macrophages is a verified strategy to mitigate FBR during tissue remodelling.

Methods : In this study, the janus scaffold was prepared by continuous electrospinning and the macrophage‐derived exosomes induced by IL‐4/IL‐13 (M2‐Exo) were extracted by ultracentrifugation. Subsequently, the M2‐Exo was immobilized on the janus scaffold via surface modification using a cationic compound polyethyleneimine. Finally, the immunomodulatory effects of M2‐Exo‐functionalized janus fibrous scaffolds on bone and muscle tissue remodelling were evaluated by rat muscle injury model and cranial bone defect model.

Results : Western blot results verified that the combination of IL‐4 and IL‐13 stimulation of bone marrow‐derived macrophages (BMDMs) can obtain regenerative M2‐Exo with stable and high expression of IL‐4. In vitro cell experiments showed that M2‐Exo could be internalized by L6 myoblast, bone mesenchymal stem cells (BMSCs), human umbilical vein endothelial cells (HUVECs), and BMDMs to promote cell migration, tube formation, myogenic differentiation, osteogenic differentiation, and anti‐inflammatory macrophage polarization. Animal experiments results indicated that M2‐Exo‐immobilized janus scaffolds accelerated vascularized bone remodelling and muscle regeneration. Histological analysis showed that M2‐Exo could regulate FBR by alleviating the excessive fibrosis caused by scaffold implantation, promoting neovascularization, and activating more pro‐regenerative CD206+ macrophages during bone and muscle remodelling.

Summary/Conclusion : M2‐Exo‐functionalized janus electrospun fibrous scaffolds have great application potential in bone and muscle remodelling. We believe that these findings will contribute to the design and new paradigm of scaffolds in orthopedics and maxillofacial surgery.

The atlas of renal cell carcinoma: analysis of tissue‐derived extracellular vesicles from tumour and surrounded healthy tissue

Gaia Bianchi 1,2 , Elisa De Lorenzis 3 ; Deborah Mattinzoli 4 ; Giuseppe Castellano 1,4 ; Giovanni Montini 1,2 ; Emanuele Montanari 1,3 ; Giancarlo Albo 1,3 ; Benedetta Bussolati 5 and Federica Collino 1,2

1 Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy 2 Laboratory of Translational Research in Paediatric Nephro‐Urology, Paediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy. 3 Department of Urology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy 4 Renal Research Laboratory, Department of Nephrology, Dialysis and Kidney Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy. 5 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy

Introduction : Renal cell carcinoma (RCC) accounts for 90% of kidney malignancies. One‐third of the RCC patients presenting with metastasis at diagnosis and high mortality rates. Extracellular vesicles (EVs) are relevant players in the tumour microenvironment. Tissue‐derived EVs mimic the different tissue components and the possible pathological mechanisms triggering a disease such as cancer. Our study aims to identify biomarkers useful for tumor diagnosis and stageing in RCC by screening tissue‐EV composition in tumour and adjacent tumour‐free tissue.

Methods : Thirty‐five patients with RCC were enrolled (each providing informed consent) and their clinical data were recorded. Fourteen patients underwent total nephrectomy, allowing the analysis of both tumour and adjacent tumour‐free tissue. Transmission electron microscopy (TEM) was used to assess the presence of EV‐like particles in the kidney tissue, and EVs were extracted from tumour and non‐tumour tissues using an optimized enzymatic protocol. Nanoparticle tracking analysis (NTA) was used to measure EV size and number, and flow cytometry analysis using the MACSPlex kit allowed the EV surface characterization. Cellular composition was assessed by MACSima Imaging Cyclic Staining (MICS). RNA from tissues and EVs was extracted and sequenced to characterize their non‐coding RNA cargo.

Results : EV‐like particles were detected within the kidney tissue accumulating in the extracellular space by TEM. No significant differences were observed in particle count and tetraspanin expression between EVs extracted from either tumour or adjacent tumour‐free tissue. Tumour‐derived EVs, however, showed an increase in immune‐related markers, mirroring the immune infiltration observed in tumour tissues by MICS. Additionally, tumour‐EV displayed reduced levels of epithelial and endothelial molecules, reflecting the altered expression of these markers in RCC as well as the loss of organized epithelial morphology detected in the tissue through spatial proteomics.

Summary/Conclusion : We optimized an EV isolation protocol for kidney biopsies. EVs from RCC and adjacent tumour‐free kidney tissue exhibit distinct patterns of immune‐ and epithelial‐related markers, reflecting the cellular alterations within the tissues. The correlation between EV markers and patients’ clinical characteristics will be instrumental in the development of an EV‐based molecular panel for RCC diagnosis and stageing.

Funding : Funding support from IMPACTsim S.p.A. (Grant P‐0038) and the University of Milano (PSR‐2022).

Dual Metalloproteases Regulate Osteoclast‐mediated Physiological and Pathological Skeletal Remodelling

Presenter: Lingxin Zhu

Wuhan University, China (People's Republic)

Introduction : Myeloid‐lineage osteoclasts actively remodel both the mineral and proteinaceous components of bone during normal growth and development as well as pathologic states ranging from osteoporosis to bone metastasis. The cysteine proteinase cathepsin K confers osteoclasts with potent type I collagenolytic activity; however, cathepsin K‐null mice, as well as cathepsin K‐mutant humans, continue to remodel bone and degrade collagen by as‐yet‐undefined effectors. Herein, we identify a cathepsin K‐independent collagenolytic system in osteoclasts that is composed of a functionally redundant network of the secreted matrix metalloproteinase MMP9 and the membrane‐anchored matrix metalloproteinase MMP14.

Methods : We generated myeloid‐specific Mmp14 conditional knockout mice in either wild‐type or Mmp9 null backgrounds. Primary bone marrow‐derived macrophages were isolated and differentiated into osteoclasts. Bone resorption was assessed in osteoclast‐bone wafers in vitro or in a calvaria organ culture model ex vivo.

Results : Unexpectedly, whereas deleting either of the proteinases individually leaves bone resorption intact, we find that Mmp9/Mmp14 conditional double‐knockout osteoclasts display major defects in bone resorption in vitro. In vivo, Mmp9/Mmp14 conditional double‐knockout mice exhibited marked increases in bone density and displayed a highly protected status against either parathyroid hormone‐ or ovariectomy‐induced pathologic bone loss. Wild‐type osteoclasts inefficiently resorbed of Col1ar/r bone ex vivo, while Col1ar/r mice femurs exhibited an increase in trabecular bone density. In an effort to define the signalling mechanisms underlying a combined requirement for MMP9 and MMP14, bioinformatic analysis illustrates that double‐knockout osteoclasts—as well as MMP‐inhibited human osteoclasts—unexpectedly display major changes in transcriptional programs in tandem with compromised RhoA activation, sealing zone formation and bone resorption. Further study revealed that osteoclast function is dependent on the ability of MMP9 and MMP14 to cooperatively proteolyze the β‐galactoside–binding lectin, galectin‐3, on the cell surface. Mass spectrometry identified the galectin‐3 receptor as low‐density lipoprotein‐related protein‐1.

Summary/Conclusion : Together, these studies characterize a previously unsuspected role for dual proteolytic signalling in regulating the turnover of galectin‐3 lattice to govern osteoclast activation in tandem with the ability of the dual metalloproteases to mediate bone collagenolytic effects, and identify the myeloid‐derived dual metalloproteases axis as a potential target for therapeutic interventions for bone‐wasting disease states.

Funding : NSFC grants 82370914 and 81970919.

Transglutaminase 2 is externalised by urinary vesicles and facilitates extracellular vesicles‐matrix and cells dynamics

Maria Pia Savoca 1 , Timothy S. Johnson 2,3 , Elisabetta AM Verderio 1,4

1 Nottingham Trent University, United Kingdom, 2 University of Sheffield, United Kingdom, 3 Mestag Therapeutics Limited, United Kingdom, 4 University of Bologna, Italy.

Introduction : Increased externalization of the crosslinking enzyme transglutaminase 2 (TG2) is linked with fibrosis progression in CKD and occurs via extracellular vesicles (EVs) (Furini, JASN 2018). We have hypothesised a novel role for EVs‐TG2 in tuning EVs‐dynamics in the extracellular space.

Methods : Clinical urine samples were obtained by informed consent from the Sheffield and Patras University biorepositories. TG2 was characterised in urinary EVs (uEVs) by enzyme‐activity assay, immune electron microscopy and single uEVs quantifications (Exoview R200). Mouse embryonic fibroblasts MEFTG2WT and MEFTG2KO were from a transgenic model.

Results : Small uEVs isolated from samples of CKD patients, stratified as “progressive” according to eGFR‐loss (> 4 mL/min/year, n = 18), displayed a significantly increased TG2 level on the uEV surface and higher transamidase activity ( p < 0.05) compared to uEVs from “stable” patients (< 1.2 mL/min/year, n = 16). Transglutaminase activity was detected in both intact uEVs and total lysates, confirming that TG2 is present on the uEV surface. To understand the role of TG2 in vesicular uptake by recipient cells, we employed PKH67‐labelled small EVs isolated from MEFTG2WT and MEFTG2KO conditioned medium. EVs‐TG2WT uptake by MEF was significantly higher than the uptake of EVs‐TG2KO ( p < 0.0001) and EVs‐TG2WT entered the cell cytosol. On the contrary, EVs‐TG2KO led to a slight punctuate fluorescence in the recipient cells. Moreover, EVs‐TG2WT led to the visualisation of TG2 cross‐linking in situ on the surface and inside MEFTG2KO, confirming EVs‐transfer of TG2 biological activity in recipient cells. In the fibronectin extracellular matrix (ECM), EVs‐TG2WT better adhered to the ECM than EVs‐TG2KO ( p = 0.024), suggesting that EVs‐TG2 could affect EV‐migration and adhesion. To identify cell‐surface targets of cross‐linking by EVs‐bound TG2, MEFTG2KO were incubated with either EVs‐TG2WT or negative control EVs‐TG2KO in the presence of the biotinylated TG2 amine‐substrate cavaderine, and the specific biotinylated substrates were revealed by mass spectrometry. Desmoplakin, vinculin, fibronectin emerged as specifically modified by EVs‐TG2.

Summary/Conclusion : We report for the first time that protein transamidation mediated by EVs‐TG2 contributes to the collabourative role of EVs and ECM/cell surface in the extracellular space.

Funding : NTU Vice Chancellor studentship to EAMV and MPS.

Extracellular vesicles of podocytes impact intraglomerular signalling and parietal epithelial cell activation

Pamella Marie Sendon 1,2 , Annika Gathmann 1,2 , Alexander Pausch 1,2 , Kilian Teicher 1,2 , Inka Homeyer 1,2 , Moritz Lassé 1,2 , Milagros N. Wong 1,2,3,4 , Anja Obser 1,2 , Hayritten Yavuz 7 , Hetty Wong 5 , Nicola Wanner 1,2 , Maja T. Lindenmeyer 1,2 , Lawrence B. Holzman 5 , Markus Rinschen 1,2 , Gunther Zahner 1,2 , Christian Krebs 1,2 , Franz L. Ricklefs 6 , Victor G. Puelles 1,2,3,4 , Uta Erdbrügger 7 , Tobias B. Huber 1,2 , Fabian Braun 1,2,8

1 III. Department of Medicine, University Medical Center Hamburg‐Eppendorf, Germany, 2 Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg‐Eppendorf, Germany, 3 Department of Clinical Medicine, Aarhus University, Denmark, 4 Department of Pathology, Aarhus University Hospital, Denmark, 5 Renal‐Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania, USA, 6 Department of Neurosurgery, University Medical Center Hamburg‐Eppendorf, Germany, 7 Department of Medicine, Division of Nephrology, University of Virginia, USA, 8 Martin Zeitz Center for Rare Diseases, University Medical Center Hamburg‐Eppendorf, Germany

Introduction : Extracellular vesicles (EVs) have the ability to impact basic pathological processes such as malignant, metabolic and autoimmune diseases through intercellular signalling. However, we lack a concise knowledge about their role in kidney health and disease. Our study aims to characterize the signalling propagated by large (lEVs) and small EVs (sEVs) shed by podocytes specialized cells of the kidney filtration unit and whether these impact the development of kidney disease.

Methods : Using differential (ultra‐)centrifugation and filtration we separated lEVs and sEVs from cell culture supernatants, kidney tissue and urine samples. Using Western Blot, immunofluorescence microscopy employing antibody stainings and pKH membrane dyes as well as image flow cytometry and cryo‐electron microscopy (cryo‐EM) we investigated the release dynamics of podocyte‐specific EVs in different models of murine podocyte damage in vitro and in vivo. Potential signalling factors contained in EVs were analysed through proteomics. Live microscopy and cross‐culture experiments were used to determine the effect of podocyte‐specific EVs on neighbouring kidney cells—parietal epithelial cells (PECs).

Results : Upon podocyte damage, we detected a unified response of an increased EV release with a size‐shift towards larger vesicles revealed in Cryo‐EM. Depending on EV size and the initial insult, podocyte‐specific EVs exerted different effects on the migratory behaviour and proliferation of PECs corresponding to distinct morphological differences in cryo‐EM. Interestingly, EV proteomics revealed limited differences in the in different stress conditions. We shortlisted the first candidate proteins potentially propagating the effect on PECs. In vivo, decreased EV release by podocytes resulted in reduced PEC activation and limited recruitment of macrophages in a model of inflammatory kidney disease.

Summary/Conclusion : We present essential insights on podocyte‐specific release of different sizes of EVs, their protein contents and functional implications in health and upon podocyte damage. Ongoing experiments focus on further elucidating the impact of podocyte‐specific release in vivo and the impact of knocking‐out identified EV candidate proteins.

Funding : German Research Foundation. German Federal Ministry of Education and Research

The role of circulating exomiRs in thermogenic adipose tissue‐skeletal muscle crosstalk during exercise

Presenter: Dominik Tischer

University Bonn, Germany

Introduction : Brown and beige adipose tissue (BAT) play a crucial role in non‐shivering thermogenesis, dissipating energy as heat. BAT activity is inversely related to age and body mass index (BMI), making it a potential therapeutic target for obesity and metabolic disorders. “Browning” of white adipose tissue (WAT) is a promising approach for improving metabolic health. Exercise may modulate browning by releasing extracellular vesicles (EVs) containing microRNAs (exomiRs), which act as cellular messengers. In animal models, exercise‐induced exomiRs have been shown to promote WAT browning, but their specific mechanisms and relevance in humans remain unclear. We hypothesized that exercise‐derived exomiRs from skeletal muscle contribute to enhanced thermogenesis through browning.

Methods : Serum samples from athletes were collected before and after an acute exercise intervention. EVs were isolated for small RNA sequencing and proteomic analysis. To simulate exercise in vitro, electrical pulse stimulation (EPS) was applied to muscle cells, allowing us to study exercise‐induced exomiR release and identify those involved in muscle‐adipose communication. Differentially expressed exomiRs were analysed for their role in browning and cellular origin.

Results : Several exomiRs were differentially expressed in response to exercise, both in vivo and in vitro. One exomiR, linked to browning, was highly expressed following EPS of muscle cells, indicating its muscle origin. Another exomiR increased the expression of thermogenic markers such as beta‐adrenergic receptors in white adipocytes and enhanced myogenic factors in muscle cells, suggesting it promotes thermogenic crosstalk. Additionally, this exomiR was negatively correlated with age and BMI, suggesting a link to improved metabolic health.

Summary/Conclusion : Our study shows that exercise, modeled by EPS in vitro, stimulates the release of muscle‐derived exomiRs that may promote browning in adipose tissue. These findings provide insights into the thermogenic crosstalk between muscle and adipose tissue, with potential implications for exomiR‐based biomarkers and therapies targeting metabolic diseases.

Technologies and Methods

Subtopic Analysis of EV‐Cargo 2

Biophysical study of extracellular vesicle mechanoporation using a nanofluidic device

Presenter: Rui Hao

Shenzhen Institute of Advanced Technology, China (People's Republic)

Introduction : Small extracellular vesicles (sEVs) are cell‐derived lipid‐bound nanoparticles typically smaller than 200 nm, serving as vital intercellular mediators in various (patho)physiological processes. Their potential in drug delivery is immense, yet efficient cargo loading remains a significant challenge. Conventional engineering loading methods, such as cuvette electroporation, suffer from limitations including mismatched feature dimensions, inconsistent stimulation, and sEV damage. Nanofluidic mechanoporation, which involves generation of transient nanopores in sEV membranes to facilitate exogenous substance uptake while minimizing disruption, offers a promising alternative. However, the underlying mechanisms governing nanopore dynamics during this process remain inadequately understood.

Methods : This study proposes a nanofluidic device tailored for the biophysical study of sEV mechanoporation via hydrodynamic stretching. Initially, a computational fluid dynamics model was developed to simulate sEV deformation and force distribution as they navigate through a contraction‐expansion nanochannel, deepening our understanding of fluid‐sEV interactions. Delving into the biophysical properties of sEVs during this process lays a crucial theoretical foundation for optimizing nanofluidic designs to provide new engineering methods. Subsequently, the simulation model was experimentally validated by observing membrane mechanoporation and quantifying cargo loading efficiency.

Results : Cryogenic transmission electron microscopy confirmed that fluid‐dynamic stresses within the nanochannels induce sEV deformation, leading to membrane permeabilization and the generation of transient nanopores. Additionally, nano‐flow cytometry analysis, using Alexa Fluor 488‐labelled dextran (10 kDa, ∼5 nm) as a model cargo, revealed that the proportion of dextran‐loaded sEVs increased with higher sample flow rates. This finding aligns with theoretical hydrodynamic studies, suggesting that elevated flow rates enhance fluid‐induced stresses, thereby promoting membrane deformation and permeability. Detailed experimental data and mechanistic insights will be elabourated upon at the conference.

Summary/Conclusion : In summary, the nanofluidic mechanoporation platform effectively permeabilizes sEV membranes by precisely modulating membrane deformation and transient nanopore formation through hydrodynamics, facilitating controlled cargo loading. This technology not only serves as a robust tool for EV engineering but also holds promise as a versatile platform for the exploring biophysical properties of EVs. By integrating theoretical modelling with experimental validation using the nanofluidic platform, we could pave the way for advancing the understanding and application of sEVs in nanomedicine and beyond.

Surface adhesion and membrane fluctuations influence the elastic modulus of extracellular vesicles

Presenter: Apurba Dev

Electrical Engineering, Sweden

Introduction : The elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. However, accurate quantification of their elastic modulus is challenging due to their nanoscale dimensions and their fluid‐like lipid bilayer.

Methods : Here we apply the Canham‐Helfrich model to quantify the elastic properties of EVs from force spectroscopy performed by an atomic force microscope. We then apply our method to EVs derived from the human embryonic kidney (HEK293T) cell line and different families obtained by genetically altering it's tetraspanins expression. Each of the EVs were carefully analysed with over 150 of force indentation to capture their fluctuations and the data were theoretically modeled. The probability distribution functions obtained were differentiated with Jensen‐Shanon divergence.

Results : We find that in contrast to previous reports the EVs behave as surface‐adhered vesicles showing a linear dependence of tether force on the tether length. The bending modulus estimated for the different EV families range from 15.7 − 33.0 kBT with large fluctuations within each family of vesicles which is consistent with inherent EV heterogeneity. Interestingly, our experiments also show large fluctuations of measured values even for a single EV when repeatedly examined under identical conditions. The data consequently show a large spread in their elastic modulus and bending modulus in the range 4‐19 mN/m and 15.7‐33 kBT, respectively, concealing any potential differences among the families of the EVs. Only the family of CD63‐knockout sample exhibited detectable differences with the wild‐type sample.

Summary/Conclusion : We show that linear dependence of tether force on tether length is consistent with the theory of adhered vesicles. We suggest that the fluctuations in the individual vesicles is driven by both thermal and compositional effects, as expected for such nanoscale objects. We also show that the probability distribution function (PDF) of elastic moduli are best described by not a single Gaussian but superposition of Gaussians, thus, necessitating appropriate statistical approach. The study suggest that protein alternations might have an influence on the elastic moduli, but they are often hidden in such large fluctuations.

Funding : The Swedish Research Council; The Swedish Cancer Foundation.

Impact of centrifugal forces on the integrity of small extracellular vesicles

Sunyoung Ham 1,2 , Xuanxuan Li 1,2 , Yue Su 1, 2 , Su‐Ho Park 1, 2 , Andreas Möller 1,2

1 Chinese University of Hong Kong, Hong Kong SAR 2 Li Ka Shing Institute of Health Sciences, Hong Kong SAR

Introduction : Ultracentrifugation is the most commonly employed method for enriching small extracellular vesicles (sEVs) due to its efficiency in pelleting small, buoyant particles. While there are established guidelines regarding optimal centrifugal force and time to prevent cell and organelle damage, limited data exist on the specific impacts of ultracentrifugation on sEV integrity. This study aims to investigate how different centrifugal forces affect the physical properties of sEVs.

Methods : To estimate the forces exerted on sEVs and liposomes during centrifugation, theoretical calculations were conducted, focusing on potential limits for shape deformation and rupture based on membrane composition. Experimentally, sEVs from different natural sources and manufactured liposomes were labelled with two different colours and subjected to different centrifugal forces and durations. The samples were analysed through nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, and super‐resolution microscopy to evaluate changes in particle size and shape.

Results : Theoretical calculations indicated that increased centrifugal forces could exceed the rupture threshold for lipid membranes. Moreover, the increased centrifugal forces are capable of altering sEV size and protein distribution, suggesting the hypothesised vesicle rupture or deformation occurring during ultracentrifugation. Notably, liposomes consisting of DOPC (Dipalmitoylphosphatidylcholine) and cholesterol demonstrated greater structural stability compared to sEVs across varied centrifugal forces and durations.

Summary/Conclusion : Understanding the impact on vesicle integrity by ultracentrifugation will enable us to better define the limits of interpretation of sEVs generated by this method as well as optimise centrifugation conditions designed to preserve sEV integrity.

The impact of isolation methods on blood‐based extracellular vesicles characterization: a nanoscale infrared spectroscopy study

Jéssica Verônica da Silva 1 , Otávio Berenguel 2 , Raquel Silva Neres‐Santos 1 , Herculano da Silva Martinho 1 and Marcela Sorelli Carneiro‐Ramos 1

1 Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC, Brazil. 2 Labouratório Nacional de Nanotecnologia (LNNano), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Brazil.

Introduction : Extracellular vesicles (EVs) contain cell‐type specific signatures and have been proposed as biomarkers in various diseases. However, due to their small dimensions, and light diffraction limits, imaging EVs samples in the “fingerprint” range of wavelengths of biomolecules makes mid‐IR spectroscopy impossible to capture images and spectra at the nanoscale by Fourier transform infrared spectroscopy (FTIR). Therefore, we aimed to use the nanospectroscopy (AFM‐IR) technique to elucidate the molecular signature of EVs isolated from blood (serum and plasma), as well as the direct correlation of their topography and biochemical signature concerning the isolation method.

Methods : For this, three isolation methods have been tested using serum or plasma from C57BL/6 male mice and evaluated, where we have carried out the standardization of drying of the isolated vesicle sample and its analysis by micro‐reflectance in FTIR followed by AFM‐IR analysis. The first isolation method used was carried out according to the Total Exosome Isolation Reagent (TEIR) from the serum kit protocol using blood serum; the second isolation method was the isolation from plasma samples by ultracentrifugation at 100,000 × g for 60 min of the plasma isolated; resuspending the pellet in 500 µL of PBS 1× exosome free; and the third and last one method was by size exclusion chromatography (SEC) and resuspending the pellet in 500 µL of PBS 1× exosome free.

Results : The results largely corroborate the schematic spectrum proposed by the literature on infrared signatures from studies dedicated to characterizing extracellular vesicles from blood. However, the spectra also overlap with albumin signature, except for bands associated with membrane‐bound oligosaccharide, lipids, and nucleic acid signatures, where a spectra red‐shift was observed according to the isolation method.

Summary/Conclusion : In this way, knowing the potential use of nanospectroscopy in the study of EVs and the lack of information about the spectral composition of blood‐based EVs, AFM‐IR proved to be a great tool to investigate the impact of different isolation methodologies and their artifacts in EV spectral characterization.

Funding : This research was funded by Brazilian agencies Fundação de Amparo à Pesquisa do Est. de S. Paulo (FAPESP), proc. 2022/00153‐0, 2023/06583‐0; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), proc. 311933/2021‐1.

Characterization of extracellular particles directly in diluted blood and plasma by ultraviolet‐visible spectroscopy and interferometric light microscopy

Presenter: Anna Romolo

University of Ljubljana, Slovenia

Introduction : Harvesting of extracellular particles (EPs) from bodily fluids and their assessment is challenging as the processing significantly impacts the samples. Ultraviolet and visual spectroscopy (UV‐vis) and Interferometric Light Microscopy (ILM) are minimally invasive methods based on the absorption of light, and light scattering and interference, respectively. UV‐vis yields the proportion of nucleic acid and protein content and ILM yields the number density n and the hydrodynamic diameter Dh of EPs (sized 80 nm to 500 nm). A threshold saturation of detected light can be reached by appropriate dilution of samples. Isolation of EPs before assessment is not necessary.

Methods : The study included 82 samples of canine blood, 73 samples of canine plasma, 230 samples of human blood, and 286 samples of human plasma. For EP assessment in blood, blood was diluted with saline. Plasma was obtained by centrifugation of blood and subsequently diluted with saline. Absorbances of light at wavelengths 260 nm and 280 nm were assessed with the Nanodrop spectrometer (Thermofischer Scientific, Waltham, MA, USA). EP parameters n and Dh were assessed with the Videodrop ILM (Myriadelab, Paris, France).

Results : A statistically significant correlation between the 280/260 ratio and number density of EPs was obtained in canine plasma ( r = 0.24, p = 0.03) and in human plasma ( r = 0.36 with ethylenediaminetetraacetic acid anticoagulant, 0.33 with trisodium citrate anticoagulant, p < 0.0001), but not in blood. In canine and human blood and plasma a strong correlation was found between absorbance at 260 and 280 nm (all r > 0.99; p < 0.00001). The average 260/280 ratio was 0.95 in human blood, 0.94 in canine blood, 0.58 in human plasma, and 0.60 in canine plasma. In both species, the average Dh in blood was statistically significantly larger than in plasma.

Summary/Conclusion : ILM and UV‐vis spectroscopy enable analysis of large populations of samples. As regards characterization with ILM and UV‐vis, human and canine samples showed similar features. In plasma of both species, UV‐vis indicated a higher protein than nucleic acid content compared to blood samples which positively correlated with the number density of EPs.

Funding : Slovenian Research Agency: J3‐3066, P3‐0388, P2‐0232, P2‐0132, P4‐0053, J2‐4427, J2‐4447, and EU Horizon 2020 MSC Staff Exchange: FarmEVs, 101131175.

How do milk‐derived extracellular vesicles cross the gut barrier?

Rachel Butler 1 , Yunyue Zhang 2 , Driton Vllasaliu 2 , Alan Goddard 1

1 Aston University, UK. 2 Kings College London, UK

Introduction : Oral delivery of biologics, like nucleic acid therapies, could transform treatment through non‐invasiveness and ease of administration, making it more cost‐effective and accessible. However, progress remains limited by enzymatic degradation and poor gastrointestinal absorption. Current platforms, such as lipid nanoparticles and liposomes, suffer from low encapsulation efficiency and instability. Extracellular vesicles (EVs) are promising alternatives due to their ability to cross biological barriers. Among these, milk‐derived EVs (mEVs) demonstrate superior stability and transport across gut epithelium compared to other EVs and synthetic systems. This work aims to uncover the mechanisms enabling mEVs' efficient gut epithelial transport.

Methods : mEVs were isolated from raw bovine milk according to MISEV2023 guidelines using ultracentrifugation and size‐exclusion chromatography, with a chymosin‐incubation step to eliminate casein micelles and improve vesicle purity. mEVs were harvested from the apical and basal sides of an in vitro intestinal epithelium model (Caco‐2 cell monolayer). Predicting the mEV surface's role in transport, we isolated mEV membranes to characterise membrane‐associated proteins. Mass spectrometry identified key components of the mEV lipidome and proteome that may be involved in intestinal permeability.

Results : Comparative analysis of permeating ‘basal’ and non‐permeating ‘apical’ mEV populations revealed distinct molecular features associated with enhanced gut permeability. Basal mEVs were enriched in certain fatty acids and acyl chains, suggesting altered mechanical membrane properties could promote transcytosis. Proteomic analysis showed a depletion of membrane proteins in basal mEVs, indicating that overall membrane characteristics, rather than specific ligand‐receptor interactions, may govern gastrointestinal stability and transport across the intestinal epithelium. Furthermore, whilst mEVs are thought to derive from a diverse mixture of cell types, basal mEVs were depleted in immune markers, hinting that basal mEVs may originate from a distinct population of non‐immune cells, such as mammary epithelial cells.

Summary/Conclusion : By identifying key intestinal‐permeating components, we provide insight for engineering gut‐permeable EVs. Future work will optimise a method of enriching the permeating mEV population by affinity purification to increase the efficiency of drug delivery for future engineered mEV therapies. This study demonstrates the potential of mEVs as scalable and cost‐effective platforms, paving the way for next‐generation oral delivery of biologics.

Funding : UKRI Engineering Biology Mission Award (BB/Y008065/1)

Multi‐Parametric Surface Plasmon Resonance—a label‐free new technology for EV corona and cell interaction studies

Presenter: Abhishek Sharma

Bionavis Ltd., Finland

Introduction : Multi‐Parametric Surface Plasmon Resonance (MP‐SPR) is a label‐free technique for detecting and quantifying biochemical interactions, such as drug‐protein binding, in real time without labels like fluorophores or radiolabels. MP‐SPR collects the complete SPR angular spectrum, enabling analysis of parameters like thickness, refractive index, and optical dispersion. It is compatible with crude samples, including serum.

Methods : MP‐SPR instruments can be used to study extracellular vesicles (EVs) and their interactions with plasma or serum. When EVs interact with plasma, they can form a protein layer known as the EV protein corona, which might affects EV function [1]. MP‐SPR has been used to measure the thickness of this protein layer on nanoparticles [2]. Otto et al., for example, measured the ‘soft’ and ‘hard’ corona layers formed on EVs by plasma [2]. Lipophilic dyes (used to label EVs), can interfere with EV measurements and uptake studies [3,4], MP‐SPR enables label‐free study of EV interactions with mammalian cells [5‐7].

Results : MP‐SPR, is also a valuable tool in EV diagnostic and therapeutic research, allowing precise measurement of binding kinetics between EVs and capture agents like antibodies or aptamers [8].

Summary/Conclusion : BioNavis's multi‐wavelength instruments enhance MP‐SPR's utility by enabling EV size and concentration measurements, especially for small EVs (< 100 nm) [9‐11]. These measurements leverage different evanescent fields produced by multiple wavelengths. BioNavis's regenerable avidin for fully reversible ligand immobilization tool for EV capture [12,13], making it a powerful, versatile tool for EV and therapeutic research.

Funding : 1. Dietz et al. https://doi.org/10.1002/jev2.12399. 2. Otto et al. https://doi.org/10.1007/s13346‐016‐0320‐0. 3. Dominkus et al. https://doi.org/10.1016/j.bbamem.2018.03.013. 4. Wan et al. https://doi.org/10.2147/IJN.S475060. 5. Koponen et al. https://doi.org/10.1016/j.bios.2020.112510. 6. Suutari et al. https://doi.org/10.1002/smLl.201601815. 7. Scurti et al. https://doi.org/10.1021/acs.langmuir.3c00728. 8. Carney et al. https://doi.org/10.1002/adbi.201600038. 9. Parkkila et al. https://doi.org/10.1016/j.colsurfa.2022.130015. 10. Norling et al. https://doi.org/10.1021/acs.langmuir.1c03096. 11. Rupert et al. https://doi.org/10.1021/acs.analchem.6b01860. 12. https://www.bionavis.com/en/publications/application‐notes/166‐regenerable‐avidin‐kit‐assay‐working‐principle 1/13. https://www.bionavis.com/en/publications/application‐notes/168‐regenerable‐avidin‐kit‐assay‐concentration‐kinetics/

Evaluation of MagReSyn SAX and ENRICH‐iST methods for liquid biopsy proteomics

Presenter: Ryo Takahashi,

Nippon Boehringer Ingelheim Co., Ltd., Japan

Introduction : One of the biggest challenges in LC‐MS‐based plasma proteomics is to reduce the dynamic range of plasma proteins to capture the deregulation of low abundant proteins. Resin Biosciences’ MagReSyn SAX technology can enrich a heterogeneous group of membrane‐associated particles from plasma. In this study, this method was compared with a physicochemical enrichment method (PreOmics’ ENRICH‐iST) for plasma proteomics.

Methods : EDTA plasma of healthy control (SD, male, n = 3) and type 2 diabetes (ZDF, male, n = 3) rat were purchased from the Jackson Laboratory Japan, Inc. This animal study was approved by the institutional review board. Twenty µL of plasma was processed by MagReSyn SAX and ENRICH‐iST magnetic beads and then digested by trypsin/Lys‐C and DIGEST solution, respectively. The digested peptides were analysed by an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) equipped with a Vanquish Neo UHPLC system (Thermo Fisher Scientific) using data‐independent acquisition mode. The raw data were processed by DIA‐NN software (Ver. 1.8.1). Statistical analysis was conducted using Perseus (Ver. 1.6.15.0) and R (Ver. 4.3.1). Deregulated pathways were investigated using Ingenuity Pathway Analysis (IPA) (QIAGEN).

Results : Through the combination with the optimized LC‐MS method, the proteome coverage reached to 5825 and 5434 in MagReSyn SAX and ENRICH‐iST methods, respectively. Each protein abundance of the known EV markers (e.g., Cd9 and Cd63) and plasma abundant proteins (e.g., Alb and A2m) between MagReSyn SAX and ENRICH‐iST samples were comparable. Whereas the tissue‐specific membrane proteins such as Asgr2 and Ncam1 were highly enriched (> 15, 20‐fold, respectively) in MagReSyn SAX samples. Using IPA, sets of 219‐down and 80‐upregulated proteins (fold change ≥ 1.5, pBH ≤ 0.05) were identified with the MagReSyn SAX method while sets of 68‐down and 43‐upregulated proteins (fold change ≥ 1.5, pBH ≤ 0.2) were identified with the ENRICH‐iST method. Some of the significantly deregulated pathways (ex. Elastic fibre formation) were uniquely identified by MagReSyn SAX method.

Summary/Conclusion : Plasma proteomics using the membrane‐associated particles enriched by MagReSyn SAX method was shown to be a powerful tool to identify deregulated proteins and pathways. Possible applications are biomarker discovery and efficacy/safety assessment in drug development.

Glycosaminoglycan disaccharide analysis of lung cancer cell line derived small extracellular vesicles

Mirjam Balbisi 1,2 , Tamás Langó 1 , Virág Horváth 1 , Domonkos Pál 1,2 , Gitta Schlosser 3 , Zoltán Varga 1 , Kinga Ilyés 1 , Nikolett Nagy 1 , Otília Tóth 1 , Tamás Visnovitz 2 , Beáta G. Vértessy 1 , Lilla Turiák 1

1 HUN‐REN Research Centre for Natural Sciences, Hungary, 2 Semmelweis University, Hungary, 3 Eötvös Loránd University, Hungary

Introduction : Proteoglycans play an important regulatory role in the extracellular matrix, the balance of which is disrupted in many cancers. Proteoglycans contain glycosaminoglycan (GAG) chains that consist of repeating disaccharide units, which can be sulfated at various positions. Here, we present the chondroitin sulfate (CS) GAG disaccharide analysis of small extracellular vesicles (sEVs) derived from A549 lung adenocarcinoma and BEAS‐2B non‐tumourigenic epithelial cell lines.

Methods : A549 cells were cultured in fetal bovine serum‐free F12 medium, while BEAS‐2B cells were cultured in BEGM medium for 72 h. Next, the supernatant was collected and after centrifugation steps, small EVs were isolated by mini‐size exclusion chromatography on in‐house prepared columns. Freeze‐thaw cycles were followed by an overnight digestion step by chondroitinase ABC enzyme. The resulting disaccharides were purified by a cotton‐graphite solid phase extraction method and analysed by HPLC‐MS(MS). 6‐6 parallel samples were measured for both cell lines from separate EV isolations.

Results : The most abundant CS disaccharides: the non‐sulfated D0a0, the monosulfated D0a4 and D0a6 (distinguished by tandem MS measurements), and the disulfated D0a10 disaccharides were successfully identified in each sample. Cancer EVs contained on average three times more CS disaccharides than non‐tumour EVs. In terms of relative amounts, the proportion of D0a4 increased and the proportion of D0a6 decreased in A549 EVs. Based on both absolute and relative amounts of the disaccharides, the two groups were well separated in principal component analysis and hierarchical clustering.

Summary/Conclusion : In the current study, we performed CS GAG disaccharide analysis of sEVs derived from lung cancer and non‐cancer cell lines. To the best of our knowledge, this is the first study to characterize the glycosaminoglycan profile of EVs, which might be a new area of biological interest.

Funding : Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences and Semmelweis 250+ Excellence PhD Scholarship is acknowledged.

Role of bacterial extracellular vesicles for immunomodulation using omics analysis

Presenter: Sofia P. Leistl

Universitätsklinikum Erlangen, Germany

Introduction : Multiple studies highlight bacterial extracellular vesicles (bEVs) as novel key player of immunomodulation within host microbiome communication. Vesicles released by the gut microbiome may significantly impact the progression of gut located inflammatory bowel diseases (IBD). A comprehensive knowledge of the bEV cargo and surface composition and cross‐species interaction is crucial to gain better understanding about immune mechanisms that influence the disease.

Methods : Cultures from different bacteria were grown for bEV isolation. Disease‐associated bacterial strains (Klebsiella pneumoniae, Proteus mirabilis), derived from patient isolates, as well as E. Coli Nissle 1917, which is approved as a probiotic for medication, and the Laboratory strain E. Coli Bl21 DE3 were used for analysis. BEVs were purified, tested for quantity and quality via NTA and TEM and further characterized by proteomic analysis via MS measurement. The results were first analysed with Perseus (http://www.perseus‐framework.org) and further with the tool DAVID (Database for Annotation, Visualization and Integrated Discovery).

Results : BEVs exhibited as expected strain‐dependent signatures in their proteomic profile. Size distribution of vesicles and TEM imaging revealed additionally distinct physical characteristics. Differences of enriched protein groups were highlighted and might explain possible sources for pathogenic effector molecules. Strain dependent pathway enrichment further suggests varying immunomodulatory effects, underscoring the potential influence of BEVs on host immune responses.

Summary/Conclusion : Our analyses suggest that the protein composition of bEVs elicit strain‐specific fingerprints that are transmitted in a bEV‐strain‐specific immune response to the host. A deeper understanding of proteomic profiles will be a crucial to fully elucidate these interactions. Multiple observations identifying K. pneumoniae as a driver of IBD, could be sourced to functional protein groups and should be undergoing deeper investigations. Moreover, bEVs could be further explored for new diagnostic and therapeutic strategies of gastroenterological diseases.

Funding : I want to acknowledge the support by Interdisziplinäres Zentrum für Klinische Forschung (IZKF, Jochen Kalden Junior Research Group N5) and by the collabourative Research Center 241 (Transregio 241: Immune‐ Epithelial Communication in Inflammatory Bowel Diseases, Group A02) to enable this scientific project.

N‐glycosylation features of A549 and BEAS‐2B cell line‐derived small extracellular vesicles

Mirjam Balbisi 1,2 , Tamás Langó 1 , Virág Horváth 1 , Zoltán Varga 1 , Kinga Ilyés 1 , Gábor Kecskeméti 3 , Zoltán Szabó 3 , Nikolett Nagy 1 , Otília Tóth 1 , Beáta G. Vértessy 1 , Lilla Turiák 1

1 HUN‐REN Research Centre for Natural Sciences, Hungary, 2 Semmelweis University, Hungary, 3 University of Szeged, Hungary

Introduction : Post‐translational modifications greatly influence protein function. However, their characterization in extracellular vesicles (EVs) has been limited. N‐glycosylation, essential in processes like cell‐cell interactions, is frequently altered in cancer. This modification occurs at asparagine residues in proteins at consensus sequences. The attached N‐glycans are classified as complex, hybrid, or oligomannose. Our aim was to develop a methodology to analyse the site‐specific N‐glycosylation of glycoproteins of small EVs isolated from A549 lung adenocarcinoma and BEAS‐2B non‐tumourigenic cell lines.

Methods : A549 and BEAS‐2B cells were cultured in non‐completed F12 and BEGM medium for 72 h, respectively. Small EVs were isolated by mini‐size exclusion chromatography in 6 6 replicates per cell line. The size distribution of EVs was characterized by microfluidic resistive pulse sensing and shapes by transmission electron microscopy. Small EVs were subjected to freeze‐thaw cycles and digested with trypsin. The peptides obtained were enriched for N‐linked glycopeptides by acetone precipitation and analysed by nanoUHPLC‐MS(MS).

Results : Altogether close to 1000 N‐glycopeptides were quantified by GlycReSoft software across the 12 samples. 72 N‐glycopeptides were overrepresented, while 161 were underrepresented in the A549 cell line‐derived sEVs compared to the BEAS‐2B cell line‐derived EVs. Overall, changes in fucosylation, sialylation, and galactosylation were observed. Among the significantly altered N‐glycopeptides, we also identified changes corresponding to vesicular marker proteins.

Summary/Conclusion : We have demonstrated that site‐specific N‐glycosylation analysis of glycoproteins present in small EVs is feasible following isolation from cell culture media. Our results lay the basis for future studies to explore the roles of glycosylation of EV proteins in understanding mechanisms behind disease progression, such as lung cancer.

Funding : Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences is acknowledged.

Quantification of HIV‐1 protein Nef extracellular release from in vitro model of translationally competent HIV‐1 reservoir by nanobody‐based ELISA

Samuel Žvanut 1 , Teja Lavrin 1 , Marija Holcar 1 , Nina Mavec 1 , Aleksandra Usenik 2,3 , Sara Pintar 2,3 , Dušan Turk 2,3 , Metka Lenassi 1

1 Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 2 Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia; 3 Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia

Introduction : Translationally competent HIV‐1 reservoirs persist despite effective antiretroviral therapy. The viral protein Nef, released via extracellular vesicles (EVs) from the reservoirs, is implicated in various HIV‐1‐associated pathologies. We recently developed a sensitive nanobody‐based ELISA (nanoNefELISA) to investigate Nef release. This work validates the method using cell culture models of HIV‐1 reservoirs, biological matrices spiked with extracellular Nef, and Nef‐containing EV‐enriched preparations.

Methods : We modeled translationally competent HIV‐1 reservoirs in vitro by co‐culturing varying ratios (0–100%) of doxycycline‐inducible Nef‐EGFP‐expressing human microglia and Jurkat cells with their counterparts lacking Nef‐EGFP. After 48 h of doxycycline induction, we quantified Nef‐EGFP‐positive cells via flow cytometry and measured Nef levels in conditioned media using nanoNefELISA. Further, conditioned media from Nef‐EGFP‐expressing cells were concentrated on 100,000 MW filter units and spiked into biological matrices (serum, plasma, cerebrospinal fluid) for analysis. EV‐enriched preparations were obtained by differential ultracentrifugation of conditioned media of Nef‐EGFP‐expressing cells and characterized with nanoNefELISA, immunoblotting, and nanoFCM.

Results : Nef concentration in conditioned media correlated with the percentage of Nef‐EGFP‐expressing microglia (r = 0.9149) and Jurkat cells (r = 0.9230). Extracellular Nef levels were significantly higher in microglia (mean = 24.88 ± 1.05 ng/10 5 cells) than in Jurkat cells (mean = 1.04 ± 0.08 ng/10 5 cells). We successfully detected extracellular Nef when spiked into the cerebrospinal fluid (detection efficiency of 96.6–104.8%), while detection in plasma and serum was hampered (detection efficiency of 17.1–31.5% and 27.0–52.9%, respectively). The 100,000 K pellets from conditioned media of Nef‐EGFP‐expressing cells tested positive for Nef and EV markers by immunoblotting, with 61.2% of EVs from microglia and 28.4% from Jurkat cells being Nef‐EGFP‐positive, as revealed by nanoFCM. Nef concentrations in 100,000 K pellets reached 51.11 ± 5.59 ng/10 6 cells in microglia and 2.71 ± 0.66 ng/10 6 cells in Jurkat cells.

Summary/Conclusion : The nanoNefELISA assay quantifies Nef release from translationally competent HIV‐1 reservoirs in vitro. High detection efficiency in cerebrospinal fluid suggests the assay's potential in studying HIV‐1‐associated neurocognitive disorders. The assay reveals significant differences in Nef EV release between the two cell types tested, informing future research into Nef EV release and HIV‐1 pathogenesis.

Funding : Slovenian Research and Innovation Agency research grant P1‐0170.

FACS‐Proteomics strategy for extracellular vesicles single‐phenotype characterization in biological fluids: insights into leukocyte‐derived EVs protein cargo and immune system machinery

Maria Concetta Cufaro, Alice Di Sebastiano, Ilaria Cicalini, Marianna Gabriella Rispoli, Giulia Catitti, Domenico De Bellis, Serena Pilato, Antonella Fontana, Valentina Tomassini, Serena Veschi, Alessandro Cama, Paola Lanuti, Luca Federici, Damiana Pieragostino, Piero Del Boccio

University ‘G.d'Annunzio’ of Chieti‐Pescara, Italy

Introduction : The isolation and proteomics characterization of Extracellular Vesicles (EVs) from biological fluids remains an open challenge due to their vast heterogeneity and their suitability for proteomics purposes, once purified. Circulating EVs can enable an extreme reduction in the high dynamic range of detectable protein concentration in whole biofluids, paving the way for the biomarker discovery in the so‐called “dark proteome”. We have recently optimized an innovative “FACS‐Proteomics” workflow for the isolation and subsequent proteomics characterization of EVs directly from untouched biological fluids taking advantage of a lipophilic cationic dye (LCD) able to probe intact EVs. Here we report an update of the workflow suitable for carrying out proteomics on specific EV populations purified by sub‐typing them with an appropriate panel of antibodies.

Methods : FACS strategy was used to purify Leukocyte‐derived EVs (Leuko‐EVs, LCD+/CD45+/Phalloidin‐ events) from blood and tears of healthy volunteers and multiple sclerosis patients. After purification, Atomic Force Microscopy and Nanoparticle Tracking Analysis were used to investigate Leuko‐EVs size distribution, morphology and number. Moreover, EVs were analysed for the expression of CD63 and CD81 by flow cytometry. Label‐free proteomics approach was performed by nanoLC‐Orbitrap‐Fusion‐Tribrid‐Mass Spectrometer; raw data were processed by MaxQuant and loaded on Ingenuity Pathway Analysis for functional investigation.

Results : proteomics study was carried out on LeuKo‐EVs sorted and characterized from blood and tears (1.4x105 and 2x106 events, respectively) displaying a high level of purity (>90%). More than 90% of proteins identified in both biofluids (78 in tears and 155 in blood) were mainly referred to the leukocyte phenotype. This strategy was applied for proteomics evaluation of Leuko‐EVs purified from tears of multiple scleorosis patients, demonstrating that they were packaged with a well‐defined encoded protein cargo, reflecting the neuroinflammatory condition of the disease (TGFB1 and NFE2L2, Upstream Regulators activated) together with an increase of vascular networks (AGNPT2 and VEGF, Upstream Regulators activated) which mirrors pro‐angiogenic processes predicted as downstream effects.

Summary/Conclusion : Our data show that the combined FACS‐Proteomics strategy for EV single‐phenotype characterization could open groundbreaking avenues for biomarker discovery, exalting the clinical value of tears EVs and helping in a better understanding of the EV‐mediated processes in vivo.

Lipidomic and proteomic analysis of tetraspanin‐deficient extracellular vesicles

Presenter: Martin Smolko

Masaryk University, Czech Republic

Introduction : Tetraspanins are integral membrane proteins crucial for the formation, release, and uptake of extracellular vesicles (EVs). While recent studies have focused on single tetraspanin knockouts, a detailed study on the knockout (KO) of the three main tetraspanins (CD9, CD63, and CD81) within the same cellular background has not been conducted yet.

Methods : We isolated EVs from DU145 wild‐type cells and three tetraspanin‐deficient cell lines (CD9, CD63, CD81 KO) generated via CRISPR‐Cas9. We performed both proteomic and lipidomic analyses on the EVs and corresponding cells of origin to identify changes in protein composition and lipid profiles associated with tetraspanin depletion. LC‐MS/MS was employed for proteomic profiling, while targeted HPLC‐MS/MS quantified phospholipids (PL), sphingolipids (SLs), and glycosphingolipids (GSLs) in the KO EVs and cells, enabling us to compare these profiles to those from the wild‐type counterparts.

Results : Our study demonstrates that the absence of tetraspanins induces significant alterations in lipid and protein composition within EVs and cells. Lipidomic data revealed distinctive lipid profiles in EVs from KO cells, notably a significant enrichment of EV GSLs. Proteomic analysis further indicated substantial shifts in cellular and EV protein cargo following tetraspanin KO. This is the first report to demonstrate such significant alterations in EV proteomic and lipidomic composition linked to tetraspanin deficiency.

Summary/Conclusion : Tetraspanins play distinct roles in the regulation of EV biogenesis, sorting, and uptake. By combining proteomic and lipidomic profiling, our analysis offers a multi‐dimensional view of how CD9, CD63, and CD81 contribute to EV biology, advancing our understanding of tetraspanin roles at both cellular and EV levels.

Funding : This work was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102)—funded by the European Union—Next Generation EU. CIISB, Instruct‐CZ Centre of Instruct‐ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127, is gratefully acknowledged for the financial support of the measurements at the CEITEC Proteomics Core Facility.

Revealing brain tumour biomarkers: Optimizing sEV proteomics for liquid biopsy applications

Gabriella Dobra 1,2* , Edina Gyukity‐Sebestyen 1,2* , Matyas Bukva 1,2 , Timea Boroczky 1,2,3 , Regina Csordas 4 , Zoltan Szabo 4 , Almos Klekner 5 and Krisztina Buzas 1,2

1 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, H 6726 Szeged, Hungary; 2 Department of Immunology, Albert Szent‐Gyorgyi Medical School, Faculty of Science and Informatics, University of Szeged, H‐6720 Szeged, Hungary 3 Doctoral School of Interdisciplinary Medicine, Albert Szent‐Gyorgyi Medical School, University of Szeged, H‐6720 Szeged, Hungary 4 Department of Medical Chemistry, Albert Szent‐Gyorgyi Medical School, University of Szeged, H‐6720 Szeged, Hungary; 5 Department of Neurosurgery, Faculty of Medicine, University of Debrecen, H‐4032 Debrecen, Hungary; * Equally contributed to this work

Introduction : Current diagnostic methods for brain tumours, such as computed tomography, MRI, and tissue biopsy, have limitations that highlight the need for less invasive testing, including molecular analysis of body fluids. Small extracellular vesicles (sEVs) are promising targets for liquid biopsy in brain tumours due to their specific properties. By analyzing the protein content of sEVs, tumour markers may be identified; however, isolation and proteomic analysis methods for sEVs need refinement. Our goal is to advance the quantitative and qualitative proteomic analysis of sEVs, enabling effective differentiation between patients with different tumour types. Our method development was focused on selecting an optimal EV isolation technique and creating a robust database.

Methods : In our study, we analysed plasma samples from 9 Glioblastoma and 9 Meningioma patients. sEVs were isolated using differential ultracentrifugation (dUC) and size exclusion chromatography (SEC), and protein coronas obtained with 1.5 M NaCl digestion (EVcorona). Regarding sEV characterization, morphology was measured with transmission electron microscopy (Tecnai G2 20 X‐Twin), size distribution with nanoparticle tracking analysis (NS300), and sEV markers with flow cytometry (Cytoflex S). Proteomic analysis was performed using liquid chromatography‐mass spectrometry (ACQUITY M‐Class UPLC, Exploris 240). Patients were clustered based on proteomes from plasma, dUC and SEC isolates, and EVcorona; while protein intensities were compared using Welch's test.

Results : We established a vesicle proteome database with 719 identified proteins: 553 in plasma, 614 in dUC samples, 681 in SEC‐3 isolates, 637 in SEC‐4, and 584 in SEC‐5 isolates. The SEC‐3 isolate contained the highest number of identified proteins (681). Abundant serum proteins not associated with sEVs, such as albumin, decreased progressively from plasma to dUC and SEC samples. Vesicle marker concentrations were the highest in the SEC‐3 fraction, while plasma markers fell below detection thresholds. Clustering and Welch's test showed optimal separation of patient groups based on SEC‐3.

Summary/Conclusion : Our results indicate that the third SEC fraction is best suited for comparative plasma EV proteomics. Based on the developed method, the two patient groups are distinguishable, though identifying tumour markers requires a larger sample size and the inclusion of healthy controls.

Funding : TKP‐EGA‐09; OTKA‐K143255; MEC_R 149857; MEC_R 149846;

An evolutionarily Conserved Mechanism for Selective Sorting of Full‐Length tRNAs Lacking Functional CCA Tails into Extracellular Vesicles

Chantal Scheepbouwer 1,2,3,4 , Ernesto Aparicio 5 , Cristina Gómez‐Martin 1,4 , Monique A. J. van Eijndhoven 1,4 , Esther E. E. Drees 1,4 , Leontien Bosch 1,4 , Daphne de Jong 1 , Thomas Wurdinger 3 , Josée Zijlstra 6 , Michael Hackenberg 5 , Alan Gerber 2,3 *, D. Michiel Pegtel 1,4*

1 Pathology, Amsterdam UMC, VU University, Amsterdam, Netherlands, 2 Cancer Center Amsterdam, Cancer Biology, Netherlands, 3 Neurosurgery, Amsterdam UMC, VU University, Amsterdam, Netherlands, 4 Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands, 5 Genetics, Faculty of Science, University of Granada, Granada, Spain, 6 Hematology, Cancer Center Amsterdam, Amsterdam UMC, VU University, Amsterdam, Netherlands. *Equal contribution .

Introduction : High‐throughput sequencing has advanced our understanding of small non‐coding RNAs (ncRNAs) shorter than 50 nucleotides within extracellular vesicles (EVs). However, profiling longer RNAs, like transfer RNAs (tRNAs), long remained challenging due to extensive chemical modifications that hinder reverse transcription and quantification. To address this, we employed ALL‐tRNAseq to profile small ncRNAs, including tRNAs, small nucleolar RNAs (snoRNAs), and Y RNAs, in EVs derived from cell lines across multiple species. This approach provided a broader perspective on the RNA composition in EVs and facilitated cross‐species comparisons.

Methods : EVs were isolated using sequential centrifugation followed by size exclusion chromatography (SEC). RNA extraction, deacylation and demethylation steps were performed before ALL‐tRNAseq library preparation. Sequencing data were processed with sRNAbench and normalized with NormSeq. Validations were conducted using Northern Blotting and qPCR.

Results : Our results demonstrated that full‐length tRNAs dominate small RNA profiles in both cells and EVs across multiple species, comprising 60‐85% of assigned reads, as confirmed by Northern Blotting. tRNA modification analysis revealed similar modification signatures between cellular and EV‐tRNAs, suggesting an origin from the mature tRNA pool. While cellular tRNAs retain conserved 3’ CCA tails, EV‐tRNAs exhibit extensive 3’ CCA tail truncations across all species. Additionally, plasma EVs from Hodgkin lymphoma patients showed comparable tRNA profiles with partial 3’ CCA truncations, reinforcing the conservation of this pattern in physiological fluids.

Summary/Conclusion : These findings suggest a selective mechanism by which EVs package full‐length tRNAs lacking functional 3′ CCA tails. While these truncated tRNAs are unlikely to participate in translation, they retain cellular tRNA modification patterns, suggesting their origin from functional tRNAs. The consistent 3’ CCA tail truncation across various tRNA types and multiple species suggests an evolutionary conserved sorting mechanism for mature tRNAs. This study highlights a previously underappreciated aspect of small EV‐RNA composition, revealing a selective tRNA sorting process that provides new insights into EV biology.

Funding : Stichting CCA, MRD Hodgkin Lymphoma, and AQrate TKI

Extracellular vesicle‐associated miR‐23∼27∼24 clusters in plasma of children with type 1 diabetes as markers of disease progression

Presenter: Giorgia Mele

University of Naples Federico II, Italy

Introduction : Traceable biomarkers able to predict the immune cell‐mediated destruction of pancreatic β‐cells in children with type 1 diabetes (T1D) may sensibly improve patient management. We previously found that, at T1D onset, the quantities of plasma‐circulating miR‐23∼27∼24 clusters are higher in T1D children who rapidly lose insulin secretion, as compared to those preserving it. The present work aims at assessing whether extracellular vesicle (EV) purification may sensibly augment plasma miR‐23∼27∼24 sensitivity and at characterizing the EV‐associated proteomic signatures.

Methods : EVs were isolated by size‐exclusion chromatography from plasma of 40 pediatric subjects at T1D onset, stratified as: low‐risk (insulin‐dose adjusted HbA1c [IDAA1c] < 8; n = 20) and high‐risk (IDAA1c > 10.5; n = 20), assessed 1 year after the diagnosis; then EVs were analysed for miR‐23a‐3p, miR‐23b‐3p, miR‐24‐3p, miR‐27a‐3p, and miR‐27b‐3p (miR‐23∼27∼24 clusters) by RT‐qPCR; in parallel, EV protein cargo was evaluated by liquid chromatography‐mass spectrometry.

Results : The quantity of miR‐23∼27∼24 clusters in isolated plasma‐circulating EVs from T1D subjects was lower compared with the whole plasma, although it was elevated in EVs from high‐ compared to low‐risk T1D children. Moreover, the EV/whole plasma delta Cts for those microRNAs significantly decreased in high‐ compared to low‐risk children, suggesting a specific enrichment in the EV compartment in the former group. In parallel, EV proteomics has demonstrated high EV protein coverage and, at present, we are analyzing the complex molecular signatures associated with different rates of insulin secretion loss.

Summary/Conclusion : The EV‐associated protein‐microRNA composite cargo holds promising potential in regard to the prediction of distinct disease trajectories and it may also uncover novel pathogenic mechanisms contributing to β‐cell dysfunction in children with T1D.

Funding : PRIN PNRR 2022: Prot. P2022H8MZ4

Comparison of the genomic and proteomic profile of PS‐EV from tumour‐associated fibroblasts derived from HNSCC tumour tissue

Monika Kratochvílová 1 , Jan Balvan 1 , Klára Hánělová 1 , Květa Pilařová 1 , Martina Raudenská 1 , Tomáš Vičar 1 , Michal Masařík 1,2,3

1 Masaryk University Brno, Czech Republic, 2 St. Anne's University Hospital Brno, Czech Republic, 3 Charles University Prague, Czech Republic.

Introduction : PS‐positive EVs obtained from CAFs from patient tissue (HNSCC) and control cell line hGFb were subjected to proteomic and genomic analysis according to standard protocols. The aim of this work was to determine whether the content of PS‐EVs at the mRNA and protein levels is related and contributes to communication within the tumour microenvironment.

Methods : Genomic analysis (Illumina next‐generation sequencing) provided lncRNA and mRNA content, proteomic analysis (LC‐MS) data on the protein composition of PS‐EVs. The mRNA and proteins were compared. Bioinformatic analysis was performed in the R programming environment, using libraries edgeR and limma, etc. Normalized data were compared to control using a moderated t‐test, with the Benjamini Hochberg adjustment method for multiple comparisons.

Results : A total of 19,239 mRNA and 1,957 proteins were determined. Most of the proteins were also identified as mRNA, with only 10 exceptions. However, when compared by the average amount in the samples, the orders of individual proteins and their corresponding mRNA did not correlate. Only a few mRNA (6 ‐ 60) were significantly altered compared to the control, while more proteins showed differences (200 ‐ 400). In the GSEA results, most of the terms enriched at the protein level (in all GO categories) were also prominent at the mRNA level. In contrast, ORA showed differences at the mRNA and protein levels for both samples across all categories when searching the GO database and others such as WIKI pathways or KEGG. For both samples, only one protein was significantly increased at both the mRNA and protein levels: IGFBP5, which is involved in the cellular response to cAMP. Both samples also shared one significantly decreased protein compared to the control (both at the mRNA and protein levels), namely LPAR1, a receptor that binds LPA.

Summary/Conclusion : Despite the large number of mRNAs identified, only a few were significantly different from the control. Proteins showed a higher proportion of significant changes. Therefore, the information that cells release into the tumour microenvironment is fundamentally different at the mRNA and protein levels.

Funding : Ministry of Health of the Czechia NU22‐03–00202, Internal grant MUNI/R/1316/2024, Martina Roeselova MF NF‐IOCB‐TECH

Proteomic profiling of fractions enriched in extracellular vesicles produces by macrophages infected with Francisella tularensis

Ivona Pavkova 1 , Jana Klimentova 1 , Adela Cermakova 1 , Jitka Zakova 1 , Dominik Pinkas 2 , Jaroslav Hanus 3

1 University of Defence, Military Faculty of Medicine, Czech Republic, 2 Electron Microscopy Core Facility, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic, 3 University of Chemistry and Technology, Prague, Czech Republic

Introduction : Extracellular vesicles (EVs) are composed of various biomolecules through which they participate in cellular crosstalk. During infection, their effect might be bidirectional. They can either promote or inhibit the infection progress. In this study we focused on the proteomic analysis of fractions enriched in EVs produced by macrophages infected with intracellular bacterium Francisela tularensis, the causative agent of severe disease tularaemia. Despite the intensive research, the knowledge of molecular mechanisms of pathogenesis are incomplete, and represents the major challenge to the development of an effective vaccine. The release and role of EVs in relation to this pathogen has not been reported so far, and is expected to play a key role in host‐pathogen interaction.

Methods : Mouse bone marrow‐derived macrophages (ca. 9x107cells) were infected with virulent strain of F. tularensis subsp. holarctica for 10 h and the cell culture supernatant was enriched for extracellular vesicles (EVs) using differential centrifugation. The presence of EVs was confirmed by TEM, NTA and immunoblot analysis. Further, proteomic analysis of EV containing fractions were performed using LC/MS/MS followed by analysis using the Metascape platform. For comparison, samples from non‐infected cells were processed in the same way.

Results : In response to infection, the macrophages were found to release EVs with mode diameter 77.1 ± 3.8 nm. The presence of EVs was further successfully validated by TEM and immunodetection of EV markers CD9, CD81 and TSG101 On the other hand, almost no vesicles could be detected in control samples prepared from non‐infected cells. The MS analysis reveals the presence of about 1270 proteins, many of them are involved in cellular processes in response to infection indicating the potential role of EVs in tularaemia pathogenesis. In addition to host cell proteins, several bacterial proteins were identified as well, most of them with known relation to virulence.

Summary/Conclusion : This study shows the production of EVs from cells infected with F. tularensis together with a dataset of their protein cargos, providing a preliminary view of their potential function in pathogenesis of tularaemia.

Funding : The work was supported by the Ministry of Defence of the Czech Republic (project no: DZRO‐FVZ22‐ZHN II).

NGS‐based miRNA profiling of salivary extracellular vesicles and whole saliva: implications for biomarker discovery

Presenter: Elodie Simphor

CNRS/ALCEN, France

Introduction : Extracellular vesicles are lipid‐bound nanoparticles that play a crucial role in cell‐to‐cell communication, influencing both physiological and pathological processes. Their presence in peripheral biofluids, including saliva, an easily accessible and non‐invasive sample source, makes them promising candidates for biomarker discovery in diagnostics and prognostics. Additionally, microRNAs (miRNAs) are increasingly recognized as valuable diagnostic tools for various diseases. Although multiple methods exist for isolating and characterizing salivary EVs (sEVs), further optimization and standardization are necessary to facilitate their clinical translation. This study aims to characterize and compare the miRNA content of salivary EVs isolated using two rapid and scalable techniques, co‐precipitation (Q) and immunoaffinity (M), against the gold standard ultracentrifugation (UC) method.

Methods : Saliva samples were collected from three healthy volunteers. sEVs were isolated using UC, Q, and M. Following a comprehensive characterization, miRNAs were extracted from both sEVs and whole saliva (WS) and subsequently sequenced using next‐generation sequencing (NGS).

Results : Our analysis revealed that while saliva and EVs share common sets of miRNAs, several miRNAs exhibit differential expression between WS and sEVs. Principal component analysis demonstrated a clear separation between WS and the three isolation methods. UC and Q showed a strong similarity, clustering primarily by individual rather than by isolation technique, whereas M exhibited a separation pattern driven more by the isolation method than by individual variation. Despite these differences, all three methods yielded highly consistent miRNA enrichment profiles when compared to WS. Gene Ontology and KEGG pathway analyses indicated significant enrichment of miRNA‐related processes, including small RNA pathways and splicing regulation, particularly in the M condition.

Summary/Conclusion : This study highlights the significance of sEVs as a source of miRNA and demonstrates that certain miRNA species are preferentially enriched in WS, while others are predominantly contained within sEVs. The three isolation techniques provided robust and consistent miRNA profiles, with UC and Q exhibiting strong similarities and M showing superior enrichment in specific pathways. These findings underscore the importance of EV isolation before miRNA analysis and confirm that all three methods are suitable for clinical salivary sample processing.

Funding : This research was founded by French Centre National de la Recherche Scientifique (CNRS) and ALCEN

Potential clinical application of salivary extracellular derived miRNAs quantified by RT‐qPCR: from accurate analytic methodology to data reproducitivty

Presenter: Thi Nhu Ngoc Van

CNRS/ALCEN, France

Introduction : Salivary extracellular vesicles (sEVs) have recently emerged as biomarkers for detection and monitoring of various diseases. Compared to whole saliva, sEVs are loaded with specific cargos such as protein, nucleic acids, lipid and metabolites, whose concentration might reflect pathological or physiological states. In our previous work (1), we demonstrated that miRNAs are consistently enriched in sEVs independent from the used isolation methods. Additionally, we highlighted, in another work (2), that specifically quantifying salivary miRNAs using RT‐qPCR is highly challenging. We addressed these challenges by establishing methodological recommendations to reduce data inconsistency and enhance data interpretation accuracy (2). This study aims to decipher further the profile of miRNAs enriched in sEVs compared to that of whole saliva. First, new RT‐qPCR assays were developed employing stem‐loop priming strategy, displaying highly specific detection of individual miRNA targets. Second, miRNA expression profiles of sEV‐ and whole‐saliva‐derived miRNAs were analysed by newly developed assays. Lastly, a comparative analysis between stem‐loop based (newly developed) and polyA (commercially available) based RT‐qPCR was conducted on sEV‐ and whole‐saliva‐derived miRNA samples.

Methods : Saliva samples were collected from healthy individuals followed by sEV isolation using co‐precipiation method. Small RNA extraction and RT‐qPCR analysis were performed in accordance with our previously optimized protocol (2).

Results : Our data showed that newly developed RT‐qPCR assays display highly specific detection of individual miRNA targets, allowing distinction between two highly homologous miRNA molecules whose sequences are differed by only one nucleotide. Comparative analysis on sEV‐ and whole‐saliva‐derive miRNAs reveals method dependent profiles of both miRNA populations. Specifically, the polyT‐based approach exhibited broader detection of target miRNAs with a pan specific tendency, whereas the Stem‐Loop based approach reports on specific quantification of individual miRNA targets.

Summary/Conclusion : These findings underscore the importance of taking into account the assay performance and its specificity into data interpretation process in order to reduce study‐dependent data in miRNA research. By enhancing the reproducibility and specificity of salivary miRNA quantification method, this study pave the way to clinical application of sEV‐associated miRNAs as reliable diagnostic and prognostic biomarkers.

Funding : Agence Nationale de la Recherche. ANR T‐ERC_STG2 278927 ReGuCel. Dr Marc Van Der Hofstadt

Subtopic Engineering and Loading EVs 2

Naturally secreted EVs VS artificial CDVs

Presenter: Pauline S. Ley

Institute for Experimental and Clinical Cell Therapy, Austria

Introduction : Extracellular vesicle (EV)‐based biomarker discovery can be more sensitive than from conventional liquid biopsies. Here we studied nano‐vesicle reference material for theranostic EV‐biomarker discovery for Parkinson's disease (PD). EV isolation methods, like tangential flow filtration (TFF) and size exclusion chromatography (SEC), showed limitations concerning time and resource consumptions. We therefore explored cell extrusion as efficient method to create artificial cell derived vesicles (CDVs) as reference material. We investigated whether CDVs are comparable to naturally produced EVs derived from the same cell lines.

Methods : We used fluorescent neuroblastoma cells SH‐SY5Y expressing GFP‐coupled alpha‐synuclein (a‐Syn), a key protein for PD development and progression. Two control cell lines HEK‐CD63‐mNEON and HEK‐WT were included for comparison. Vesicles were enriched by TFF and cell extrusion. We determined EV quantity and size by tunable resistive pulse sensing (TRPS), and EV quality by tetraspanin and a‐Syn expression in dot blots and, fluorescence nano‐tracking analysis (fNTA). CDVs and EVs were imaged by cryo‐transmission electron microscopy (cryoTEM).

Results : We found that CDVs were comparable to EVs in size with a mode diameter of 88±11 nm and 93±16 nm, respectively. We obtained mean 1E11 CDVs and 1E10 EVs per two million starting cells. Both expressed a‐Syn and tetraspanins CD9/63/81 but CDVs were mean 10‐fold less fluorescent than corresponding EVs. Bilayer membrane‐coated EVs and CDVs of appropriate size were readily detected by cryoTEM in a frequency corresponding to input concentration.

Summary/Conclusion : Testing additional extrusion parameters will be necessary to improve reference nano‐particle production.

Optimization of the isolation method for industrial production of synthetic bacterial vesicles

Negar Ordouzadeh 1,3 , Rossella Crescitelli 1 , Agnes Zimmer 2 , Petra Tjärnlund 2 , Cecilia Lässer 1 , Jan Lötvall 1 , and Kyong‐Su Park 1

1 University of Gothenburg, Sweden. 2 NorthX Biologics, Sweden. 3 Exocure Sweden AB, Sweden.

Introduction : Bacterial outer membrane vesicles (OMV) have emerged as compelling naturally produced future therapeutic drug candidates in a vast array of diseases, including cancer. However, the yield of OMV production is limited, and they are known to be toxic nature, by expressing multiple pattern recognition molecules such as LPS and beyond. To overcome these concerns, our group has recently developed semi‐synthetic bacterial vesicles (SyBV) from isolated bacterial cell membranes, as a potential alternative to natural OMV, in view of the higher yield and increased safety profile. This project thus aims to create a scalable manufacturing approach to produce SyBV, with the aim of treating patients with cancer with the product.

Methods : In this project, we developed a detergent‐free modified from our previous methods. Thus, to develop an approach suitable for good manufacturing practice (GMP) we have undesirable procedures for large‐scale production, such as ultracentrifugation or sonication, as well as certain detergents. Specifically, we have applied osmotic pressure combined with benzonase to break cells and remove nucleic acid and exposed the cells with high pH to remove cytosolic components. We have also been able to replace ultracentrifugation with a lower‐speed centrifugation step to collect purified membrane sheets, and we have named these “SyBV_New”. Furthermore, we examined any possible adverse effects in different reporter cell lines and the therapeutic functionality.

Results : Vesicles obtained from non‐pathogenic Escherichia coli exhibited a higher yield than the previously developed SyBV isolation methods, with similar purity and a comparable reduced toxicity profile. Additionally, SyBV_New significantly activates dendritic cells and inhibits melanoma progression in vivo. Furthermore, it has been confirmed that the optimized process can be applied to GMP‐compatible E. coli, enabling the production of SyBVs under pre‐GMP conditions.

Summary/Conclusion : Together, these findings demonstrate a dependable method for the large‐scale production of SyBVs while maintaining their therapeutic potential, thereby allowing the integration of these bioactive SyBV into clinical application.

Using the proteins CD9 and TSP‐1 to develop fully synthetic exosome‐mimetics

Nahide Zeren Arda Ozturk 1 , Oliwia Majchrzak 2 , Gianluca Ulivi 2 , Viorica Patrulea 2 , Petek Ballar Kirmizibayrak 3 , Gerrit Borchard 2 , Ozgen Ozer 3

1 Fenerbahce University, Turkiye, 2 University of Geneva, Switzerland, 3 Ege University, Turkiye Petek Ballar Kirmizibayrak, Gerrit Borchard and Ozgen Ozer are co‐senior authors .

Introduction : Exosomes are extracellular vesicles that contribute to several kinds of biological processes. Their ability to provide intercellular communication, one of their most important functions, has inspired the use of exosomes as drug carriers. Fully synthetic exosome‐mimetics (FSEM) is a drug delivery technology that takes its inspiration from the natural exosome. FSEM membrane proteins and lipids enhance adhesion and fusion. They also have a low toxicity profile. It is crucial that the proteins used in FSEM retain their 3D structure and conformation in the membrane.TSP‐1 and CD9 proteins are membrane proteins that function during intercellular communication. The CD9 protein is inserted into the exosome membrane, while the TSP‐1 protein is bound to the outside of the exosome membrane. Different methods are used to produce synthetic exosome mimetics, depending on the position of the proteins in the membrane.

Methods : The FSEMs were prepared using DMPC, DOPE, DOPS, sphingomylin and cholesterin. To incorporate CD9 protein, the liposomes were treated with Triton X‐100, followed by incubation with CD9 protein. Then the Triton X‐100 was removed using a detergent removal column and the membrane incorporation was completed. To bind the TSP‐1 protein, liposomes were prepared using a modified DOPE molecule.The binding of the TSP‐1 protein to the liposome membrane was achieved by the EDC‐NHS reaction.

Results : Particle size, zeta potential and PDI measurements of FSEM were as targeted and compatible with natural exosomes. Protein presence and conformation in the formulations were established by SDS‐PAGE analysis and the bands were compatible with commercial proteins alone and markers. FSEM also showed higher cellular uptake in SK‐MEL3 cells compared to DOTAP and phosphatidylcholine liposomes.

Summary/Conclusion : Enhanced cellular uptake is achieved by using lipids present in natural exosomes and liposomes functionalised with membrane proteins in the preparation of FSEM. This drug delivery system could provide a new alternative for non‐toxic drug and gene delivery.

Funding : Ege University Office of Scientific Research Projects, University of Geneva, TUBITAK 2244 project

Effective Strategies for Delivering Exosomes into the Skin

Ji Hoon Ha 1 , Sung Eun Kim 1 , Jin Young Kim 1 , Jin Mo Kim 1 , Sang‐Geun Han 1 , Yeon Gyun Jung 2 , Yoon Soo Cho 2†

1 Kolmar Korea, Korea, Republic of. 2 Hanllim university, Hangang Sacred Heart Hospital, Korea, Republic of.

Introduction : This study includes various strategies and results for exosome penetration into the skin. Unlike blood vessels, the skin is composed of a hard stratum corneum, the outermost layer of the skin, making it difficult for exosomes to pass through the skin barrier and reach the cell layer. Although exosomes are known to be very small, about 100 nm in size, and can pass through the gap of the skin barrier. However, exosomes are not affected by gravity because of it's size, so they cannot move spontaneously under the skin, and are greatly affected by the concentration gradient.

Methods : We adjusted three methods to properly insert exosomes into the skin. Surface engineering of exosomes Hybridization of exosomes with soy lecithin by extrusion and surface modification with cations and anions. Loading support control Increasing the skin adhesion of polymers in formulations containing exosomes and evaluating the delivery ability of exosomes Opening the skin barrier with negative pressure Applying exosome formulations to octopus suction cup‐mimicking patches using biomimetic technology and evaluating them.

Results : More exosomes were delivered when the surface of the exosome was positive rather than negative, and when the polymer in the polymer support solution containing exosomes increased adhesion to the skin, the concentration of exosomes increased as they moved to the skin surface, passing through the skin. In particular, when the skin barrier was relaxed by negative pressure, the space in the stratum corneum opened, accelerating the movement of exosomes. It showed excellent effects on improving wrinkles and moisturizing the skin of women aged 40‐60s.

Summary/Conclusion : Exosomes have various effects on the skin, such as wrinkle improvement, whitening, and moisturizing. However, due to the structural limitations of the skin barrier and the side effects caused by the small size of exosomes, they are difficult to pass through. The various strategies presented in this study will be important indicators for increasing the efficacy of exosomes in the skin.

Funding : “This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (grant number : HP23C0061)”.

Glycopolymer‐Engineered Extracellular Vesicles to Enhance Precisely Targeted Regeneration in Acute Kidney Injury

Presenter: So Hee Kim

Sungkyunkwan University, Republic of Korea

Introduction : Acute kidney injury (AKI), commonly induced by nephrotoxic agents, leads to high morbidity due to limited repair treatments. Interventions like dialysis or transplantation relieve symptoms but do not restore kidney function. Effective recovery requires protecting renal tubular epithelial cells (RTECs) and regulating M1 macrophages. Stem cell‐derived extracellular vesicles (SC‐EVs), rich in bioactive molecules, offer a cell‐free regenerative approach. However, since most injected EVs accumulate in the liver, surface modifications are needed. This study developed glycopolymer‐engineered EVs(PH‐EVs) with PEGylated hyaluronic acid(PEG‐HA) to target CD44 and TLR4 on RTECs and macrophages, achieving precise kidney targeting and promoting renal protection.

Methods : To synthesize PEG‐HA, PEG was conjugated to HA (5:100 ratio), followed by DBCO‐PEG‐HA synthesis for SC surface engineering via click chemistry. PH‐EVs were isolated from PEG‐HA‐modified SCs using TFF. The physicochemical properties of PH‐EVs were confirmed by TEM and ELISA. In vivo bio‐distribution of PH‐EVs were compared to unmodified‐EVs. The therapeutic efficacy of PH‐EVs was verified in both cellular level and cisplatin(CP)‐induced AKI mice model.

Results : Surface modification of EVs was confirmed by TEM, with HA polymer quantification via ELISA. Cisplatin (CP) was used to model drug‐induced AKI in vitro and in vivo. PH‐EVs showed enhanced uptake compared to unmodified‐EVs, only in CP‐treated HK‐2 cells. PH‐EVs protected RTECs from apoptosis and inhibited macrophage polarization into a pro‐inflammatory state in vitro. In CP‐induced AKI mice, PH‐EVs selectively accumulated in injured kidneys, co‐localizing with CD44, thereby significantly reducing AKI markers, including creatinine, BUN, and NGAL levels. Also, immunohistochemistry showed PH‐EV‐mediated renal protection by reducing apoptotic cells and inducing proliferation of cells.

Summary/Conclusion : Our PH‐EVs demonstrate strong potential as a regenerative AKI treatment by selectively targeting injured kidneys via AKI‐related receptors (CD44 and TLR4), thereby alleviating AKI symptoms. This precisely targeted approach highlights the therapeutic promise of extracellular vesicle engineering, offering a cell‐free strategy to overcome limitations in current AKI therapies and enhance kidney repair.

Biotinylated RBCEVs as a promising tool for surface functionalization and targeting

Perla Elena 1 , Biagiotti Sara 1 , Tiboni Mattia 1 , Agostini Rachele 1 , Nozza Antonio 1 , Canonico Barbara 1 , Guescini Michele 1 , Rossi Luigia 1 , Magnani Mauro 1

1 Department of Biomolecular Sciences, University of Urbino, Italy

Introduction : Red blood cells‐derived extracellular vesicles (RBCEVs) have recently emerged as promising tools for targeted drug and biologics delivery. Cargo‐loaded RBCEVs can be efficiently produced starting from preloaded RBCs using the innovative “soft extrusion” technique. To date, the produced RBCEVs have been employed in both in vitro and in vivo studies, leverageing their inherent uptake and biodistribution characteristics. Additionally, biotinylation is a fruitful strategy for attaching various peptides and antibodies to cell or EV surfaces, enabling specific targeting of particular cell types or tissues. This method is fully biocompatible and safe, so it has already found applications in clinical settings. The present work investigates the biotinylation of RBCEVs as a method for membrane engineering and targeted delivery, focusing on enhancing specificity for cardiomyocytes.

Methods : Two biotin derivatives, NSH‐biotin and NSH‐LC‐biotin, were employed to biotinylate RBCEVs. Both the efficiency of biotinylation and cell recovery were assessed. Furthermore, flow cytometry analyses with Alexa Fluor‐streptavidin were conducted to quantify the degree of biotin incorporation. A three‐step delivery system was employed to target cardiomyocytes. It consists in the administration of the biotin‐Cx43 Ab followed by the introduction of avidin, which forms a stable complex with the Ab. Finally, biotinylated RBCEVs were decorated with the specific antibody biotin‐Cx43 using an avidin bridge and administered to allow them to attach via avidin's biotin‐binding sites.

Results : Both biotinylation strategies demonstrated high efficiency and satisfying cell recovery. Flow cytometry analysis confirmed successful biotin incorporation on RBCEVs, facilitating specific targeting of cardiomyocytes via the biotin Cx43 Ab‐avidin bridge. The functionalization of RBCEVs with the antibody resulted in enhanced binding to target cells, highlighting the effectiveness of this approach for directed delivery.

Summary/Conclusion : Both biotinylation strategies demonstrated high efficiency and satisfying cell recovery rates. Flow cytometry analysis confirmed successful biotin incorporation on RBCEVs, facilitating specific targeting of cardiomyocytes via the biotin Cx43 Ab‐avidin bridge. The functionalization of RBCEVs with the antibody resulted in enhanced binding to target cells, highlighting the effectiveness of this approach for directed delivery.

Engineered red blood cell‐derived extracellular vesicles: unlocking new avenues in drug delivery systems

Michela Arena 1,2 , Alessia Brossa 1, Elena Ceccotti 3 , Francesca Orso 4 , Giuseppina Poppa 5 , Vincenza Dolo 5 , Daniela Taverna 1 , Stefania Bruno 3 , Benedetta Bussolati 3

1 Department of molecular biotechnology and Health Sciences, University of Turin, Turin, Italy; 2 University of Naples Federico II, Naples, Italy, 3 Department of Medical Sciences, University of Turin, Turin, Italy; 4 Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy; 5 Clinical Pathology Unit, Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Introduction : Current RNA‐based drug therapies are facing significant limitations in the clinical setting, largely due to low cellular uptake efficiency and potential toxicity. Red blood cell‐derived extracellular vesicles (RBC‐EVs) represent a promising alternative as natural delivery vehicles, offering key advantages over non‐RBC‐EVs and synthetic nanovesicles, including DNA‐free composition, biocompatibility, and accessibility from blood banks. The aim of this study is to evaluate the engineering of RBC‐EVs with therapeutic RNAs as a novel drug delivery system.

Methods : RBC‐EVs were isolated from healthy donors by ultracentrifugation and characterised by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), western blot analysis and single EV cytofluorometric analysis. To assess the loading efficiency of engineered RBC‐EVs, a scrambled FITC sequence was used as control. RBC‐EVs were subsequently engineered by electroporation with anti‐cancer or anti‐fibrotic therapeutic miRNAs and then tested in different cellular models to determine their efficacy as therapeutic delivery vehicles.

Results : RBC‐EVs were successfully engineered with various RNA species and evaluated across different cellular models. Notably, RBC‐EVs loaded with siRNA targeting luciferase led to a reduction in luciferase gene expression in target cells. Additionally, RBC‐EVs loaded with therapeutic RNAs, such as the anti‐tumour miR200b, anti‐miR214 and miR148b, and the antifibrotic miRLet7b and miR29a were tested respectively on renal cancer stem cells, breast and melanoma cell lines, and an in vitro liver fibrosis model. Quantitative PCR analysis confirmed the presence of therapeutic RNA within the RBC‐EVs and their delivery in target cells after the treatment. In addition, we demonstrated their biological effect in target cells.

Summary/Conclusion : This study establishes an efficient protocol for RBC‐EVs loading with therapeutic RNAs, demonstrating successful delivery to target cells. These findings underscore the potential of RBC‐EVs as versatile drug carriers, expanding their applicability in future RNA‐based delivery systems.

Funding : This project is funded by PNRR; National Center for Gene Therapy and Drugs based on RNA technology, CN3_Spoke8.

Long‐term storage and transdermal delivery of extracellular vesicles using hyaluronic acid‐based microneedles

Presenter: Soyoung Son

Sungkyunkwan University, Republic of Korea

Introduction : The clinical application of extracellular vesicles (EVs) is constrained by their poor stability during storage. The current preservation methods, including cryopreservation and lyophilization, either fail to maintain stability at low concentrations or cause cytotoxicity at high levels. Furthermore, effective EV delivery remains a significant challenge due to the skin barrier and the necessity for precise intradermal administration. Microneedles (MN) offer a minimally invasive, self‐administered solution for stable, controlled intradermal delivery. They can retain the activity of encapsulated biomolecules over extended storage and under extreme conditions. In this study, we developed hyaluronic acid (HA)‐based MN loaded with EVs (EV@MN) to explore their potential for the long‐term storage and effective delivery of EVs.

Methods : After EV@MN was prepared, the biological activity of EVs in EV@MN were evaluated at various stages of the fabrication process and over 6 months of storage by examining their functional impact on fibroblasts. CLSM and in vivo imaging were employed to analyse the precise delivery and sustained release of EVs within the dermis. Moreover, the biological effects of EV@MN on dermal tissue in vivo were investigated.

Results : EV@MN demonstrated the ability to maintain EV stability, exhibiting high particle retention and bioactivity over a 6‐month period at 4°C. The mechanical testing confirmed that the EV@MN could penetrate the skin, thereby achieving the targeted release of EVs into the dermis. In vivo, EV@MN demonstrated sustained EV retention at the injection site for seven days, significantly longer than intradermal injections, with extended fluorescence. Functional analysis revealed that EV@MN‐treated skin exhibited increased dermal thickness, enhanced collagen and elastin levels, and promoted fibroblast proliferation.

Summary/Conclusion : EVs were effectively isolated and incorporated into EV@MN, with their biological activity preserved for over 6 months under mild storage conditions. Furthermore, EV@MN facilitated precise and straightforward intradermal administration, allowing for sustained EV release within the dermal layer. This formulation augmented the functional effects of EVs on dermal fibroblasts. This study presents a viable approach for EV clinical applications, addressing challenges in storage and delivery while enhancing usability for potential skin care applications.

Ceftriaxone Encapsulation in Erythrocyte‐Derived Pharmacocytes: A Novel Approach for Sustained Antibiotic Release

Kulzhan Berikkhanova, Alexandr Gulyayev, Ernur Zakirov, Shynggys Sergazy, Zarina Shulgau

Nazarbayev University, Kazakhstan

Introduction : Encapsulating therapeutic agents within erythrocyte‐derived pharmacocytes provides an innovative approach to achieve sustained drug release while potentially minimizing systemic toxicity. This study explores the encapsulation and controlled release profile of ceftriaxone loaded into erythrocyte‐derived pharmacocytes through a hypoosmotic hemolysis technique, offering a new avenue for antibiotic delivery.

Methods : Blood samples were collected and erythrocytes isolated under sterile conditions. Using a hypoosmotic solution, erythrocytes underwent hemolysis, allowing for ceftriaxone loading (1.0 g per 7.8 mL pharmacocyte suspension). Post‐loading, the pharmacocytes were resealed and subjected to a temperature‐stabilized environment. Controlled release of ceftriaxone from pharmacocytes was assessed over 24 h using equilibrium dialysis in NaCl (0.9%) solution at intervals (15 min to 24 h). Spectrophotometric analysis determined ceftriaxone concentration at each interval.

Results : The pharmacocytes demonstrated consistent ceftriaxone release over the 24‐h period, with concentrations ranging from 1.8534 ± 0.033 mg/mL at 15 min to 0.0130 ± 0.007 mg/mL at 24 h. Peak release was observed within the first 3 h, with a progressive decline, indicating a stable release profile. Average ceftriaxone release across five pharmacocyte samples totaled 5.75–6.41 mg/mL over 24 h, confirming efficient loading and sustained release potential.

Summary/Conclusion : This study establishes erythrocyte‐derived pharmacocytes as effective carriers for ceftriaxone, providing a controlled release profile that could enhance therapeutic outcomes for antibiotic therapy. Future investigations will focus on pharmacokinetic modelling and in vivo evaluation of ceftriaxone pharmacocyte efficacy and biocompatibility.

Funding : This research is funded by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Enhanced loading of miRNA into extracellular vesicles utilizing mechano‐nanoporation reduces by 10x the dose required for anti‐inflammatory effects

Presenter: Michaël Francotte

XomeXBio SRL, Belgium

Introduction : Small extracellular vesicles (sEVs) have emerged as natural nanocarriers for the delivery of therapeutic agents, including miRNAs due to their intrinsic properties as intercellular mediators. However, efficient loading of miRNAs into sEVs remains a challenge, and current electroporation method damage both the sEVs and their cargo. Mechano‐nanoporation offers a novel physical approach for miRNA loading by applying mechanical forces to transiently permeabilize the sEV membrane without compromising their structural integrity and while preserving both biological function. This work demonstrates the efficiency of miRNA loading by nanofluidic mechano‐nanoporation, highlighting key factors that influence miRNA loading efficiency and purification of the loaded sEVs.

Methods : We optimized loading parameters (flow rate, pressure, chip design, sEVs and miRNA concentrations) to encapsulate a selected miRNA into sEVs from HEK293 cells and convEyXO immortalized WJ‐MSCs. The nanofluidic chips were designed and manufactured by Somestech. Various parameters of the loading process were evaluated, including flow rates from 100 µL/min to 4 mL/min and fluidic pressures up to 50 bars with high stability. After purification steps, the sEVs were characterized by size distribution, protein, cholesterol, and residual nucleotides concentrations. The absence of GM130 and presence of tetraspanins CD9/CD63/CD81 were also confirmed. The loading efficiency was assessed by specific qRT‐PCR, and the preservation and combination of biological function of EVs and miRNA was assessed by cytokines quantification from RAW 264.7 macrophages.

Results : The nanofluidic chips were optimized for reproducible drug loading into sEVs at high flow rates. The purification steps effectively removed contaminants and unloaded molecules. If current electroporation method damage both the sEVs and their miRNA, the anti‐inflammatory assay showed that unloaded HEK‐EVs had no anti‐inflammatory effects, while mechano‐nanoporation’ loaded HEK‐EVs showed very potent anti‐inflammatory effects, demonstrating the efficiency of the loading. Unloaded sEVs from WJ‐MSCs presented anti‐inflammatory effects, but loaded sEVs presented similar effects when used at a 10x lower concentration, demonstrating preservation of both biological function.

Summary/Conclusion : Mechano‐nanoporation represents a scalable, efficient, and minimally invasive strategy to improve the loading of miRNA into sEVs. The loading efficiency in this controlled and robust approach enables the production of high‐quality drug‐loaded sEVs with yields compatible for future clinical applications.

Functionalization of extracellular vesicles utilizing a microfluidic platform

Presenter: Yanhang Hong

Shenzhen Institute of Advanced Technology, China (People's Republic)

Introduction : Extracellular vesicles (EVs) exhibit diverse functional components and sources, leading to challenges in developing universal engineering approaches. Traditional methods for modifying and loading isolated EVs are often complex and inefficient, limiting the development of EV‐based delivery systems. Here, we have developed a novel microfluidic platform for EV functionalization. This platform enables the engineering modification of EVs isolated from various sources and supports the surface modification with a variety of functional components, including proteins and peptides.

Methods : EVs derived from bovine milk and HEK293T cells were isolated via ultracentrifugation. DSPE‐PEG2000‐MAL was embedded into the membrane of EVs using a microfluidic device. The device incorporates with Tesla valve‐shape structures, which facilitate rapid conjugation of thiol‐containing peptides and proteins to the maleimide groups on the EV surface. The characterization of EVs were performed using NanoFCM, fluorescence spectroscopy, TEM and WB. To investigate the functionality of the modified EVs, a glioblastoma‐targeting peptide was introduced into HEK293T‐derived EVs, their targeting efficiency and blood‐brain‐barrier (BBB) penetration were evaluated using flow cytometry and confocal microscopy.

Results : Results demonstrated that the embedding efficiency of DSPE‐PEG2000‐MAL in EVs derived from HEK293T cells and milk was 97.93% and 98.47%, respectively, using the ExoNP chips, compared to 13.97% and 46.10% with co‐incubation. Within the same reaction time, the microfluidic chip facilitated the binding of 677 peptides per EV, compared to only 50 with co‐incubation. The binding efficiency of FITC‐BSA‐SH was also increased by 3.1‐fold. Furthermore, EVs modified with peptides exhibited significantly improved internalization in tumour cells and enhanced penetration capability across the BBB.

Summary/Conclusion : In conclusion, a novel platform for EVs functionalization, based on thiol‐ene reaction and microfluidic manipulation, offers a universal approach for modifying EVs from various sources. This platform efficiently introduces multiple biofunctional structures onto the EV surface, paving the way for large‐scale EV production and clinical applications.

Engineering commensal bacterial extracellular vesicles for cancer therapy

Peivand Sadat Mousavi, Lucie Thomas, John C. Bell, Carolina S. Ilkow

University of Ottawa, Ottawa Hospital Research Institute, Canada

ABSTRACT UNAVAILABLE

Engineered Extracellular vesicles for protein delivery into the Hematopoietic Stem and Progenitor Cells

Keerthiga Ariudainambi 1,2 , Saravanabhavan Thangavel 1

1 Centre for stem cell research (a unit of instem, Bengaluru), Christian medical college campus, Bagayam, Vellore, Tamil nadu, India. 2 Manipal Academy of Higher Education, Manipal, Karnataka, India

Introduction : Hematopoietic stem and progenitor cells (HSPCs) are essential for lifelong blood and immune cell production, making them key targets for gene therapy. Our studies have shown that CRISPR‐Cas9 editing of patient‐derived HSPCs can effectively reverse Sickle Cell Disease, β‐thalassemia, and HIV in lab and animal models. Electroporation of Cas9 RNP was used for delivery, but this approach poses challenges in manufacturing gene‐edited HSPCs at scale. The field urgently seeks non‐viral delivery systems, as few lipid nanoparticles can efficiently deliver proteins into primary cells like HSPCs.

Methods : Electroporation‐based methods were employed for the exogenous loading of cargoes into extracellular vesicles (EVs), while endogenous loading was achieved by tagging cargo proteins with extracellular vesicle sorting scaffolds. For the endogenous loading approach, we tagged a GFP reporter protein with established EV‐sorting scaffolds from the literature, as well as newly engineered scaffold variants. These constructs were expressed in 293T cells, and the optimal scaffold was selected based on its delivery efficiency to 293T cells. Following this selection, the best‐performing scaffold was fused with other proteins, including mCherry, NanoLuciferase, and CRE. The engineered EVs were subsequently purified and characterized using NTA and TEM. HSPCs were then treated with the purified EVs, and delivery efficiency to HSPCs was assessed by flow cytometry.

Results : Exogenously engineered EVs were utilized to deliver Cas9 RNP into HSPCs, achieving gene editing efficiencies exceeding 70% at the CCR5 locus. However, considerable variability in editing efficiency was observed between experiments. In contrast, EVs endogenously engineered with novel scaffold variants demonstrated a delivery efficiency of over 80% for reporter proteins into 293T cells. Additionally, EVs loaded with CRE recombinase successfully mediated genome editing upon delivery to LOX‐P‐stable HEK293T cells. HSPC culture conditions, including cytokine supplementation and cell density, were optimized to facilitate EV treatment. The optimal non‐cytotoxic dose of the engineered EVs was efficient in delivering the reporter proteins into the HSPCs with an efficiency of >50%.

Summary/Conclusion : We demonstrate the development of EV‐based protein delivery to the HSPCs. This system will be used for delivering therapeutically relevant proteins into HSPCs.

RBCs‐derived carriers for targeted delivery of hepatoprotector ademethionine to the liver

Kulzhan Berikkhanova, Alexandr Gulyayev, Ernur Zakirov, Askhat Zhilkaidarov, Shynggys Sergazy

Nazarbayev University, Kazakhstan

Introduction : Patients with abdominal sepsis face toxemia and bacteremia, requiring continuous detoxification and liver support. Targeted drug delivery offers new ways to manage liver dysfunction. The hepatoprotector ademethionine distributes non‐selectively with low liver accumulation. Red blood cells (RBCs) are the primary vesicle‐secreting cells in blood circulation. RBCs‐derived carriers (RBCCs) have been created for targeted delivery of ademethionine to the liver. Ademethionine‐loaded RBCCs are recognized by liver macrophages as damaged RBCs, resulting in their capture and digestion. This process extends the duration of peak ademethionine concentration in the liver following phagocytosis of RBCCs. In this study, ademethionine‐loaded RBCCs were created, and in vitro release of ademethionine from RBCCs was described.

Methods : This study was approved by institutional ethics committee. Blood samples were taken from volunteers after receiving signed informed consent. RBCCs loaded with 500 mg of ademethionine were developed using hypoosmotic hemolysis method, with our original modifications. To evaluate degree of ademethionine release from RBCCs, equilibrium dialysis with semipermeable membrane was performed. Samples were taken at intervals of 15 min, 30 min, 1 h, 3 h, 17 h, and 24 h. The concentration of ademethionine in these samples was determined using spectrophotometric analysis.

Results : The release of ademethionine from RBCCs occurred gradually over a 24‐h period. The highest release was observed at 15 min (17.02 µg/mL ± 0.21), attributed to the release of both the internally deposited and superficially adsorbed drug. This was followed by gradual release of ademethionine over the next 3 h, maintaining levels of 5‐8 µg/mL. By 17 h, concentration of released ademethionine had decreased to 1.14 µg/mL ± 0.13 and reached zero by the 24th h.

Summary/Conclusion : RBCCs demonstrated ability to encapsulate the ademethionine and release it gradually into the external environment over 24 h, achieving the highest concentration within first 17 h. These findings can be applied to study pharmacokinetics of ademethionine‐loaded RBCCs in animal models. This approach could provide promising therapeutic strategy for manageing liver disorders in abdominal sepsis.

Funding : This research is funded by Science Committee of Ministry of Science and Higher Education of Kazakhstan, Grant №AP19676272 and by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Nanoerythrosomes: erythrocyte‐derived drug delivery systems for treatment of purulent diseases

Kulzhan Berikkhanova, Alexandr Gulyayev, Ernur Zakirov, Nurzhan Bikhanov, Askhat Zhilkaidarov

Nazarbayev University, Kazakhstan

Introduction : Red blood cell‐derived nanocarriers, known as nanoerythrosomes, are remarkable nanovesicles that enable controlled drug release and targeted delivery. Administering antibiotic‐loaded nanoerythrosomes to an inflammatory site achieves higher and more prolonged drug concentrations compared to conventional free‐drug administration. This strategy aligns with physiological nature of the inflammatory process, as purulent tissues have increased blood flow and numerous macrophages. The antibiotic‐loaded nanoerythrosomes are selectively captured by macrophages. In purulent‐inflammatory areas, extensive phagocytosis leads to the release of a high concentration of the encapsulated drug. Nanoerythrosomes are believed to enhance targeted antibiotic delivery to sites of microbial inflammation, providing sustained drug release into the affected tissue. To model this release process, an in vitro study was conducted to observe the release of ceftazidime from these carriers.

Methods : Nanoerythrosomes loaded with 1 g of Ceftazidime were obtained by the hypoosmotic hemolysis method in our original modification. This study received approval from institutional ethics committee. Venous blood samples were collected from volunteers after obtaining their signed informed consent. To assess the degree of ceftazidime release from nanoerythrosomes, method of equilibrium dialysis with semipermeable membrane was used. Samples were collected after 15 min, 30 min, 1 h, 3 h, 17 h, 24 h. The concentration of ceftazidime in the collected samples was determined spectrophotometrically.

Results : The maximum release of ceftazidime from nanoerythrosomes was observed at 15 min (1.72 µg/mL ± 0.15), then until 17 h there was a period of stable release, in average of 1.5 µg/mL in each sample. From 17 h to 24 h concentration of antibiotic released from nanoerythrosomes decreased from 0.17 µg/mL ± 0.07 to zero.

Summary/Conclusion : The release of ceftazidime from nanoerythrosomes occurs gradually over 24 h, with peak concentrations sustained for up to 17 h. The results suggest that nanoerythrosomes are effective carriers for targeted ceftazidime delivery to inflammatory sites, potentially improving treatment of purulent diseases. These data will be considered in pharmacokinetic preclinical in vivo study of ceftazidime deposited in nanoerythrosomes.

Funding : This research is funded by Science Committee of Ministry of Science and Higher Education of Kazakhstan, Grant №AP19676272 and by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Engineered extracellular vesicles from Mesenchymal Stromal Cells as nano‐shuttles for viral angiogenic oligopeptides

Presenter: Gabriele Scattini

University of Perugia, Italy

Introduction : A number of diseases in humans and animals are favoured by inadequate angiogenesis. The administration of angiogenic proteins or oligopeptides is considered one of the most promising strategies to support tissue reperfusion. Unfortunately, it currently faces many critical challenges, such as inefficiency in targeting the desired site, rapid clearance and degradation. Extracellular vesicles (EVs) can serve as natural delivery systems due to their biological properties. Mesenchymal stromal cell‐derived EVs (MSC‐EVs) have been investigated as drug carriers, taking advantage of their ability to target injured tissues and resist degradation in unfavourable environments. Here, we investigated the angiogenic potential of oligopeptides (OPs) derived from the HIV‐1 matrix protein p17. Human and equine MSCs were engineered to produce EVs decorated with the angiogenic p17‐derived OPs. To this end, the sequences of OPs were fused to CD63, a tetraspanin protein that naturally targets EVs. The sequence of the green fluorescent protein (GFP) was also incorporated as a reporter.

Methods : p17‐derived OPs were tested in vitro for their angiogenic potential on human and equine endothelial cells. Tube formation, wound healing and proliferation assays were performed. Sequences coding p17‐derived OPs that showed angiogenic properties were successfully ligated into a vector for the expression of CD63‐GFP. MSCs from horse fat and human MSCs were transfected to obtain the expression of OPs on EVs membrane. EVs were enriched by ultrafiltration from the cell culture media and analysed by nanoparticle tracking assay (NTA), electron microscopy (EM) and Western blotting (WB).

Results : Engineered EVs showed a typical dimension and morphology. EM and NTA made it possible to determine a size range from 70 to 500 nm, with a size pick variable among different OPs. At WB analysis, the enriched EVs showed a positive reaction to anti‐GFP and expressed typical markers.

Summary/Conclusion : These preliminary results show that the CD63‐mediated EVs engineering system is suitable for obtaining functionalized EVs. However, it still needs to be optimized to achieve higher transfection efficiency and vesicle production. Further in vitro tests on endothelial cells will confirm whether EVs engineering preserves the angiogenic activity of p17‐derived OPs.

Funding : PRIN 2022 – Finanziato dall'Unione Europea—Next Generation EU ID 2022BJB5FP.

Development of a prototype of an automated software‐controlled complex based on a new technology for automatic loading of RBCs‐derived carriers with selected drugs

Kulzhan Berikkhanova, German Seredin

Nazarbayev University, Nur‐Sultan, Kazakhstan

Introduction : The targeted transport of drugs encapsulated within autologous erythrocytes leads to a substantial enhancement in treatment efficacy and a marked reduction in drug toxicity. Based on extensive experimental and clinical trials, we have proven the possibility of using RBCs‐derived carriers containing a deposited antibiotic to improve the results of treatment of surgical infections. However, in process of creating transport systems based on pharmacocytes, obvious limiting challenge is time‐consuming drug loading process. Difficulties of loading are associated with maintaining strictly sterile conditions for preparation of pharmacocytes, which is carried out manually. This situation is a significant obstacle to widespread introduction of innovative treatment method into clinical practice.

Methods : The engineering design and construction, experimental methods for creating a prototype model of a new technology, assembly and installation, configuration and calibration of a prototype of a new technology for automatic loading of RBCs‐derived carriers with selected drugs has been carried out.

Results : A model of new technology has been developed for automatic loading of erythrocytes with selected drugs with precision dispensers and special software. Based on the technology of targeted delivery of antibiotics based on RBCs‐derived carriers using hypo‐osmotic haemolysis for the treatment of surgical infection, a hardware and software complex has been developed to automate the process of preparing RBCs‐derived carriers.

Summary/Conclusion : Automation, standardization of the process of loading drugs into pharmacocytes and saving the doctor's working time. We believe that automation of the technology for obtaining a transport system based on RBCs‐derived carriers ensures optimization of the process of targeted delivery of drugs in the body. This will lead to the widespread introduction of this innovative treatment method into clinical practice, which will significantly expand the effectiveness of treatment both in surgery and in all areas of medicine.

Funding : This research is funded by Science Committee of Ministry of Science and Higher Education of Kazakhstan, Grant № AP19676272 and by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Engineered extracellular vesicles for targeting and activation of VEGFR‐3 for lymphatic vascular regeneration

Johannes Oesterreicher 1,2 , Tanja Zauner 1,2 , Lukas Narendja 1,2 , Sivun Dmitry 1,3 , Madhusudhan Reddy Bobbili 1, 2 , Jaroslaw Jacak 1,3 , Michael Jeltsch 4 , David Hercher 1 , Johannes Grillari 1,2 , Wolfgang Holnthoner 1

1 Ludwig Boltzmann Institute for Traumatology, Austria; 2 BOKU University, Austria; 3 University of Applied Sciences Upper Austria, Austria; 4 University of Helsinki, Finland

Introduction : Historically, the lymphatic vasculature has received limited scientific attention, despite being crucial role in development and homeostasis. For successful tissue homeostasis after injuries and diseases, therapeutic strategies must include lymphatic vascular regeneration. VEGF‐C is key in lymphangiogenesis, acting through lymphatic endothelial‐specific VEGFR‐3. VEGF‐C has been studied in lymphedema and cancer, but many applications overlook its complex biosynthesis. Proteolytic processing of VEGF‐C results in variants with different receptor specificity and affinity. To enhance VEGF‐C's pro‐lymphangiogenic effects and systemic availability, we suggest linking it to proteins like tetraspanins (e.g., CD81), thus creating extracellular vesicles (EVs) with both targeting and activating capacity.

Methods : We established telomerase‐immortalized adipose‐derived stromal cell (ASC/TERT300) lines expressing different VEGF‐C variants fused to CD81 using lentiviral transduction. The expression and localization of the construct were tested by Western blotting and immunofluorescence microscopy. EVs were enriched via tangential flow filtration and characterized via nanoparticle tracking analysis, bead‐based flow cytometry, atomic force microscopy, and dot blot. A Ba/F3 cell line expressing a VEGFR3/EpoR chimera was used to test the variants for receptor activation. Pro‐lymphangiogenic function was assessed by vascular network formation and migration assays using human lymphatic endothelial cells.

Results : We successfully confirmed expression of CD81/VEGF‐C in the ASCs and respective EV preparations. The VEGFR3 activation assay showed specific receptor activation by VEGF‐C variants. Network formation and migration assays revealed significant differences in the effects of CD81‐VEGF‐C EVs compared to vesicles without VEGF‐C.

Summary/Conclusion : This study demonstrates the successful generation of pro‐lymphatic EVs in relevant assays and evaluate their potential use for lymphatic vascular regeneration.

Mesenchymal stem cell‐derived extracellular vesicles (MSC‐sEVs) as a nanodelivery system for enhanced drug penetration in chondrosarcoma

Presenter: Enza Torino

University of Naples Federico II, Italy

Introduction : Chondrosarcomas (CHS) account for approximately 20% of all primary malignant bone tumours, characterized by slow progression and minimal early symptoms. Despite their heterogeneity, high‐grade and dedifferentiated CHS subtypes pose significant therapeutic challenges due to their resistance to conventional chemotherapy and radiotherapy. Consequently, there is an urgent need for innovative treatment strategies, particularly for aggressive CHS forms with metastatic potential.

Methods : In this study, we explore the use of mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEVs) as a nanocarrier system to enhance drug delivery in a 3D in vitro CHS model. Using high‐pressure homogenization (HPH), we achieved exceptional encapsulation efficiency of doxorubicin (DXR) within MSC‐sEVs (DXR‐MSC‐EVs).

Results : A comparative analysis of free DXR versus DXR‐MSC‐EVs revealed significantly improved drug penetration and cellular uptake in the 3D tumour model. Notably, DXR‐MSC‐EVs induced a higher rate of necrosis and exhibited greater cytotoxic effects than free DXR, underscoring their potential as an advanced drug delivery platform for treating aggressive CHS.

Summary/Conclusion : These findings highlight MSC‐sEVs as a promising strategy for overcoming drug resistance and improving therapeutic outcomes in CHS patients.

An efficient tool for nucleic acids loading into extracellular vesicles

Presenter: Raffaella Daniele

EVis Bioscience AG, Switzerland

Abstract unavailable

Extracellular vesicle‐based delivery platform for the targeted administration of therapeutic RNA by inhalation

Presenter: Alex Eli Pottash

Weizmann Institute of Science, Israel

Introduction : Recent advances have highlighted RNA as a promising therapeutic modality. Yet, challenges in efficient targeting and delivery remain. Extracellular vesicles (EVs) are naturally secreted nanoparticles that, owing to their biocompatibility and innate role in encapsulating RNA, hold significant potential as delivery vehicles. In this study, we developed an EV‐based platform for the targeted delivery of a therapeutic RNA species via nebulization and inhalation, enabling localized administration to the lungs while minimizing off‐target effects.

Methods : Producer HEK293T cells were genetically engineered to express a membrane‐bound, high‐affinity binder of human ACE2 that localizes to the EV surface. EVs were isolated by ultracentrifugation, characterized, and subsequently loaded with a therapeutic RNA molecule using a commercial reagent. The RNA‐loaded EVs were applied either directly to cell cultures or nebulized onto cells. In animal studies, the EVs were delivered via an inhalation tower. Gene and protein expression assays were employed to assess biological function.

Results : Our findings indicate that the EVs preferentially deliver their RNA payload to ACE2‐expressing cells. We optimized a protocol for RNA encapsulation into EVs and demonstrated effective modulation of target gene expression in vitro following nebulization. Furthermore, when administered to animals, the nebulized, RNA‐loaded EVs produced significant modulation of target gene expression, correlating with a measurable therapeutic effect in a model of induced inflammation.

Summary/Conclusion : This work demonstrates the feasibility of using EVs for targeted delivery of therapeutic RNA to lung cells, with the potential to modulate disease‐relevant pathways. The versatility of this platform, allowing for the interchangeability of both the RNA cargo and the targeting moiety, opens the door to a wide array of therapeutic applications.

Designing a plug‐and‐play system to produce engineered extracellular vesicles

Presenter: Kapadia Devarshi

Lonza, USA

Introduction : Extracellular vesicles (EVs), including exosomes, are being studied extensively and have emerged as a promising novel technology for the delivery of complex therapeutic payloads such as nucleic acids or proteins. EVs are naturally occurring within the body and have several potential advantages over other delivery vehicles such as LNPs or viral vectors, such as their ability to evade the immune system response and therefore support repeat dosing, as well as their capacity to access biological barriers and deliver a wide range of cargo to specific target tissue types given their tropism profiles. These attributes make EVs favourable candidates to engineer as drug delivery systems.

Methods : Here, we discuss a plug‐and‐play platform approach to express biomolecules using engineered EVs. Therapeutic cargo may be loaded onto or within EVs via identified scaffold proteins using novel plasmid design. This concept was explored in the development of therapeutics such as exoSTING, exoASO‐STAT6 and exoIL‐12, which have completed Phase 1 clinical studies within oncology applications. Alternatively, small molecules may be linked to EVs using a proprietary linker technology. A dedicated cell line for EV production was also generated, which resulted in a clone capable of high particle titer, integrity, and purity.

Results : Results are to be discussed.

Summary/Conclusion : These technologies will be made available to the wider scientific community under a research license, or via Lonza's CDMO service offerings.

Subtopic EV Detection 2

Linking extracellular vesicle markers to cancer‐specific biomarkers: a correlative study

Presenter: Juan P. Hinestrosa

Xzom, USA

Introduction : Extracellular vesicles (EVs) offer immense potential in disease detection, treatment choices, and screening by reflecting disease states through biomolecules like proteins, nucleic acids, and metabolites. Although EV markers are well‐studied (per ISEV guidelines), their link to specific cancer biomarkers remains underexplored, with most research focused on nucleic acids and free proteins. Our work seeks to bridge this gap, examining the relationship between known EV markers and cancer‐related proteins.

Methods : EVs were isolated via ultracentrifugation from cancer cell lines H1975, A549, AsPC‐1, and SKOV‐3. Using multiplex ELISA, we measured classic EV markers (CD63, CD9, CD81, TSG101) and 52 cancer‐associated markers, analysing correlations with cancer biomarkers while adjusting for EV particle concentration and size. Biomarkers were tested across multiple concentration levels. To confirm results, EVs were spiked into buffer and human plasma, then assessed on a microelectrode array platform, closely reflecting their native state.

Results : As anticipated, EV markers (CD9, CD63, CD81) correlated strongly across cell lines and directly with particle input. Each cell line type displayed a unique EV protein profile aligned with its cancer type. Among the 52 cancer biomarkers tested, 15 were detected in H1975, 4 in AsPC‐1, 2 in A549, and 4 in SKOV‐3. Notably, the lung cancer biomarker CYFRA21‐1 was present in both lung cancer cell lines and showed correlation with CD63 and CD81, indicating its presence on the EV surface. Full correlation data will be presented between all the EV biomarkers and the cancer biomarkers detected. Following EV spiking into buffer and plasma, biomarker signals were confirmed in an environment as close as possible to their native state. Further analyses regarding EV concentration, morphology, and findings post‐lysis for cytosolic cancer biomarkers will also be shared.

Summary/Conclusion : The promise of EVs for early cancer detection is closer than ever to becoming a reality. Demonstrating that EVs are directly linked to cancer biomarkers and that this information can be easily accessed brings us a step nearer to achieving reliable, near‐patient diagnostics. Here, we illustrate the specific correlations between EV markers and cancer biomarkers across cell lines, showing their feasibility for simple, direct detection.

Funding : This study was sponsored by Xzom, Inc.

Functional nucleic acid probes‐enabled in situ detection of extracellular vesicle proteins and micrornas for cancer diagnostics

Pengfei Wang, Jing Ye, Fei Zhou, Li Pan, Donglei Yang, Weihong Tan

Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Introduction : Tumour cell‐derived small extracellular vesicles (sEVs) contain various molecular ingredients such as membrane proteins and microRNAs (miRNAs) that can serve as critical biomarkers for cancer diagnostics. Nevertheless, conventional detection methods involve extracting proteins and miRNAs out of sEVs, which not only may cause damage/loss of biomarkers, but also demand large amount of effort. There is an increasing need to develop novel methods to enable in situ detection of membrane proteins and miRNAs with sEVs remaining intact.

Methods : We developed a sEV membrane protein detection method (PRECISE) by encoding individual protein‐aptamer molecular recognition events into DNA strands of unique sequences via proximity‐induced DNA extension. DNA strands carrying encoded information are subject to amplification, sequencing, and sequence decoding for revealing protein identities and counts. For miRNA detection, we conjugated hydrophobic tethers onto molecular beacons (MB) to promote its transportation efficiency into sEVs to enable in situ miRNA detection.

Results : Membrane protein profiles of serum‐derived sEVs from a total of 174 individuals with cancers have been acquired by using PRECISE. Employing machine learning algorithms, we were able to build diagnostic models for distinguishing prostate cancer, gastric cancer, and breast cancer patients from noncancer controls, with a diagnostic accuracy of 100%. Furthermore, we built a multiclass cancer classifier that exhibited potent performance in the classification of three cancer types with a classification accuracy of 82.4%. Cholesterol‐tethered MBs can enable simultaneous detection of multiple miRNA species within serum‐derived sEVs for the diagnosis of prostate cancer, breast cancer, and gastric cancer with an accuracy of 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals.

Summary/Conclusion : Our works provide a feasible, robust, and precise technical platform for sEV surface protein and miRNAs fingerprinting that shall hold great application potential in cancer diagnostics and beyond.

Specific labelling of outer membrane vesicles with antibiotic‐conjugated molecular probe reveals early bacterial infection signatures in blood

Presenter: Qianbei Li

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Bacterial outer membrane vesicles (OMVs) are nano‐sized structures derived from the outer membrane of Gram‐negative bacteria. These vesicles have emerged as key players in host‐pathogen interactions, but their potential as biomarkers remains largely unexplored due to the difficulty of identifying them in complex biological samples.

Methods : We used the polymyxin B‐fluorescein probe (PmbF) to label bacterial OMVs in blood samples and analysed them using nano‐flow cytometry. In a mouse model of pneumonia, we collected serum samples at various time points post‐infection to monitor changes in PmbF+ EVs. To confirm the origin of these EVs, we utilized an engineered mCherry‐expressing E. coli strain. Additionally, we collected clinical samples and compared the performance of PmbF+ EVs with C‐reactive protein (CRP) and procalcitonin (PCT) in diagnosing bacterial infections and differentiating them from viral or mycoplasma infections. Finally, we conducted co‐culture experiments to assess the effects of OMVs on macrophages.

Results : In the mouse model of pneumonia, we observed a significant increase in serum PmbF+ EVs as early as 6 h post‐infection, preceding positive blood cultures. In clinical samples, PmbF+ EVs outperformed CRP and PCT in diagnosing bacterial infections and distinguishing them from viral or mycoplasma infections. Co‐culture experiments revealed that OMVs triggered macrophage apoptosis by inducing mitochondrial dysfunction and reactive oxygen species (ROS) production.

Summary/Conclusion : Our findings highlight the potential of circulating OMVs as promising biomarkers and mediators of bacterial infections. By using the PmbF probe and nano‐flow cytometry, we can efficiently detect and quantify these OMVs, providing a new tool for the early diagnosis and monitoring of bacterial infections. Furthermore, the role of OMVs in host immune responses offers new insights into the pathogenesis of bacterial infections.

Funding : This work was supported by the National Science Fund for Distinguished Young Scholars (82025024); the Key project of the National Natural Science Foundation of China (82230080); the National Natural Science Foundation of China (82302593 and 82272438); Guangdong Natural Science Fund for Distinguished Young Scholars (2023B1515020058); the Natural Science Foundation of Guangdong Province (2023A1515012512); the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2022J001).

Multiplexed sensitive detection of small extracellular vesicles using an electrokinetic based microfluidic device

Presenter: Moein Talebian Gevari

Uppsala University, Uppsala, Sweden

Introduction : Here, we demonstrate the fabrication, characterization, and application of a novel silicon microchip‐based biosensor that exploits streaming current (I_s) for the detection and characterization of small extracellular vesicles (sEVs). The method was further optimized and compared with a fluorescence‐based single EV technology for a semi‐quantitative assessment of EV count, membrane‐protein expression, and relative abundance of various EV‐subpopulations.

Methods : sEVs were isolated from cell culture media of the non‐small cell lung cancer H1975 cell line cultured in RPMI‐media with FBS depleted from exogenous exosomes. The media was concentrated and sEVs were isolated by SEC. The sEVs were characterized by NTA and earlier western blot confirmed expression of the targeted proteins. The CD81 and CD9 membrane proteins of the sEVs were profiled using a novel microchip technology. The microchips, composed of four interconnected microchannels, were designed with an open‐top geometry where each of the channels could be functionalized with different antibodies for multiplexed measurement. The expression level of target markers was detected by immunocapture of sEVs on the sensor surface and measuring the changes in I_s.

Results : We assessed poly‐L‐lysine and silane‐based surface functionalization strategies to capture sEVs on silica. As a proof of concept, CD81 and CD9 tetraspanins from H1975‐derived sEVs were targeted and compared to state‐of‐the‐art methods. The results showed an LoD of 9.5 × 10 3 sEVs/mL and 7.6 × 10 4 sEVs/mL for CD81 and CD9 tetraspanins, respectively. Using membrane‐sensitive peptides, our platform achieved an LoD of 1 × 10 4 sEVs/mL. sEVs were isolated by size exclusion chromatography, and their surface proteins were profiled using single‐EV fluorescence. Nanoparticle tracking analysis (NTA) characterized size and charge distribution prior to biosensing.

Summary/Conclusion : This biosensing platform demonstrated sensitive, multiplexed detection of sEVs, outperforming other sensitive platforms. With a quick 30‐min detection time and minimal sample requirement of 30 µL, this device holds promise for clinical and research applications in studying sEVs.

Funding : This study was supported by the Swedish Research Council; The Swedish Cancer Foundation

Refining flow cytometry‐based sorting of plasma‐derived extracellular vesicles

Presenter: Daniele Reverberi

IRCCS Ospedale Policlinico San Martino, Genoa, Italy

Introduction : Extracellular vesicles (EVs) are membrane‐bound particles released by cells, playing a key role in intercellular communication and serving as promising biomarkers for various diseases. The ability to isolate and characterize specific EV subpopulations, particularly those circulating in plasma/serum, holds great potential for enhancing biomarker precision and developing targeted therapies. Cancer‐derived EVs often express unique surface markers, enabling their distinction from EVs of other cellular origin. Thus, the accurate sorting of tumour EVs could provide valuable insights into cancer progression, metastasis, and treatment response.

Methods : We developed a robust method for isolating and sorting CD9‐positive plasma EVs as a proof‐of‐concept for broader EV subpopulation analyses. Plasma EVs were isolated using sucrose cushion ultracentrifugation, balancing purity and yield. After a bulk EV characterization, an unconventional flow cytometry strategy based on fluorescence threshold triggering was initially applied to assess the percentage of CD9‐positive EVs and subsequently to sort them. CD9 is one of the most highly expressed tetraspanins by plasma‐derived EVs and was selected here as a proof‐of‐concept antigen to describe our standardized sorting protocol.

Results : To determine the minimal prerequisites for EV detection and sorting, instrument setup in terms of sheath and sample pressure, as well as particle concentration, were thoroughly considered. The effect on event rates per second, side light scattering, and fluorescent signals obtained during the analysis of different EV numbers was assessed using various nozzles, demonstrating that particle concentration can significantly affect their accurate quantification and characterization. Analyzing an excessive number of EVs leads to the coincident detection of nano‐sized vesicles, resulting in an unwanted swarming effect. Moreover, we demonstrated how the removal of unbound reagents (dyes, antibodies), by size exclusion chromatography, after EV staining profoundly impacts the percentage of CD9‐positive EVs and consequently affects their sorting efficiency in terms of event rate/second and volume of sorted material.

Summary/Conclusion : The advancements here proposed could enable high‐purity isolation of EV subpopulations, facilitating their use in downstream applications, such as the identification of cancer‐specific biomarkers or development of EV‐based targeted therapies.

Funding : This work was supported by Worldwide Cancer Research under Grant (Reference 24‐0042).

Harnessing microfluidic chips and metamaterial sensors for isolation and detection of extracellular vesicles

Eylul Gulsen Yilmaz 1,2 , Esma Derin 1,2 , Fatih Inci 1,2

1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey

Introduction : Acute kidney injury (AKI) affects approximately 13 million individuals worldwide and is responsible for 1.7 million deaths annually. AKI poses a significant healthcare challenge due to limited biomarkers. Extracellular vesicles (EVs) offer promising potential for diagnostics and therapy, though isolating and characterizing EVs from complex fluids remains challenging in clinical settings. This study aims to isolate and characterize surface markers of EVs released in the urine of AKI patients using a streamlined (5‐step protocol), rapid(1.5‐h), and cost‐effective ($5) method. Herein, we developed an ultrafiltration‐based chip to isolate EVs from both cultured samples and urine samples from healthy individuals and AKI patients. Additionally, we repurposed nanograting plastic surfaces as sensitive metamaterial sensors, further functionalized with EV‐specific antibodies to enable EV detection.

Methods : In all experiments, MISEV criteria is closely followed. Through an ultrafiltration chip and a metamaterial sensor, we isolated and detected EVs from both cultured samples and urine samples from healthy controls and AKI patients. EVs were further validated using nanoparticle tracking analysis (NTA), fluorescence NTA, Western blotting, scanning electron microscopy, and x‐ray photoelectron spectroscopy. To evaluate the effectiveness of our platforms, we compared them with standard ultracentrifugation and commercial ELISA methods, followed by statistical analysis to assess performance differences.

Results : The ultrafiltration chip yielded results comparable to ultracentrifugation, isolating up to 10 9 EVs/µL. Compared to ELISA, our platform achieved approximately 100‐fold greater sensitivity and a wider dynamic range (spanning four orders of magnitude). Using the metamaterial sensor, we further validated samples from patients and controls, analyzing a broad AKI biomarker panel—including ATF‐3, NGAL, THP, AQP‐1, CD133, and Fetuin A—in addition to tetraspanins.

Summary/Conclusion : Our metamaterial sensor exhibited exceptional sensitivity down to 10 3 EVs/µL, complemented by our user‐friendly, cost‐effective, and rapid ultrafiltration chip. In the near future, both platforms have the potential to be seamLessly implemented in clinical settings, significantly advancing the application of EV markers in point‐of‐care diagnostics.

Funding : The authors gratefully acknowledge support from the Scientific and Technological Research Council of Turkey 2232 International Fellowship for Outstanding Researchers (118C254), Outstanding Young Scientists Award(GEBİP) from Turkish Academy of Sciences, Young Scientist Awards Program(BAGEP) form Science Academy, and the exRNA‐PATH COST Action CA20110.

Model to correct extracellular vesicle concentrations for false‐positive fluorescent events

Presenter: Joyce Rops

Amsterdam UMC, Amsterdam, The Netherlands

Introduction : Flow cytometry is a widely used method to measure concentrations of extracellular vesicles (EVs) in blood plasma and other biofluids. With flow cytometry, fluorescent labelling is used to identify EVs in plasma, but non‐specific binding of labels to lipoproteins may result in overestimation of the measured EV concentration. Here we developed a model to correct the measured EV concentration for non‐specific binding to lipoproteins.

Methods : A logistic regression model was developed and fitted to flow cytometry data from the Antiplatelet Therapy Effect on Extracellular Vesicles (AFFECT EV) study, which uses the labels cluster of differentiation (CD)31, CD45, CD61, CD62p, CD146, CD235a, lactadherin and fibrinogen. To validate the model, EVs and lipoproteins were identified using the flow cytometry scatter ratio (Flow‐SR), a label‐free method that can distinguish EVs from lipoproteins based on their difference in refractive index (RI).

Results : We tested the model on all data from the AFFECT EV study. Here, preliminary data from CD45‐positive particles will be used as a representative example. CD45‐positive EV concentration values, corrected for false‐positive events using both the model and Flow‐SR, were in close agreement with each other (0.98 ± 0.38). Of all CD45‐positive particles with a diameter > 200 nm that were identified as CD45‐EVs, 70% were classified as EVs by the model and 72% according to Flow‐SR. The results confirm that concentrations of labelled particles in plasma should be corrected for false‐positive events.

Summary/Conclusion : Commonly used fluorescent labels, including monoclonal antibodies, do not only label EVs but also lipoproteins in plasma. The developed model corrects for false‐positive events and can therefore be used to improve the accuracy of EV concentration measurements with flow cytometry. In contrast to Flow‐SR, which can only be applied to EVs > 200 nm using a limited number of flow cytometers, our new model can be applied to all EV sizes measured by all flow cytometers. After validation using spike‐in measurements with lipoproteins, this model can be used in clinical research studies and possibly applied to other biofluids containing EVs and non‐EV particles.

Funding : The authors acknowledge the Dutch Heart Foundation for funding this work.

Cancer cell‐derived extracellular vesicle detection on a surface‐functionalized power‐free microfluidic chip

R. Ishihara 1 , H. Yokohari 1 , R. Ogata 1 , T. Nakajima 2 , E. Shimura 1 , and T. Baba 1

1 Juntendo University, Tokyo, Japan; 2 Yokohama City University, Yokohama, Japan

Introduction : Extracellular vesicles (EVs) present in body fluids have garnered significant attention as novel biomarkers for various diseases, including cancers. However, the inherently low concentration of EVs in these fluids poses challenges for their detection. To facilitate facile and highly sensitive EV detection for cancer point‐of‐care diagnostics, we have developed portable surface‐functionalized power‐free poly(dimethylsiloxane) microfluidic chips (SF‐PF microchips). In this study, we present the development of a novel SF‐PF microchip and demonstrate its application in detecting cancer cell‐derived EVs.

Methods : The SF‐PF microchip for EV detection was prepared in the following four steps: (1) A PDMS microchip was fabricated using a soft lithography technique. (2) The inner surfaces of the microchannels of the microchip were modified with poly(2‐aminoethyl methacrylate) (PAEMA) via UV graft polymerization. (3) Anti‐integrin β1 antibodies were subsequently immobilized into the grafted polymer phase. (4) The antibody‐immobilized microchip was attached to a glass slide and degassed. Subsequently, EVs were detected using laminar‐flow‐assisted dendritic amplification on the SF‐PF microchip.

Results : The surface modification of PAEMA was confirmed via ATR‐FTIR spectroscopy. Anti‐integrin β1 antibody was selected as a targeting antibody for antigens highly expressed on EVs derived from malignant breast cancer cells. Immobilization of a sufficient amount of anti‐integrin β1 antibodies on the microchannel surface was verified using a fluorescent‐labelled secondary antibody. EV signals were evaluated based on the signal‐to‐blank ratio, which was significantly higher for EVs than for the control ( p < 0.05, two‐tailed t ‐test). Anti‐integrin β1 antibodies immobilized on the inner surface of the microchannel effectively recognized EV surface antigens, enabling selective EV detection. The limit of detection (LOD) of the SF‐PF microchip was calculated according to the 3σ criterion from a calibration curve, yielding an LOD of 2.1 × 10 10 EVs/mL. This value is comparable to the EV concentration in blood. The required sample volume and assay time were 2.0 µL and 20 min, respectively.

Summary/Conclusion : A portable SF‐PF microchip that enables facile and highly sensitive EV detection was successfully developed. This SF‐PF microchip has the potential to contribute to the establishment of EV‐based point‐of‐care cancer diagnostics.

Funding : This research was partly supported by JSPS KAKENHI (20K12697 and 23K11905).

Biosensors engineered with extracellular vesicle imprints for breast cancer diagnosis

Eylul Gulsen Yilmaz 1,2 , Fatih Inci 1,2 , Yeşeren Saylan 3†

1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey; 3 Hacettepe University, Department of Chemistry, Ankara, Turkey

Introduction : Early detection of breast cancer improves therapy and results, however 3D in vitro models are difficult to use to investigate biophysical influences on cancer cells. The microfluidic chip we show isolates extracellular vesicles (EVs) from MCF7 breast cancer cells, with an emphasis on EVs as cancer progression indicators. We will conduct 3D cell cultures with an extracellular matrix to investigate the impact of EVs on MCF‐12A normal breast cells and their function in alterations to the tumour microenvironment. For more accurate, up‐to‐the‐minute diagnoses, optical biosensors will be integrated with EV‐imprinted nanoparticles to enhance detection. This technique improves our knowledge of cancer biology and allows for more accurate non‐invasive cancer detection.

Methods : The microfluidic chip were prepared in order to mimic continuous flow in dynamic cell culture. We cultured MCF‐7 and MCF‐12A. Isolated EVs have been examined by Western blotting, NTA, and SEM after they were extracted from MCF‐7 cells by ultrafiltration. EV‐imprinted nanoparticles were synthesized using methacrylic acid and micro‐emulsion polymerization, and their size and morphology were analysed using a Nano Zetasizer and TEM.

Results : EVs extracted from a collagen‐coated microfluidic chip exhibited a size of 129.2 ± 4.5 nm and a concentration of 6.49 × 10 9 ± 3.56 × 10 8 particles/mL, as validated by NTA. SEM confirmed the elimination of bigger particles, isolating those within the 50–150 nm range, which exhibited a spherical morphology. Western Blot verified the existence of tetraspanin proteins CD63 and CD81. EV‐imprinted nanoparticles had a size of 62.78 nm with low polydispersity (PdI = 0.116), signifying uniformity in size and shape, corroborated by TEM analysis. The nanoparticles were uniform, spherical, and consistent with the size distribution obtained from zeta size analysis.

Summary/Conclusion : This research seeks to create a molecularly imprinted optical biosensor for the sensitive, selective, and real‐time detection of EVs. This innovative method entails the extraction of MCF7 EVs from 3D cell cultures utilizing microfluidic chips incorporated with an extracellular matrix. The gathered EVs will be examined to ascertain their effects on MCF‐12A, yielding insights into alterations in the cancer microenvironment and the cancerization process.

Funding : All authors acknowledge the support from The Scientific and Technological Research Council of Türkiye (TÜBİTAK) (Project Numbers:121C226).

Characterization and validation of GFP farnesylation as a suitable labelling strategy in tracking engineered exosomes during uptake studies

Lucrezia Luisotti 1 , Rebecca Piccarducci 1 , Lorenzo Germelli 1 , Alessandra Falleni 2 , Benedetta Masciulli 3 , Ranieri Bizzarri 3,4,5 , Martina De Felice 6 , Francesca Cella Zanacchi 4,6 , Chiara Giacomelli 1,4 , Laura Marchetti 1,4*, and Claudia Martini 1,4

1 Department of Pharmacy, University of Pisa, Pisa, Italy; 2 Department of Experimental and Clinical Medicine, University of Pisa, Pisa, Italy; 3 Department of Surgical, Medical and Molecular Pathology, and Critical Care Medicine, University of Pisa, Pisa, Italy; 4 Center for Instrument Sharing, University of Pisa, 56126, Pisa, Italy; 5 NEST, Istituto Nanoscienze‐CNR and Scuola Normale Superiore, Pisa, Italy; 6 Department of Physics, University of Pisa, Pisa, Italy

Introduction : Exosomes are cell‐derived small extracellular vesicles (EVs) essential for cell‐to‐cell communication; indeed, exosome bioengineering is an appealing approach to tune the exosome cargo as well as modify the exosome surface to target desired recipient cells. Consequently, exosome journey monitoring is essential. Although fluorescent lipophilic dyes are the gold standards for exosome labelling, they lack selectivity, due to their unconditional affinity for lipids. An alternative exosome fluorescent labelling strategy is herein evaluated, by taking advantage of green fluorescent protein farnesylation (GFP‐f), a post‐translational modification that allows the direct anchor of GFP to the exosome membrane. The current study analyses the performance of GFP‐f, in terms of selectivity and efficiency, in tracking engineered exosomes during uptake studies.

Methods : Naïve or engineered HEK293T‐derived exosomes were isolated by size exclusion chromatography and physically and biochemically characterised. Then, they were employed for uptake studies.

Results : Firstly, the advantage of directly anchoring the GFP to the exosome membrane for labelling exosomes was demonstrated. Then, GFP‐f‐bearing exosomes were further compared to VybrantTM DiD lipophilic dye labelled ones in uptake studies, by capturing intracellular fluorescence in a time‐ and concentration‐dependent manner. The internalization assay revealed a particular ability of GFP‐f to monitor the uptake of tagged exosomes into recipient cells, with a significant peak of intensity reached 12 h after administration by GFP‐f, while it was negligible in VybrantTM DiD labelled exosomes. Finally, the GFP‐f tagging capability was tested in the presence of a surface‐modification of exosomes, that is, LAMP2B‐Exenatide, a GLP1 analogue moiety tailored for targeted delivery into pancreatic b‐cells. Results showed that GFP‐f performance is influenced by surface engineering compared to naïve GFP‐f exosomes, but the fluorescence is importantly preserved. Therefore, the ability of LAMP2B‐Exenatide to selectively target human pancreatic b‐cells is currently under investigation, by monitoring the internalization of targeted exosomes into different recipient cells through GFP‐f labelling.

Summary/Conclusion : Overall, these data provide a direct insight into the advantages and limitations of GFP‐f as a tagging protein for selectively and accurately tracking exosome routes from isolation to uptake in recipient cells, also in the context of EV bioengineering applications.

Funding : This study was funded by the European Union‐NextGenerationEU: Mission 4‐Component 2‐CUP B93D21010860004.

A versatile droplet microfluidic sensor for simultaneous sensitive detection of proteins and mRNA at single EV resolution

Presenter: Huixian Lin

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Simultaneous analysis of proteins and mRNA at single extracellular vesicle (EV) resolution enables comprehensive analysis of specific EVs subpopulations holding promise for accurate cancer diagnostics. However, developing a sensitive and specific approach for simultaneous analysis of multidimensional biomarkers in single EV is still challenging.

Methods : The antibody‐DNA conjugates were first prepared as the EVs protein biomarkers recognition conjugates and signal transformer. Coupling with the assembled triplex droplet digital PCR system, a versatile droplet digital assay for simultaneous detection of membrane protein and mRNA at a single EV level was developed.

Results : The versatile droplet microfluidic sensor displayed high sensitivity and specificity. This assay also has demonstrated superior performance in distinguishing breast cancer from healthy individuals and benign controls through combined detection of EVs protein and mRNA markers, especially for patients with breast cancer at early stage.

Summary/Conclusion : Consequently, this study proposes a promising strategy for accurately identifying and analyzing specific EV subgroups through the co‐detection of proteins and mRNA at the single EV level, holding significant potential for tumour diagnosis and offering potential for extension into the investigation of various diseases, contributing to advancements in EV research.

Are intraluminal dyes a better tracking strategy than lipophilic membrane dyes to assess extracellular vesicles biodistribution within corneal tissues?

Manon Jammes 1 , Monika Sypecka 2,3 , Yedizza Rautavaara 4 , Seyedmohammad Moosavizadeh 1,5 , Trung Bach 1 , Abbas Tabasi 1 , Jiemin Wang 1 , Neil Lagali 4 , Thomas Ritter 1,5

1 Regenerative Medicine Institute, School of Medicine, University of Galway, Galway, Ireland; 2 Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland; 3 Doctoral School of Translational Medicine, Centre of Postgraduate Medical Education, Warsaw, Poland; 4 Department of Biomedical and Clinical Sciences, Faculty of Medicine, Linköping University, Linköping, Sweden; 5 CURAM Centre for Research in Medical Devices, University of Galway, Galway, Ireland

Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs; MSC‐EVs) recently emerged as a promising approach to address corneal diseases, leveraging the anti‐inflammatory, immunomodulatory, and pro‐regenerative properties of MSCs while bypassing the challenges inherent to cell therapy. Exploring the uptake and biodistribution of MSC‐EVs within the tissue typically involves using lipophilic membrane dyes, like PKH, inevitably increasing EV size and modifying their surface composition which potentially affect the interactions with their targets. In this context, using intraluminal dyes presents a suitable alternative to standard protocols to ensure the preservation of EV mechanism of action and the reliability of generated data.

Methods : EVs were isolated from human bone marrow MSCs using size exclusion chromatography. Fresh EVs were labelled with CFSE or CellTrace Far Red (CTFR), and the stability of the fluorescence signal was investigated by flow cytometry after ‐80°C storage (three different timepoints) followed by 37°C incubation to mimic in vivo administration. Human corneal epithelial cells (CEpiCs) and keratocytes were treated with CFSE‐ or CTFR‐EVs at several concentrations and the EV uptake was followed by fluorescence live imaging over four days.

Results : The EV size and morphology remained unchanged after labelling with both dyes. Although most of the fluorescence was preserved, CFSE‐EVs exhibited signal reduction after 37°C incubation, while CTFR‐EVs were impacted by −80°C storage. Given the natural strong green fluorescence background in cell cultures, CFSE‐EV uptake was more arduous to track in vitro than CTFR‐EVs.

Summary/Conclusion : CTFR staining is an effective method to label EVs and track them in vitro, avoiding the structural alterations associated with PKH‐like dyes. EV tracking on corneal explants after CTFR‐EV topical or subconjunctival administration is currently investigated to ensure the translation of the method to in vivo studies.

A diagnostic tool to distinguish between acute ischaemic stroke and intracerebral haemorrhage in a prehospital setting

Lee‐Ann Clegg 1,2 , Jesper Just 3 , Jan B. Valentin 4 , Rikke Bæk 1 , Birgitte H. Mumm 5 , Malene M. Jørgensen 1,2 , Grethe Andersen 6 , Rolf A. Blauenfeldt 6,7 , Kim R. Drasbek 5

1 Aalborg University Hospital, Aalborg, Denmark; 2 Aalborg University, Aalborg, Denmark; 3 Aarhus University Hospital, Aarhus, Denmark; 4 Danish Center for Clinical Health Services Research, Aarhus, Denmark; 5 Center of Functionally Integrative Neuroscience, Aarhus, Denmark; 6 Aarhus University Hospital, Aarhus, Denmark; 7 Aarhus University, Aarhus, Denmark Lee‐Ann Clegg is the presenting author, l.clegg@rn.dk (patent pending—limited disclosure) .

Introduction : Stroke is a serious condition that affects 12 million persons each year. Stroke encompasses both acute ischaemic stroke (AIS) or intracerebral hemorrhage (ICH), and currently these cannot be distinguished based on symptoms alone. Timely evaluation, regardless of stroke type, is essential for improving stroke outcome. Primary stroke centres can treat most ICH while AIS patients will benefit from transfer to a comprehensive stroke centre. Early diagnosis is therefore paramount for optimal choice of treatment for acute patients to preserve their brain functionality and minimize permanent disabilities. This study aims to develop a diagnostic tool to distinguish between AIS and ICH patients in the hyper‐acute phase after stroke onset. We included plasma samples from stroke patients, readily available patient characteristics and a panel of 48 specific extracellular vesicle (EV) biomarkers.

Methods : This was a prediction study, mainly exploratory, which adhere to the transparent reporting of a multivariable prediction model for individual prognosis or diagnosis. The study population was a subset of the RESIST trial and constituted 424 patients independent in their daily activities with a putative stroke. Danish regional ethics committees (ID:1‐10‐72‐97‐17) approved this study and a written consent was obtained by the patient or a relative. Stroke diagnosis was based on clinical and neuroimaging findings. To test 48 biomarkers ability to distinguish between no stroke, AIS or ICH, plasma from blood drawn in the ambulance and at the hospital were applied for EV analysis with a high‐throughput protein microarray method called EV Array. All models were trained and tested on these data.

Results : Among the 424 patients, 34% of AIS patients and 42% of ICH patients were male, while 66% and 58% were female, respectively. A benchmark model was developed based on key patient characteristics, including age, sex, and PreSS scores. The AUC for the benchmark models for both large AIS and large ICH were above 0.55.

Summary/Conclusion : We suggest that markers on EVs from plasma, detected with EV Array, are candidates for acute stroke diagnostics with the potential to improve treatment initiation in a pre‐hospital setting. Further investigation of stroke related biomarkers on EVs should be pursued.

Funding : This study was supported by the Novo Nordisk Foundation

Detection of extracellular vesicles by lipophilic self‐extinguishing fluorescent molecules

Dorina Lenzinger 1 , Anna Almási 1 , Anna Koncz 1,2 , Attila Csomos 3 , Kelsey Fletcher 1 , Krisztina V. Vukman 1 , Krisztina Németh 1,2 , Zoltán Mucsi 4 , Edit I Buzás 1,2,5 , Tamás Visnovitz 1

1 Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary; 2 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Budapest, Hungary; 3 Eötvös Loránd University, Faculty of Sciences, György Hevesy PhD School, Budapest, Hungary; 4 University of Miskolc, Faculty of Materials and Chemical Engineering, Institute of Chemistry, Miskolc, Hungary; 5 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary

Introduction : The defining component of extracellular vesicles (EVs) is their phospholipid bilayer membrane, distinguishing them from co‐purified non‐vesicular extracellular particles (NVEPs) such as protein aggregates or lipoproteins. Most commonly used methods for EV characterization include nanoparticle tracking analysis (NTA) and resistive pulse sensing. They both rely on particle counting and are often complemented by immune detection‐based techniques such as flow cytometry or SP‐IRIS, super‐resolution microscopy and Western blot. Particle enumeration is often challenged by co‐isolated NVEPs, while antibody‐based detection is often prone to signal interference from unbound antibodies or antibody aggregates. Additionally, antibody‐based detection approaches might be limited to specific EV subpopulations.

Methods : In this study, we tested novel self‐extinguishing fluorescent lipophilic (SFL) dyes for improved EV detection and quantification, aiming to better distinguish EVs from NVEPs. We used SFL dyes containing Cy2, Cy3, and Cy5 fluorophores and evaluated the fluorescent signals across multiple detection platforms, including a 96‐well fluorescence plate reader, NTA, high‐resolution flow cytometry and confocal microscopy. Initial optimization was conducted using DOPC liposomes along with EVs.

Results : Flow cytometry results demonstrated that SFL dyes were effective in differentiating EVs from most NVEPs based on the presence or absence of an intact EV membrane. Triton X‐100 lysis of EVs further validated these results. In NTA measurements, approximately 10%–50% of the detected particles were identified as EVs using the above SFL dyes. Taking advantage of the self‐extinguishing property of these dyes, we were able to detect as little as 100 ng of EV‐derived lipids, although fluorescence‐based lipid quantification showed lower accuracy than the sulpho‐phospho‐vanillin (SPV) lipid assay. The SFL dyes were also suitable for live cell imaging via confocal microscopy but based on our findings, they are not recommended for EV uptake studies due to the self‐extinguishing effect.

Summary/Conclusion : Our results suggest that SFL dyes can enhance EV characterization, particularly in settings where eliminating unbound fluorescently labelled antibodies is challenging or not feasible.

Funding : NVKP_16‐1‐2016‐0004, VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, TKP2021‐EGA‐23. RRF‐2.3.121‐2022‐00003, 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 No. 739593, János Bolyai Research Scholarship of the Hungarian Academy of Sciences

Tracking CD63 exocytosis in live cells with a newly developed fluorescent biosensor

Anna Lischnig 1 , Yusuf C. Erdoğan 1 , Wolfgang F. Graier 1,3 , Roland Malli 2,3*

1 Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; 2 CF Bioimaging, Center of Medical Research, Medical University of Graz, Graz, Austria; 3 BioTechMed Graz, Graz, Austria

Introduction : The tetraspanin CD63 is commonly associated with extracellular vesicles (EV) and plays an important role in EV biogenesis and secretion. CD63 is being used for the real‐time visualization of multivesicular body (MVP) and plasma membrane (PM) fusion events. The available reporters for CD63 secretion rely on pH‐sensitive fluorescent proteins (FPs), which show an intensity increase during MVB fusion due to pH change. However, current CD63‐based reporters have limited dynamic range and background from CD63‐FP accumulation at the PM, posing a challenge to detect fast, transient exocytosis events. Therefore, advanced strategies are essential to expand and refine the toolkit for EV secretion visualization.

Methods : We fused a genetically encoded single‐FP‐based Ca 2+ biosensor, GreenT‐EC.c, to the first extracellular loop of CD63 as an alternative to only pH‐sensitive FPs. The new design also consists of protease‐cutting sites flanking the biosensor to lower the PM background signal and a red reference FP fused to the intracellular C terminus of CD63 to normalize the readout. Upon protein structure validation in silico, we tested the new reporter in mammalian cell lines (HeLa S3, EA.hy926). We characterized the sensor dynamics and the visualization of CD63 secretion events via live cell imaging.

Results : We demonstrate that the CD63‐fused calcium biosensor emits strong green fluorescence upon exocytosis, as elevated extracellular Ca 2 ⁺ and neutral pH enhance calcium binding to the sensor. This increase in green fluorescence, without elevation in red fluorescence, enhances the GFP/RFP ratio, allowing clear visualization of secretion events. Protease treatment reduces background GFP signal at the PM, improving the dynamic range and enabling precise detection of transient exocytosis events. Overall, the protease‐mediated removal of externalized FP significantly enhances signal quality.

Summary/Conclusion : Our findings demonstrate that Ca 2 + biosensors can effectively be used to visualize secretion events in live‐cell imaging. We expect that with this new reporter system, EV secretion can be monitored in high spatial‐temporal resolution, thus being very suitable for live cell imaging applications.

Detection of extracellular vesicles by fluorescently tagged anti‐CD9 nanobodies

Vladislav Semak 1 , Manuel Hessenberger 2 , Marwa Mostageer 1 , Tanja Eichhorn 1 , Gerald J. Obermair 2 , Viktoria Weber 1

1 University for Continuing Education Krems, Krems, Austria; 2 Karl Landsteiner University of Health Sciences, Krems, Austria

Introduction : Fluorescently labelled antibodies for detecting cell surface epitopes may fail to detect these epitopes on extracellular vesicles (EVs). These limitations are related to the small size and low number of antigens on EVs, as well as to self‐aggregation and batch‐to‐batch variations in the fluorochrome‐to‐protein ratio of the conjugated antibodies. We hypothesise that nanobodies (single‐domain antibodies) can improve the detection of epitopes on EVs due to their smaller molecular size and consistent labelling. Here, we present the initial results of our study, demonstrating the use of fluorescently labelled anti‐CD9 nanobodies for the detection of EVs.

Methods : Anti‐CD9‐nanobodies fused with green fluorescent protein (GFP) were recombinantly expressed in E. coli and HPLC purified. Aliquots of 100 µL platelet‐derived EVs (protein conc. 1 µg/mL) stained with anti‐CD9‐GFP‐nanobody, anti‐CD41‐PC7‐antibody, and/or AnnexinV‐APC were analysed using a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA) and nanoparticle tracking analysis ZetaView TWIN NTA (Particle Metrix, Inning am Ammersee, Germany).

Results : Anti‐CD9‐GFP‐nanobodies were successfully expressed and purified. Flow cytometry confirmed CD9 expression on AnxV+ CD41+ platelet‐derived EVs, as detected using the anti‐CD9‐GFP‐nanobody vs. anti‐CD33‐GFP‐nanobody as control. Nanoparticle tracking analysis (NTA) of platelet‐derived EVs stained in parallel with anti‐CD9‐GFP‐nanobody and CellMask green (CMG) in particle‐free PBS yielded 20% CD9+ particles and 57% CMG+ particles.

Summary/Conclusion : We show that nanobodies are suitable for EV detection by flow cytometry and NTA. We will further optimise the flow cytometric and NTA protocols for detecting CD9+ EVs and compare anti‐CD9‐GFP‐nanobody to commercially available fluorescently labelled CD9‐antibodies. Furthermore, we will screen existing nanobody sequences for additional targets, such as more specific EV markers and test additional fluorescent proteins (e.g., mCherry).

Funding : This research was funded by the Technology Fund of Lower Austria (grant number WST3‐F‐5030664/032‐2022).

An aptamer approach to detect bacterial EVs

Maysaa Al‐Mohammedawi, Jacqueline Ross, Priscila Dauros‐Singorenko, Simon Swift

University of Auckland, Auckland, New Zealand

Introduction : The presence of bacterial EVs can indicate the presence of live bacteria. Various clinical and environmental scenarios exist where the detection of bacterial EVs may indicate the presence of problem organisms, for example, an infection after surgery, a relapsing infection, an organism in a water system, or a pathogen in a food production factory. Aptamers are short, specific nucleic acid sequences that form a 3D structure with specificity for specific molecules, similar to an antibody, but far simpler to make once the sequence for a particular molecule is determined. We identified an aptamer sequence described in the literature with an affinity for Escherichia coli vesicles and tested whether this would be able to detect E. coli EVs.

Methods : Bacterial EVs were isolated from cell‐free spent culture supernatants, concentrated using sequential tangential flow filtration and ultrafiltration (100 kDa cut off) and isolated using size exclusion chromatography. EV‐rich fractions were confirmed by protein assay, nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy. EVs were stained with Acoerela dye Aco‐600 (Excitation = 488 or 561 nm (blue or yellow laser), Emission = 586–635 nm) and an aptamer tagged with the Cy5 fluorophore. The binding to bacteria was determined by fluorescence in a microplate fluorimeter after collection and washing of stained bacteria by centrifugation. Staining and analysis of EVs from E. coli was determined using fluorescence NTA and Stimulated emission depletion (STED) microscopy.

Results : Aptamer binding to bacterial cells was demonstrated, but we were not able to demonstrate co‐localisation of Acc‐600/Aptamer stained EVs. Binding of the Cy5‐aptamer to Aco‐600 stained EVs was not observed.

Summary/Conclusion : Using aptamers to detect EVs is an attractive concept but the reality is dependent on selecting aptamers with a strong affinity for EVs. Fluorescence NTA and STED are good techniques to identify co‐localisation of an EV‐dye and an aptamer. Our experience is that if using published work to select aptamer sequences, make sure the EVs‐preparation used to select the aptamers is purified according to MISEV guidelines and not simply a crude particle preparation from the cell‐free supernatant.

Funding : This study was supported by the Maurice and Phyllis Paykel Trust.

Investigation of plasma half‐life and biodistribution of Cy5‐labelled extracellular vesicles generated via metabolic tagging technology and click chemistry

Presenter: Yingchun Xie

Sun Yat‐Sen University, Guangzhou, People's Republic of China

Introduction : Extracellular vesicles (EVs) have emerged as promising therapeutic agents due to their ability to carry parental cell characteristics and potential for targeted modification and drug loading. Various EV labelling methods are often limited by spontaneous dye aggregation or low sensitivity. Achieving reliable in vivo tracking without altering physiological distribution remains a critical challenge for translating EV research into clinical applications. This study pioneered the use of metabolic glycan labelling and click chemistry to generate Cy5‐labelled EVs, and for the first time, investigated the plasma half‐life and biodistribution of umbilical cord mesenchymal stem cell‐derived small EVs (UC‐MSC‐sEVs) using this novel labelling approach in mice.

Methods : UC‐MSC‐sEVs were isolated using anion exchange chromatography and characterized. The EVs were conjugated with Cy5 fluorophore through metabolic tagging technology and click chemistry. Azido membrane expression was verified by flow cytometry and fluorescence microscopy, while successful fluorophore conjugation was confirmed by nano‐flow cytometry. Following tail vein injection of 2 × 10 10 EVs, blood samples and organs were collected at predetermined time points (0 min–24 h). EV concentration in tissue homogenates was calculated based on Cy5 fluorescence intensity, and results were validated through ex vivo imaging.

Results : UC‐MSC‐sEV‐Cy5 maintained characteristic EV properties with no significant changes in particle size or membrane potential. The labelled EVs were efficiently internalized by BEAS‐2B epithelial cells. Following intravenous administration, sEVs showed a rapid clearance with over 50% elimination within 5 min. Biodistribution analysis revealed predominant accumulation in the liver and spleen, moderate distribution in the kidneys and lungs, and minimal presence in the brain, heart, and intestines. Ex vivo imaging results correlated well with tissue homogenate data.

Summary/Conclusion : We successfully generated Cy5‐labelled EVs without affecting their composition, size, or membrane potential based on metabolic glycoengineering and bio‐orthogonal click chemistry. And for the first time, investigated the plasma half‐life and biodistribution of umbilical cord mesenchymal stem cell‐derived small EVs (UC‐MSC‐sEVs) using this novel labelling approach in mice. UC‐MSC‐sEV‐Cy5 demonstrated rapid blood clearance with a half‐life of less than 5 min following intravenous administration.

Funding : This work was supported by grants from National Natural Science Foundation of China (No. 82271144).

A radiolabelling strategy of small extracellular vesicles for prolonged in vivo monitoring using PET imaging

Presenter: Lino Ferreira

University Coimbra, Coimbra, Portugal

Introduction : Both for diagnosis and for regenerative medicine, it is critical to develop imaging platforms that allow monitoring the biodistribution of sEVs, as currently, existing solutions have limited resolution and sensitivity. In our previous study, we developed a strategy to radiolabel EVs with 64CuCl 2 for PET/MRI imaging. Radiolabelled EVs could be detected in organs with low accumulation such as the brain (0.4%–0.5% ID g −1 ). Unfortunately, the preparation of 64CuCl 2 is expensive, complex and has a short half‐life (12.7 h) which makes it difficult for the longer monitoring. Here, we have investigated a new labelling strategy based on 89Zr that has a half‐life of 78.4 h, is safer to handle, cheaper to produce, and more stable in vivo. For this purpose, we have developed a new chelator for 89Zr4+ that could form a stable complex at room temperature and be used in PET imaging.

Methods : sEVs were isolated from different sources including human mesenchymal stromal cells, urine and mononuclear blood cells. The chelate of zirconia is new and has been synthesized by us. sEV biodistribution studies were performed by PET imaging in C57Bl6 mice.

Results : The new chelator was able to complex 89Zr4+ in one h. The EVs were radiolabelled in two steps, the first of which included conjugating the chelator with the EVs and the second of which involved radiolabelling with 89Zr4+. EV‐chelator‐Zr had a radio‐chemical yield of ∼60% and ∼ 80% stability in plasma for 7 days. Our modification did not affect the morphology, surface protein, internal RNA content and bioactivity of sEVs. Finally, we successfully imaged EVs at very low dose in healthy and myocardial infarction mice after injecting EV‐chelator‐89Zr intravenously. We further confirmed the EV accumulation to the heart using a ex‐vivo Langendorff rat heart perfusion model.

Summary/Conclusion : We have developed a new labelling strategy of sEVs that can monitored very small number of sEVs that accumulate in the heart.

Funding : The authors would like to acknowledge the funding obtained by the Portuguese funding research institution (FCT) through the projects 2022.07615.PTDC and 2022.02803.PTDC, by the PRR project HfPT‐ Health from Portugal (ref. 02/C05‐i01.01/2022.PC644937233‐00000047) and by the European project REBORN (Ref. 101091852).

Thermoresponsive polymer integrated metasurface sensor for capture and release of extracellular vesicles in real‐time

Beyza Nur Küçük 1,2 , Eylul Gulsen Yilmaz 1,2 , Yusuf Aslan 1,2 , Özgecan Erdem 1 , Bengi Özgün Öztürk 3 , Fatih Inci 1,2

1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey; 3 Hacettepe University, Department of Chemistry, Ankara, Turkey

Introduction : Extracellular vesicles (EVs) are key in cell communication, carrying biomolecules that reflect their origin cell. Pure isolation of EVs is essential but challenging with traditional methods, which risks non‐vesicular impurities and EV damage. This study introduces a smart thermoresponsive polymer on a metasurface sensor decorated with anti‐CD63 antibodies, enabling the sensitive capture and release of EVs from MCF‐7 breast cancer cells under surface plasmon resonance (SPR). By raising the local temperature above the polymer's lower critical temperature (LCST), EVs are isolated without contamination. The sensor's nanostructure offers real‐time, label‐free EV detection.

Methods : Poly(N‐isopropylacrylamide) (PNIPAM) was polymerized for its thermoresponsive and biocompatible properties. EVs from MCF‐7 cells were isolated by centrifugation, filtered, and introduced into a microfluidic device. Characterized using NTA and SEM. EVs were captured on PNIPAM‐modified metasurface at 25°C, then released at 37°C, with SPR signal tracking capture and release events.

Results : SEM results confirmed the microfluidic device's effectiveness with 50–150 nm particles that have expected spherical morphology. NTA analysis revealed exosomes sized at 137.7 ± 3.6 nm with a concentration of 1.01 × 10 1 ⁰ ± 5.23 × 10⁸ particles/mL. Following, 1 × 10 9 particles/mL were introduced to the microfluidic chip under SPR at 25°C and washed. Temperature increased to 37°C for release, and cooled to room temperature.A 1.07 ± 0.4 nm signal shift occurred after capture, slightly reduced by washing. After heating, a 0.71 ± 0.4 nm downward shift was observed, indicating approximately 34% EV release. Western blot analysis of released EVs was confirmed with Anti‐CD63 antibody.

Summary/Conclusion : Pure EV solution was collected in real‐time via SPR with 34% efficiency and confirmed with Western Blot and SEM. This study is paving the way for detailed EV studies, overcoming limitations of traditional isolation methods.

Funding : All authors acknowledge the support of Turkish Academy of Sciences (TÜBA) and The Scientific and Technological Research Council of Türkiye (TÜBİTAK) 2232 International Fellowship for Outstanding Researchers (Project No: 118C254).

Micromixer‐facilitated synthesis of extracellular vesicle‐imprinted nanoparticles for rapid detection

Özgecan Erdem 1 , Kadriye Ölmez 1,2 , Beyza Nur Küçük 1,2 , Eylul Gulsen Yilmaz 1,2 , Fatih Inci 1,2

1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey

Introduction : Molecularly imprinted polymers (MIPs) mimic antibodies or enzymes, incorporating binding sites for target analytes. Conventional MIP production is slow, typically relying on closed, batch systems. Microfluidics offers a modern approach to nanoparticle synthesis, enabling precise mixing and increased productivity. Extracellular vesicles (EVs), critical biomarkers for kidney disease diagnosis, provide more specific information than traditional markers like serum creatinine, which may lack sensitivity. This study introduces an innovative convergence‐divergence micromixer for synthesizing EV‐MIPs with high precision. Additionally, we developed a nanoplasmonic sensor using EV‐MIPs for real‐time detection of kidney‐related EVs, offering excellent specificity and binding affinity.

Methods : EVs were first isolated from HEK 293 cells using on‐chip ultrafiltration and then characterized via Nanoparticle Tracking Analysis (NTA) and Scanning Electron Microscopy (SEM) before being applied to EV‐MIP synthesis. Soft lithography‐fabricated microfluidic channels, shaped as ellipses (600 × 500 µm) merging into rectangular (100 × 300 µm) and smaller ellipsoidal (300 × 200 µm) channels, facilitated the flow. Mixing efficiency was optimized through COMSOL Multiphysics simulations at varied flow rates, resulting in successful nanoparticle synthesis. EV‐MIPs underwent analysis with NTA, Dynamic Light Scattering (DLS), SEM, and Fourier‐transform infrared spectroscopy (FTIR). A plasmonic metasurface was also modified with EV‐MIPs for EV detection.

Results : The size of the isolated EVs were measured around 180 nm with a concentration of 7 × 10⁹ particles/mL using NTA. SEM images confirmed their spherical shape and dimensions. COMSOL simulations at flow rates of 100, 120, and 150 µL/min identified 150 µL/min as optimal for producing nanoparticles with narrower size distribution. EV‐MIPs were measured around 84 nm, with a concentration 1 × 10⁹ particles/mL. EV detection by the microfluidic plasmonic sensor was indicated by wavelength shifts signalling EV‐MIP binding on the surface. Binding of EVs shifted the baseline signal to 1.34 nm, confirming EV attachment to EV‐MIPs on the sensor.

Summary/Conclusion : This study demonstrates the efficient use of a micromixer for continuous‐flow EV‐MIP synthesis and detection of kidney disease‐related EVs using a nanoplasmonic sensor. This approach shows promise for real‐time diagnostics in renal health applications.

Funding : Supported by TÜBİTAK (Project No: 118C254), Turkish Academy of Sciences (GEBİP), and Science Academy's (BAGEP) awards.

Fluorescent extracellular vesicles: a tool for imaging and investigating tumour cells communication

Lara Pierantoni, Teresa Lage, Lorena Diéguez, Carlos Honrado, Sara Abalde‐Cela

INL‐International Iberian Nanotechnology Laboratory, Braga, Portugal

Introduction : Liquid biopsies are emerging as non‐invasive methods for novel biomarkers discovery in cancer research. Extracellular vesicles (EVs) have gained significant interest due to their capacity to carry valuable cargo derived from active tumours. This can provide critical information about tumour phenotype and metastasis initiation. For this reason, it is important to find reliable methods to label and image EVs, not only for tracking during isolation protocols, but also to better understand cellular communication processes. Typical EVs dyes, like PKH67, self‐form dye micelles, thus interfering with EVs staining and making it imperative to find alternative methods.

Methods : Here we propose two different strategies for EVs fluorescent labelling. The first uses internalized cell‐permeant fluorescent dyes (e.g., calcein) added during cell culturing. The second uses cell transfection with plasmid DNA encoding a fluorescently tagged EV membrane marker (e.g., CD63). For both cases, cancer cell lines (Hec1A, SW480) are seeded, cultured with EVs‐depleted media for 72 h, and the conditioned media is collected. EVs are isolated by centrifugation (10 min 500 × g ; 10 min 2000 × g ; 20 min 10,000 × g at 4°C) and ultracentrifugation (90 min 100,000 × g at 4°C, wash with PBS, and repeat). The generated EVs are characterized by nanoparticle tracking analysis (NTA), confocal and total internal reflection fluorescence (TIRF) microscopy imaging.

Results : Preliminary results showed the successful isolation by ultracentrifugation of EVs from both labelling strategies. Confocal and TIRF imaging confirmed that EVs maintained the acquired fluorescence. To assess the fluorescence efficiency, and whether the two methods influence EVs production, further analyses are being performed, namely checking size and yield by NTA, protein presence by BCA and Western blot, and DNA quantification and characterization by Qubit assays and BioAnalyzer DNA integrity assessment.

Summary/Conclusion : With fluorescent EVs we will better visualize processes such as microfluidic‐based isolation, permitting immediate assessment of capture efficiency; and intercellular communication within complex organ‐on‐chip models, such as the interplay between cancer and stroma cells (e.g., cancer‐associated fibroblasts).

Funding : This study was supported by the Health from Portugal (C630926586‐00465198), through the NextGenerationEU Fund.

Extracellular vesicles detection through electrochemical devices: a new conservative approach to liquid biopsy

Presenter: Maria Serena Chiriacò

CNR NANOTEC, Rome, Italy

Introduction : Over the past decade, scientific evidence on extracellular vesicles (EVs) has grown exponentially, leading the scientific community to recognize their substantial importance in both medical and biological fields. The lipid bilayer of Extracellular vesicles (EVs) encapsulates bioactive components such as proteins, nucleic acids, lipids, and metabolites which can be correlated to EVs size and function.

Methods : Our approach includes the integration into LoC biosensing devices obtained by microfabrication techniques and electrochemical detection for EVs enrichment and characterization. For comparison, Large‐ and Small‐EVs were isolated by ultracentrifugation and characterized by high‐resolution flow cytometry, western‐blot and transmission electron microscopy.

Results : Functionalized microelectrodes were used for Differential Pulse Voltammetry measurements, and we were able to build a calibration plot considering different EVs membrane proteins. Multiple assays were performed including Tetraspanin antigens (CD9, CD63 and CD81), typical from small‐EVs and Flotilin and Annexin antigens, recognizing a larger category of EVs (including Large‐EVs). Moreover, Alix antigen, typical from cargo was detected only after EVs lysis, confirming that our system allows non‐destructive analysis of samples. An assessment of unspecific signal was also performed, related to culture medium, in order to establish if our system is able to detect EVs from non‐treated samples.

Summary/Conclusion : With this method we were able to demonstrate both the possibility to detect very low concentrations of EVs and the preservation of sample integrity. A key point of LoC technologies is the effort of removing highly impacting procedures, reducing time and cost, and preserving EV integrity.

Funding : This work was supported by PRIN 2022 Project—financed by the European Union—Next Genera‐tion EU‐PNRR M4.C2.1.1. RESOLVE—an innovative platfoRm based on field‐flow fractionation and sample on‐chip detection to unravel extracellular vesicles heterogeneity (grant number: 202233FTW8; CUP: B53D23013360006).

Single extracellular vesicle profiling: harnessing NanoFlow cytometry for quantitative surface marker analysis

Madeline Cramer, Stephen Lenzini, Elie Zakhem, Jon A. Rowley

RoosterBio, Inc., Frederick, Maryland, USA

Introduction : Heterogeneity of EV populations complicates their study, as traditional bulk analysis methods fail to capture individual differences in size, content, and surface markers. Single‐vesicle analysis offers a more precise approach, enabling detailed characterization of EV subpopulations. We developed protocols for calibrated NanoFlow cytometry to quantify single‐vesicle surface marker expression and applied them to purified MSC‐EVs from three sources.

Methods : We generated purified MSC‐EVs through RoosterBio established production and purification protocols. EV tetraspanin identity markers (CD81, CD63) and MSC identity marker (CD73) were selected for single‐vesicle analysis. For each antibody, optimization steps were performed to identify the optimal staining protocol. Beads with known equivalent reference fluorophore (ERF) values were obtained from NanoFCM. Data was acquired on the NanoFCM Flow NanoAnalyzer and single‐vesicle analysis methods were established to quantify the number of CD molecules per particle. The lower limit of detection was determined by background fluorescence. We then applied this analysis to purified EVs from bone marrow (BM), umbilical cord (UC), and adipose (AD) MSC‐EVs.

Results : Antibody titration was used to select a dilution of 800X for samples at 1E10 P/mL. A standard curve was generated from ERF beads to calibrate arbitrary fluorescence intensity to units of Alexa Fluor 488. The antibody‐specific limit of detection for positive expression was between 4 and 10 molecules per particle. Across all tissue sources, particles were 50%–70% CD81+, 40%–60% CD63+, and 15%–35% CD73+. Single‐vesicle analysis suggested CD81 and CD63 were enriched on AD‐EVs (40 CD81, 53 CD63 per particle) compared to UC‐EVs (33 CD81, 44 CD63 per particle) and BM‐EVs (30 CD81, 41 CD63 per particle). CD73 was preferentially expressed on larger EVs and showed ∼20 CD73 per particle for all tissue sources.

Summary/Conclusion : We successfully developed single‐vesicle quantitation of EV surface markers by calibrated NanoFlow cytometry. Application of this novel analysis not only showed retention of CD81, CD63, and CD73 in our highly purified EVs, but also discerned tissue‐ and marker‐dependent differences in EV properties. This high‐resolution characterization of EVs can advance our understanding of EV diversity, aid interpretation of EV bioactivity, and support development of methods to enrich specific EV subtypes.

An EV‐based approach to optimize the multiparametric analysis of extracellular vesicles through spectral flow cytometry: from instrument setup to fluorochrome selection

Daniela Boselli 1 , Francesca Clemente 1 , Simona Di Terlizzi 1 , Chiara Villa 1,4 , Giuseppe Alvise Ramirez 2,3 Norma Maugeri 2,4 , Angelo A. Manfredi 2,3,4 , Achille Anselmo 1,4

1 FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory, IRCCS Ospedale San Raffaele, Milan, Italy; 2 Division of Immunology, Transplantation and Infectious Diseases, IRCCS Ospedale San Raffaele, Milan, Italy; 3 Unit of Immunology, Rheumatology, Allergy and Rare Diseases, IRCCS Ospedale San Raffaele, Milan, Italy; 4 Università Vita‐Salute San Raffaele, Milan, Italy

Introduction : Extracellular vesicles (EVs) are nanoscale carriers released by cells, playing critical roles in intercellular communication and reflecting the physiological state of their origin. They hold promise as biomarkers for complex diseases. However, challenges in their characterization and quantification limit their clinical application, underscoring the need for advanced methodologies to harness their value as circulating biomarkers.

Methods : Flow cytometry is widely used for EV analysis, but it faces challenges due to the small size of EVs and their low antigen abundance. Therefore, the development of advanced tools to optimize instrument settings thus improving EV detection, is of paramount interest to the scientific community. Additionally, the limited knowledge of fluorochrome properties that support EV immunophenotyping limits the development of robust multiparametric analyses aimed at identifying rare or yet‐undescribed EV subsets.

Results : In this study, we describe a comprehensive workflow for high‐dimensional immunophenotyping and quantification of EVs using spectral flow cytometry. The first step was the optimization of light scatter parameters and fluorescence settings thereby improving instrument sensitivity. To address this topic, we used authentic EVs obtained from either in vitro stimulated neutrophils or PBMCs, rather than the more commonly used synthetic microparticles thereby potentially minimizing the detrimental effects caused by differences in the refractive index between the latter and EVs. In the second step, we assessed fluorochrome performance for EV immunophenotyping by testing 50 different fluorochromes conjugated to anti‐human CD4 mAb on EVs derived from in vitro‐stimulated PBMCs. Notably, the correlation between the absolute count of CD4+ EVs and fluorochrome brightness allowed us to rank the fluorochromes and select the most effective ones.

Summary/Conclusion : Overall, our workflow offers an innovative approach to instrument setup and panel design for high‐dimensional EV analysis, thereby potentially enabling the simultaneous use of multiple fluorochromes. This study advances the field of flow cytometry EV analysis by providing a reliable and optimized method for EV immunophenotyping, facilitating their use in translational research.

Funding : This work was supported by the European Union—Next Generation EU—NRRP M6C2 ‐ Investment 2.1 Enhancement and strengthening of biomedical research in the NHS. Project N° PNRR‐MR1‐2022‐12376638, PI Prof. Angelo Manfredi.

Separation of blood‐derived extracellular vesicles using droplet‐based sorting

Maria Gracia Garcia Mendoza 1 , Monica DeLay 1 , John Nolan 2 and Haley Pugsley 1

1 Cytek Biosciences, Inc., Seattle, Washington, USA; 2 Cellarcus Biosciences, Inc., La Jolla, California, USA

Introduction : Extracellular vesicles (EVs) are cell‐derived, membrane‐bound particles less than a micron in size and are valuable biomarkers and therapeutic agents. Due to their size and heterogeneity, EVs are difficult to characterize. Single Vesicle Flow Cytometry (vFC) is a method to detect and analyse EVs. There is interest in droplet‐based sorting to isolate EVs for further characterization. We present a proof‐of‐concept assay that purifies specific EV populations using a droplet‐based sorter.

Methods : A mixture of platelet‐ (PLT‐EVs) and red blood cell‐derived (RBC‐EVs) EVs were analysed using the vFC workflow, following MIFlowCyt‐EV guidelines. A Cytek Aurora CS system with Enhanced Small Particle (ESP) Detection Option was set up with a 70 µm nozzle. The sheath pressure was set at 30 psi to slow the sheath velocity and improve resolution. The threshold was set to membrane dye‐positive EVs. CD41‐positive PLT‐EVs and CD235ab‐positive RBC‐EVs were sorted using multiway sort mode at an average sort rate of 500 events/second.

Results : The results of three independent experiments were similar with an average efficiency and purity >98%. The average time to sort 1 million EVs was 25 min.

Summary/Conclusion : This feasibility study demonstrates the purification of two EV populations using cell‐of‐origin surface markers and a high‐sensitivity sorter. EV sorting may help address challenges in EV analysis and advance therapy and diagnostic development. Further experimentation will determine desired downstream workflows.

Extracellular vesicles detection via surface enhanced IR spectroscopy using plasmonic gold nanoparticles

Presenter: Judith Mihály

HUN‐REN Research Centre for Natural Sciences, Budapest, Hungary

Introduction : Infrared (IR) spectroscopy, especially attenuated total reflection (ATR), is a rapidly emerging label‐free tool for molecular profiling of EVs. The relatively low number of vesicles in biological fluids and the complex content of the EVs milieu (protein aggregates, lipoproteins, buffer molecules) might result in poor signal‐to‐noise ratio in the IR analysis. Exploiting the increment of the electromagnetic field at the surface of plasmonic nanomaterials, surface‐enhanced infrared spectroscopy (SEIRS) might provide an amplification of characteristic IR signals of EV samples.

Methods : Negatively charged citrate‐capped and positively charged cysteamine‐capped gold nanoparticles with around 10 nm diameter were synthesized and tested with blood‐derived EVs. Since the interaction between EVs and AuNPs is a key factor in obtaining SEIRS phenomenon, changes in plasmonic properties and possible aggregation of AuNPs were investigated by UV‐Vis spectroscopy, dynamic light scattering (DLS) and freeze‐fracture transmission electron microscopy (FF‐TEM). SEIRS measurements were performed at different EVs:AuNPs ratio using ATR‐IR technique.

Results : Both negatively and positively charged AuNPs contributed to an enhancement of the EV's IR signature. For the most common citrate‐stabilized AuNPs, a concentration dependent interaction was established between AuNPs and the lipid bilayers. This strongly affected both the plasmonic behaviour of AuNPs and the bilayers lipid organization. In case of cysteamine‐capped AuNPs, due to the electrostatic interaction between the positively charged AuNPs and negatively charged EVs, a higher enhancement was achieved concerning both lipid‐ and protein‐related bands.

Summary/Conclusion : Our preliminary SEIRS results, using easy‐to‐prepare gold nanoobjects, are promising for IR spectroscopy of EVs. Comparing the citrate‐capped and cysteamine‐capped AuNPs, the positively charged cysteamine‐capped AuNPs proved to be more advantageous for SEIRS measurements: i) higher enhancement was achieved for both lipid‐ and protein‐related IR bands; ii) unlike citrate‐capped AuNPs, there is no spectral interference with amide I and amide II protein bands. Fine‐tuning the surface properties of gold nanoparticles can further lead to selective enhancement of EVs' infrared signals. These results contribute primarily to the characterization of EVs but also pioneer the field of surface‐enhanced IR spectroscopy.

Funding : The research was funded by the National Research, Development and Innovation Office (NKFIH) under grants K‐131594, K‐131657, ADVANCED‐150077, STARTING‐150629 and projects 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021 and TKP2021‐EGA‐31.

Subtopic EV Enrichment 2

Comparison of different methods for the concentration of urine and isolation of urinary extracellular vesicles

Presenter: Misba Khan

University of Turku, Turku, Finland

Introduction : Urinary extracellular vesicles (EVs) are promising source of biomarkers for non‐invasive disease detection. However, their isolation is challenged by the high abundance of non‐EV proteins like uromodulin/Tamm‐Horsfall protein (THP), which can compromise the purity and recovery of EVs. Additionally, the low concentration and heterogeneity of urinary EVs complicate standardization across different isolation methods. Optimizing pre‐isolation concentration methods and THP removal strategies is crucial to enhance EV yield, purity, and reproducibility for reliable biomarker discovery.

Methods : Healthy urine samples were pooled and concentrated using 4 different Vivaspin Turbo centrifugal filters‐ with molecular weight cutoffs of 10, 30, 50, and 100 kDa. Tetraspanin and THP expression levels were analysed using a time‐resolved fluorescence‐based sandwich immunoassays (TRFIA), in which biotinylated tetraspanins were immobilized on streptavidin coated microtiter wells. The captured proteins were detected using europium doped nanoparticles conjugated with tetraspanins or THP‐specific antibody. THP was removed using NaCl treatment and filtration, both before and after the isolation of EVs. EVs were then isolated from the 10 kDa filtered urine fractions using size exclusion chromatography (SEC).

Results : Among four filtration methods, urine fraction concentrated using10 and 30 kDa filters resulted in higher uEVs yields, as confirmed by tetraspanin and THP expression measured by TRFIA. Notably, the 10 kDa filter led to a reduction in THP content in the concentrated urine fractions. Comparison of THP removal strategies indicated that salt treatment was more effective than filtration. These findings were further validated in the SEC‐isolated EV fractions.

Summary/Conclusion : This study introduces a comparison of four different filtration methods for concentrating urine samples and two different methods of removing THP from the urine and SEC isolated EV fractions. The results highlight the advantages of using 10 kDa filtration in combination with salt treatment for improved EV yield and purity. However, to get better yield‐driven protocol, other EV isolation methods will be included for comparison in our future experiments.

Funding : This research is funded by a grant received from the Southwest Finland Cancer Society.

PEG‐based isolation method for high‐purity small extracellular vesicles (sEVs) from cells and blood fractions

Leticia A. Fernandes 1 , Pedro E. S. Lima 1 , Milton Y. Nishiyama‐Jr 2 , Umut A. Gurkan 3 , Ana Claudia O. Carreira 1,4

1 University of São Paulo, São Paulo, Brazil; 2 Butantan Institute, São Paulo, Brazil; 3 Case Western Reverse University, Cleveland, Ohio, USA; 4 Federal University of ABC, Santo André, Brazil Milton Y. Nishiyama‐Jr and Umut A. Gurkan are Co‐senior authors .

Introduction : Extracellular vesicles (EVs) have revolutionized the biological field, prompting the development of diverse isolation methods based on size, density, immune assays, polymer precipitation, and microfluidics. However, a single method that achieves high yield, quality, cost‐effectiveness, easy hands‐on, and clinical applicability remains elusive. We developed an all‐in‐one PEG‐based methodology for EV isolation from multiple sources to address this gap.

Methods : EVs were isolated using the Polyethylene Glycol (PEG) mixture protocol. Sources standardized included (a) human bone marrow mesenchymal stem cells (hBM‐MSC); (b) human umbilical vein endothelial cells (HUVEC); (c) breast cancer cells (MDA.MB.231); and blood components (Ethical committee (05‐14‐07C) as (d) poor‐platelet plasma (PPP), (e) red blood cells (RBC), and (f) white blood cells (WBC). Blood fractions were prepared from 2 mL of fresh whole blood, concentrated using Amicon Ultra‐15 Centrifugal Filter 10 kDa cut‐off, and incubated with our PEG mixture. Conditioned media from cell lines were processed without foetal bovine serum (FBS) or exosome‐depleted serum. EV characterization included Nanoparticle Tracking (NTA), Cryo‐electron Microscopy, and label‐free quantitative liquid chromatography‐mass spectrometry (LC‐MS). The ultracentrifugation (UC) method was the comparative standard protocol.

Results : Our PEG method consistently preserved EV integrity, maintaining their intact lipid bilayer, and yielded enhanced enrichment compared to UC. HUVEC and hMB‐MSC EVs yields were nearly five times higher using our method. Interestingly, our methodology yielded 15 times more than the UC protocol for all blood components EV isolation. To challenge the effectiveness of our method, we performed the isolation using only 50 uL of plasma, exceeding a yield of over 2 × 10 11 particles per mL. Cryo‐EM confirmed better structural preservation across all blood samples using PEG. LC‐MS demonstrated, in HUVEC, that PEG method enriched 1763 proteins demonstrating that PEG maintains protein cargo integrity.

Summary/Conclusion : Our PEG precipitation method represents a significant advancement with outstanding performance in terms of yield ensuring scalability and reproducibility across diverse biological samples. Its user‐friendly principle and cost‐effectiveness make it a robust candidate for widespread application in EV research and diagnostics.

Funding : Coordination for the Improvement of Higher Education Personnel (CAPES)—Grant no. 88887.666887/2022‐00 and 88887.916425/2023‐00 (Fernandes, L.A. fellowship).

Isolation of extracellular vesicles using tannic acid‐functionalized magnetic beads

Presenter: Alexey Yashchenok,

Skolkovo Institute of Sciecne and Technology, Moscow, Russia

Introduction : The isolation of EVs from complex biological fluids with high yield and sufficient purity remains a challenge. In recent years a number of approaches have been introduced to increase the efficiency of EVs enrichment preserving their structural integrity and biological activity. Magnetic separation has received particular attention for EVs enrichment due to its availability, simplicity, high efficiency and low‐cost. In this study we developed magnetically responsive beads, functionalized with natural polyphenol tannic acid (TA) to isolate EVs derived from two cancer cell lines [N. A. Grishaev et al. 2024, Journal of Materials Chemistry B, 12, 6678]. The efficiency of the approach is evaluated for isolating of EVs from cell culture media in comparison with the size exclusion chromatography (SEC) method.

Methods : EVs were isolated from cell culture media of MCF7 and HCT116 cells by using ultrafiltration combined with size‐exclusion chromatography (UF‐SEC). To modify the surface of magnetic beads, TA was deposited using layer‐by‐layer (LbL) method. EVs capture efficiency was assessed using nanoparticle tracking analysis, micro BCA protein assay and electron microscopy, while nuclear magnetic resonance and matrix‐assisted laser desorption/ionization mass spectrometry were used to reveal the possible mechanism of interaction between EVs and TA.

Results : It shown that the binding of EVs to the surface of magnetic beads, which reaches 60 %, occurs due to the interaction of tannic acid and EVs membrane. TA binds to phospholipids through hydrogen bonds and by forming a complex between the benzene ring of gallic acid and the quaternary amine of the choline residue. Moreover, membrane‐embedded proteins can also form a complex with TA. EVs present in the supernatant of the human colon cancer cell line HCT116 were captured by the magnetic beads with recovery yield comparable with SEC method.

Summary/Conclusion : This work demonstrates a simple, rapid and novel strategy for isolating EVs with tannic acid without complex instruments. This approach would be of useful for application in basic research requiring the isolation of EVs.

Funding : This work was supported by the Russian Science Foundation (RSF) grant no. 23‐74‐00022.

Isolation of sEVs on benchtop centrifuge

Victoria O. Shender 1 , Polina V. Shnaider 1 , Arseniy Lashkin 1 , Alexandra Shirikova Zlenko 3 , Olga I. Aleshikova 2 , Elena Khomyakova 3

1 Lopukhin Federal Research and Clinical Center of Physical‐Chemical Medicine of FMBA, Moscow, Russian Federation; 2 National Medical Scientific Centre of Obstetrics, Gynaecology and Perinatal Medicine named after V.I. Kulakov, Moscow, Russian Federation; Exosome Analytics, Paris, France

Introduction : EVs, being very stable and abundant in body fluids, hold great promise in diagnostics. Clinical applications require simple, efficient and cost‐effective approaches for EV isolation. Ultracentrifugation, the gold standard for EV isolation with close to 100% yield under properly selected centrifugation conditions, is not suitable for clinical applications because it is labour‐intensive, requires expensive equipment and does not allow for simultaneous isolation of EVs from large numbers of clinical samples. Ultrafiltration is not suitable for EV isolation from cell cultures or body fluids with high protein/lipoprotein content, such as cell cultures supplied with FBS, serum, plasma, ascites and in most cases from urine of patients with advanced cancer. Polymer‐based kits and SEC columns are expensive and both have important drawbacks. Express kits require an additional purification the viscous polymer, and SEC columns are unable to purify EVs from most lipoproteins. Thus, new approaches to EV isolation are needed.

Methods : Based on the theoretical approach we have developed in Livshits et al., doi: 10.1038/srep21447, we adapted the sEV common ultracentrifugation protocol to a conventional benchtop centrifuge. According to our calculations, sEV isolation takes 3–4.5 h depending on the RCF (20,000 × g –30,000 × g ) and the size of the tube. Experimental evidence for the efficiency of isolation at 25,000 × g on a benchtop centrifuge was demonstrated using SKOV‐3 cell culture, serum of healthy people, and ascites and urine from ovarian cancer patients. EV size distribution was analysed by NTA. The expression of CD9, CD81, and CD117 membrane proteins on sEVs isolated by 25,000 × g centrifugation on a benchtop centrifuge and conventional isolation methods were analysed by bead‐based ELISA and bead‐based FACS.

Results : We compared the sEV isolation yield by centrifugation at 25,000 × g on a benchtop centrifuge with that of 100,000 × g ultracentrifugation and 100 kDa ultrafiltration. Our experimental data proved that sEV isolation on benchtop centrifuge has a yield similar to or even better than 100,000 × g ultracentrifugation and 100 kDa ultrafiltration.

Summary/Conclusion : sEVs isolation on benchtop centrifuge is high yield and cost‐effective method that can be used in research and clinics.

Optimization of extracellular vesicle isolation from ovarian tumour tissues

Élodie Barthout 1 , Björg Kristjansdottir 2 , Cecilia Lässer 3 , Emma Symonds 1 , Roger Olofsson Bagge 1,4 , Karin Sundfeldt 2 , Jan Lötvall 3 , Rossella Crescitelli 1

1 Sahlgrenska Center for Cancer Research, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 2 Department of Obstetrics and Gynecology, Sahlgrenska Center for Cancer Research, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 3 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 4 Department of Surgery, Sahlgrenska University Hospital, Region Västra Götaland, Gothenburg, Sweden

Introduction : Ovarian cancer is the most lethal gynaecological malignancy. For 70% of patients, diagnosis occurs when the tumour cells have already invaded the peritoneum, reducing the 5‐year survival rate to 30%. Disease‐specific biomarkers are lacking but extracellular vesicles (EVs) have shown promise as a valuable source for identifying such biomarkers. Based on an EVs isolation protocol previously optimized for melanoma, this study aimed to optimize an EVs isolation protocol for human ovarian tumour tissues.

Methods : Four samples were collected from eight patients (epithelial derived tumours): primary tumour, non‐tumour peritoneal tissue, peritoneal metastases and omental metastases. EVs were isolated by enzymatic treatment followed by serial centrifugation and iodixanol density cushion. Transmission electron microscopy (TEM) visualized an evident contamination with collagen fibres. To remove these fibres, the EVs were then either filtered (0.22 µm) or treated with collagenase. Tissue‐derived EVs were characterized by TEM, nanoparticle tracking analysis (NTA) and protein measurement. To evaluate the sample purity, the ratio particle/protein was calculated.

Results : Without filtration, tissue‐derived EVs revealed a contamination with collagen fibres by TEM. The level of contamination differed between patients and tumour sites. The contaminated samples were the metastases (5/8 omentum, 7/8 peritoneum), and 3/6 non‐tumour peritoneal tissues, whereas nothing was visible in the primary tumours (0/7). Interestingly, two bands were visualized for two peritoneal metastases samples after density cushion centrifugation, and TEM analysis revealed that most of the fibres ended up in the lower band with higher density. Treatment with collagenase did not eliminate the fibres, while filtration considerably reduced their quantity. A few small fibres were still observable after filtration, and the presence of collagen was also confirmed by western blot. NTA analysis demonstrated that filtration did not reduce particle number, but reduced EVs size, decreased EVs protein levels and increased EVs purity.

Summary/Conclusion : Tissue‐derived EVs isolated from both non‐tumour peritoneum and metastases located to omentum and peritoneum are contaminated with collagen fibres, not observed when isolating EVs from other tissue sites. Therefore, an optimized isolation protocol is needed for these tissues, and one possibility is a 0.22 µm pore size filtration step, which reduces but does not entirely eliminate the collagen contamination.

Micro‐TFF: a promising approach for the isolation of extracellular vesicles

Presenter: Elena Hoffmann

Fraunhofer IMM, Mainz, Germany

Introduction : In recent years, extracellular vesicles (EVs) have attracted considerable attention within medical research due to their critical role in intercellular communication and their potential as diagnostic and therapeutic avenues. EVs are released by various cell types, and they are known to carry a wide range of bioactive molecules, including proteins, lipids, and nucleic acids. These components enable EVs to convey information between cells, thereby influencing numerous physiological and pathological processes. Despite their biological significance, the isolation of EVs remains a considerable challenge. The most widely published method for EV isolation is ultracentrifugation, which, while effective, presents several drawbacks that have also been documented in the MISEV guidelines. This technique requires specialized and expensive equipment, making it less accessible to many laboratories. Additionally, ultracentrifugation is a time‐consuming process that involves multiple steps, leading to potential losses of relevant EVs and their associated cargo.

Methods : To address these limitations, the present study focuses on the isolation of EVs using a micro‐tangential flow filtration (Micro‐TFF) system, which has been specifically designed by us for this purpose. The Micro‐TFF technique offers a more efficient and versatile alternative to ultracentrifugation. It allows for the customization of the filtration process according to the specific cell type under investigation by utilizing various filters with differing membrane permeabilities. This adaptability facilitates the effec‐tive isolation of EVs of different types and sizes. Though adjusting the filtration rate, it is possible to isolate the EVs under gentle conditions. Additionally, this method saves a lot of time and space.

Results : Preliminary results demonstrate that the Micro‐TFF method has successfully isolated EVs from a A549 cell line, highlighting its potential as a reliable and scalable approach for EV isolation in both research and clinical settings.

Summary/Conclusion : By advancing the methodologies for EV isolation, this study aims to contribute to a more gentle and faster way to isolate EVs for improved ac‐cessibility. This enables an acceleration of scientific EVs research, particularly in their application in the medical field, as therapeutics or bi‐omarkers.

Development of international standards at ISO/TC 276 (Biotechnology) for extracellular vesicles

Presenter: Ikuo Kawauchi

FUJIFILM Holdings Corporation, Tokyo, Japan

Introduction : Standards are documented guideline established by consensus aiming ‘achievement of the optimum degree of order in a given context.’ Unlike regulations, it is not binding, but its use can make activities more efficient. International standards for cells are mainly developed by ISO/TC 276 (biotechnology) from the perspectives of ‘analytical methods,’ ‘manufacturing,’ and ‘supporting industries,’ and their use is also progressing.

Methods : Regarding extracellular vesicle (EV), the manufacturing process up to EV production is same as cell culture, so the standards for cells can be used. On the other hand, there are needs for standardization of the purification process after EV production and the characterization of EV, as these processes are not yet technically established. At ISEV 2024, the development plan for international standards specific to EV was introduced.

Results : After ISEV 2024, ISO/TC 276 initiated a vote for ‘new work item proposal’ regarding EV purification and the project was approved in September 2024. This standard, currently as ISO/WD 25347, defines the followings, based on the intended purification purpose : (1) clarification of the components to be extracted, removed, or allowed to be mixed in, and those that shall not be mixed in, in the cell culture fluid; (2) determination of the purification method that is considered feasible; (3) validation of the selected method; and (4) documentation. It also recommended that providers of purification equipment provide the necessary information to support the determination of the purification method. With regard to characterization, the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) has published ICH‐Q6 series. However, ISO 23033 (General requirements and considerations for the testing and characterization of cellular therapy products) has been published for cells, because cell is outside of the scope of ICH‐Q6. It is considered that development of standard for “characterization of EVs” in a similar way is also necessary. The work has also begun for development of EV characterization standard.

Summary/Conclusion : The development plan for international standards specific to EV, introduced in ISEV2024, is proceeding and purification standard was accepted as new work item.

Nano flow cytometry and sedimentation velocity analytical ultracentrifugation evaluation of cellular EV subpopulations isolated using rate zonal density gradient ultracentrifugation

Presenter: Carley D. Ross

Beckman Coulter Inc., Brea, California, USA

Introduction : Biological EVs occur in low concentrations as heterogeneous populations making these biological particles challenging to study and analyse. To overcome this challenge, ultracentrifugation was used to concentrate cell culture EVs from green fluorescent protein (GFP) expressing Jurkat and Chinese Hamster Ovary (CHO) cells. The concentrated EVs were subjected to rate zonal density gradient ultracentrifugation (DGUC) and fractions collected allowing further characterization and analysis using nano flow cytometry and sedimentation velocity analytical ultracentrifugation (SV‐AUC).

Methods : Extracellular vesicles derived from CHO and Jurkat cells expressing CD81‐GFP were purified and concentrated using two cycles of ultracentrifugal pelleting and resuspension. EVs from both purification cycles were characterized by nano flow cytometry and sedimentation velocity analytical ultracentrifugation (SV‐AUC). To separate EV subpopulations, rate zonal DGUC was used and resulting bands were fractionated and dialyzed. These fractions were analysed by both SV‐AUC and nano flow cytometry.

Results : Distinct populations of EVs were observed by nano flow cytometry analysis on scatter and fluorescent channels. The AUC results suggest that the first purification cycle resulted in a mixture of small and potentially low‐density species, with the majority sedimenting below 20 Svedberg units (S). The second purification cycle resulted in a wider distribution of larger and potentially higher‐density species, with the majority sedimenting below 100 S. Thus, the second cycle of pelleting/resuspension appears to have enriched the proportion of larger and denser species. Rate zonal DGUC separated the heterogeneous pellet populations into distinct subpopulations.

Summary/Conclusion : The rate zonal ultracentrifugation step reduced EV purification time from overnight to 90 min and separated unique subpopulations. These subpopulations were further characterized by nano flow cytometry and SV‐AUC.

Funding : This study was supported by the Beckman Coulter Life Sciences.

Lectin‐based enrichment of extracellular vesicles derived from rat renal proximal tubular cells

Haruka Sei, Fumie Nakashima, Takahiro Shibata

Nagoya University, Nagoya, Japan

Introduction : Urinary extracellular vesicles (uEVs) are secreted into urine from a variety of cells in the kidneys and urinary tract and their contents reflect changes in the kidneys. Therefore, uEVs have been thought to be a potential source of biomarkers for kidney diseases. However, it is difficult to predict the original cells of uEVs because the original segment‐dependent sorting methods have not yet been developed well. In this presentation, since the surface sugar chain patterns differ among the nephron segments, we attempted to enrich nephron segment‐specific EVs from uEVs using biotinylated lectins.

Methods : EVs were isolated by ultracentrifugation from rat urine. The lectin pull‐down assay of uEVs was performed to enrich nephron segment‐specific EVs. The flow‐through and pull‐down fractions were subjected to immunoblotting with antibodies to nephron segment‐specific proteins. Histochemical staining of the kidney sections was also performed using biotinylated lectins and nephron segment‐specific proteins.

Results : The lectin pull‐down assay revealed that Lens culinaris lectin (LCA) interacted with uEVs derived from proximal tubular cells (PT‐uEVs) but not with uEVs derived from collecting duct cells (CD‐uEVs). Histochemical staining of kidney sections showed that LCA‐positive cells were colocalized with the proximal tubular marker‐positive cells in the cortex, but not with the collecting duct marker‐positive cells.

Summary/Conclusion : Biotinylated LCA was a suitable lectin to enrich PT‐uEVs from rat uEVs. This method will be valuable in biomarker research involving rat uEVs.

Construction of a new method of PD‐L1/IL‐8 combined in situ detection of single tumour‐derived extracellular vesicles and its application in predicting the efficacy of immune checkpoint inhibitors

Presenter: Yue Qiao

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Immunotherapy, especially immune checkpoint inhibitor (ICI)‐based immunotherapy, has brought new hope for cancer treatment. However, many patients show limited response, so there is a need for effective immunotherapy response prediction methods. Tumour‐derived extracellular vesicles (TEVs), one of the biomarkers of liquid biopsy, carry PD‐L1 and IL‐8 that are associated with immunotherapy efficacy in the immunogenicity and tumour immune microenvironment, respectively. In order to improve the accuracy of TEV detection, nano‐flow analyser is often used to detect single TEV, but the clinical penetration rate of the instrument is insufficient. Therefore, this study aims to use flow cytometer to detect single TEV PD‐L1 and IL‐8 at the same time, so as to predict the efficacy of immunotherapy.

Methods : We captured GPC‐1/CD63 double‐positive TEVs using proximity ligation assay (PLA) and constructed engineered liposomes encapsulating RCA and CRISPR‐Cas13a reaction systems. Then we detected PD‐L1 on the TEV membrane surface and IL‐8 protein inside through membrane fusion. Finally, this method was applied to detect plasma from 33 lung cancer patients undergoing immunotherapy, then we built machine learning models to predict the efficacy of immunotherapy.

Results : We successfully captured GPC‐1/CD63 double‐positive TEVs, and the capture efficiency reached approximate 83.87%, with each magnetic bead providing an average of 1425 EVs captured. Besides, the prediction performance of PD‐L1 or IL‐8 alone was less powerful than the combination of both markers before and after immunotherapy, yielding a value of the area under the curve (AUC) of 0.916.

Summary/Conclusion : In this study, the simultaneous in situ detection technology of PD‐L1/IL‐8 for a single TEV was successfully constructed and applied to detect patient plasma. Additionally, we developed a prospective cohort study and built an efficient immunotherapy prediction method.

Scalable continuous flow zonal ultracentrifugation for purification of therapeutic extracellular vesicles

Presenter: Viktoria Mayer

acib GmbH, Graz, Austria

Introduction : Density gradient ultracentrifugation (DGUC) is the gold standard method for purification of extracellular vesicles (EVs). DGUC allows purification of EVs with high resolution and purity, allowing the separation of different EV populations. Sucrose or iodixanol gradients are used and EV‐enriched bands found in densities of 1.15–1.19 g/mL. DGUC is commonly performed in lab‐scale ultracentrifuges using tubes, manually prepared gradients and manual collection of the sample. This leads to a widespread assumption that DGUC is difficult to scale‐up, time consuming and user dependent. However, DGUC can be performed in scalable continuous flow ultracentrifuges with rotor sizes up to 8400 mL. These can be used in continuous flow in which sample is continuously loaded at full speed. In fact, it is estimated that the majority of the worldwide flu vaccine production relies on this type of systems.

Methods : In this study, we evaluated the performance of continuous flow zonal ultracentrifugation using a sucrose density gradient for the enrichment of HEK293‐EVs after clarification by centrifugation or filtration. The developed DGUC method was compared to traditional EV purification methods as tangential flow filtration (TFF). Particle size and concentration of EVs was assessed by nanoparticle tracking analysis. Purity was determined by western blot for calnexin and EV biogenesis markers such as TSG101, Alix & Syntenin. EV surface marker profile was assessed by bead‐based flow cytometry. Moreover, the biological activity of separated EVs populations was investigated.

Results : Continuous flow zonal ultracentrifugation using a sucrose density gradient allowed the recovery and purification of different EV populations. One litre of clarified cell culture supernatant was processed per run, and the method has the potential to be easily scaled up to several hundred litres as the technology allows for linear scalability.

Summary/Conclusion : Our work shows the potential of continuous flow zonal ultracentrifugation for purification of therapeutic EVs with high purity and resolution in a scalable manner. Moreover, the developed process is serotype‐independent allowing for a platform process to advance the transition of EV‐based therapies to late‐stage trials and commercialization. Finally, the method can easily be combined with filtration‐ and chromatography‐based methods for hybrid processes.

Funding : This work was supported by Solventum Corporation.

Facile synthesis of tuneable metal‐organic frameworks for extracellular vesicle enrichment

Presenter: Cong M. Nguyen

Griffith University, Brisbane, Queensland, Australia

Introduction : Zinc‐based metal‐organic frameworks (ZnMOF) have gained significant interest for their tunable properties and potential applications in biology. In this study, we synthesized ZnMOFs using adenine as the organic linker and explored their potential for aptamer‐based targeting and extracellular vesicle (EVs) enrichment.

Methods : The synthesized ZnMOF was characterized using UV‐Vis spectroscopy, dynamic light scattering, and zeta potential measurements. Post‐synthesis treatments, such as polystyrene sulfonate and sonication, were employed to modulate the particles' physicochemical properties. Functionalization of ZnMOF with an aptamer was assessed through fluorescence microscopy. Nanoparticle tracking analysis was used to evaluate the EV capture efficiency. Downstream analysis of captured EVs included western blotting and loop‐mediated isothermal amplification.

Results : Among the tested nucleobases, Adenine was the only nucleobase spontaneously incorporated into ZnMOF, with Zn:Adenine ratio of 2:1. The particle size and zeta potential of ZnMOF could be modulated using polystyrene sulfonate treatment and sonication. The fluorescently‐tagged VCAM‐1 aptamer with poly adenine‐tailing was successfully incorporated into ZnMOF supramolecular structure, amplifying signal in our immunofluorescence assay. CD63‐aptamer incorporated ZnMOF efficiently cleared EVs from the input sample. The utility of reverse‐complement oligo could release EVs from ZnMOF particles. The rescued extracellular vesicles were confirmed to express endosomal markers (CD63 and CD9) and negative for markers of other sub‐cellular compartments. RNA samples from hypoxic SVEC endothelial EVs isolated with CD63‐aptamer@ZnMOF were significantly amplified, compared to the normoxic SVEC EVs.

Summary/Conclusion : In conclusion, we successfully synthesized ZnMOF using adenine as a nucleobase, resulting in tunable particles that exhibit promising properties for biological applications. Our results demonstrated that post‐synthesis treatments, including polystyrene sulfonate (PSS) and sonication, can be employed to modulate the size and surface charge of the ZnMOF particles, providing versatility for various applications. The incorporation of aptamers such as VCAM‐1 and CD63 aptamers into ZnMOF particles allowed for the development of bio‐functionalized materials with enhanced capabilities in immunofluorescence and EVs isolation.

Extracellular vesicle manufacture via GEMEXO: a fully automated large‐scale isolation platform for clinical translational applications

Presenter: Xing‐Chi Liu

Wuhan University, Wuhan, People's Republic of China

Introduction : Extracellular vesicles (EVs) have emerged as promising natural therapeutic agents and drug carriers, showing significant potential in the treatment of various diseases. However, the clinical application of EVs is currently hindered by existing isolation methods, which are often labour‐intensive and not suitable for large‐scale production. This limitation poses challenges in meeting the quality requirements necessary for effective clinical translation.

Methods : To address these challenges, we developed an automated large‐scale isolation platform named GEMEXO. This innovative system integrates continuous flow centrifugation and tangential flow ultrafiltration technologies, all regulated by a central control system. The GEMEXO platform enables the efficient isolation of high‐quality EVs from large volumes of cell culture supernatant, ensuring a seamless process for impurity removal and EV enrichment.

Results : Utilizing the GEMEXO platform, we successfully isolated a substantial quantity of EVs derived from umbilical cord stem cells. The isolated EVs were systematically characterized for their specific markers, biological functions, and therapeutic effects. Our findings demonstrate that the GEMEXO platform significantly enhances the quality and consistency of EVs, making them sterile, mycoplasma‐free, and low in endotoxin levels.

Summary/Conclusion : The development of the GEMEXO platform represents a significant advancement in the clinical translation of EVs as therapeutic agents. By facilitating the large‐scale production of high‐quality EVs, this platform lays a solid foundation for their application in disease treatment, promoting the potential of EVs in modern medicine and contributing to the advancement of healthcare solutions.

Funding : This work was supported by the National Natural Science Foundation of China (82341023, 82372102), The Interdisciplinary Research Project of School of Stomatology Wuhan University (XNJC202305), The Fundamental Research Funds for the Central Universities (2042022dx0003, 2042023kfyq02).

An efficient extracellular vesicle isolation platform with artificial intelligence analysis for improved colorectal cancer diagnostics

Presenter: Bonhan Koo

Yonsei University, Seoul, Republic of Korea

Introduction : Colorectal cancer (CRC) is a leading cause of cancer‐related mortality worldwide, primarily due to late‐stage diagnosis. Early detection is crucial for improving survival rates, but current methods like colonoscopy are invasive and have limited patient adherence. Liquid biopsy through blood‐derived extracellular vesicle (EV) analysis offers a promising non‐invasive alternative. However, existing EV isolation techniques are time‐consuming and yield low purity, limiting their clinical use.

Methods : We developed a novel system that integrates an EV isolation platform based on an amine‐functionalized zeolite and homobifunctional hydrazide with artificial intelligence (AI)‐driven analysis to enhance CRC diagnostics. This platform efficiently isolates and concentrates EVs from blood plasma within 15 min, enabling immediate extraction of EV‐derived miRNAs and circRNAs without the need for separate lysis steps required by traditional methods. From an initial pool of 11 miRNAs and 3 circRNAs, four miRNAs (miR‐23a‐3p, miR‐92a‐3p, miR‐125a‐3p, and miR‐150‐5p) were identified and combined with the conventional marker carcinoembryonic antigen (CEA). AI was employed to evaluate 31 biomarker combinations to determine the optimal set for CRC diagnosis.

Results : The optimal biomarker combination—miR‐23a‐3p, miR‐92a‐3p, miR‐150‐5p, and CEA—achieved an area under the curve (AUC) of 0.9861 for overall CRC detection, with a sensitivity of 95.83% and specificity of 100%. This performance significantly surpassed traditional CEA testing and the use of individual markers. The system demonstrated exceptional diagnostic accuracy across various CRC stages. For early‐stage CRC (stages 0–2), it identified optimal biomarker sets achieving an AUC of 0.9861, with a sensitivity of 91.67% and specificity of 100%. Specifically, for stages 0–1, certain biomarker sets achieved perfect diagnostic performance with an AUC of 1.0. In stage 2, the system achieved an AUC of 0.9722, with a sensitivity of 83.33% and specificity of 100%.

Summary/Conclusion : Our system provides a rapid, reliable, and non‐invasive tool for CRC diagnostics, enhancing early detection through AI‐driven biomarker combination analysis. By integrating an efficient EV isolation platform with deep learning, this system offers substantial clinical potential for improving CRC outcomes.

Scalable strategies for the production and purification of mesenchymal stromal cell‐derived extracellular vesicles

Ricardo Silva 1,2 , Sara Rosa 1,2 , Raquel Cunha 1 , Cecília Calado 1,3 , Carmen Garcia 4 , José Santos 1,2 , Ana Azevedo 1,2 , Cláudia Lobato da Silva 1,2 , Ana Fernandes‐Platzgummer 1,2

1 Department of Bioengineering and iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Lisbon, Portugal; 2 Associate Laboratory i4HB—Institute for Health and Bioeconomy, Lisbon, Portugal; 3 Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, Lisbon, Portugal; 4 Bionet Servicios Técnicos S.L., Madrid, Spain

Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are key mediators of the MSC paracrine effect and have shown great promise as natural cell‐free therapeutics and drug delivery vehicles. However, the clinical translation of EV‐based therapies requires scalable and efficient production and purification processes that can reliably deliver high‐quality EVs in therapeutically relevant quantities.

Methods : In this study, we established scalable culture systems for MSC expansion and EV production using 250 mL (DASbox, Eppendorf) and 2 L (F0, Bionet) controlled stirred tank reactors (STR), with xenogeneic‐free, microcarrier‐based fed‐batch combined with continuous perfusion (FB/CP) mode. For the 250 mL STR cultures, a continuous 3‐day EV production phase was also evaluated. For EV purification, conditioned media from STR cultures underwent tangential flow filtration and nuclease digestion, followed by anion exchange (AEX) chromatography. Various nucleases and AEX resins were tested to optimize EV recovery and purity.

Results : Both 250 mL and 2 L STR systems enabled efficient MSC expansion and high EV yields. In the 2 L STR, maximal cell densities reached (5.3 ± 0.32) × 10⁸ cells under FB/CP mode, with MSCs maintaining their immunophenotype across scales. During the continuous EV production phase in the 250 mL STR, total EV yields reached approximately (1.92 ± 0.38) × 10 1 2 particles, corresponding to productivities of (1.21 ± 0.31) × 10⁴ EVs/cell/day. EVs from both scales demonstrated consistent characteristics, including uniform size distribution (∼120–150 nm in diameter), surface charge of ∼−23 mV, and positive expression of markers (CD9, CD63, syntenin‐1) with the characteristic cup‐shaped morphology, as observed via TEM. The purification process achieved high purity levels, removing over 98% of proteins and host cell DNA, with recovery rates exceeding 85%.

Summary/Conclusion : This combined culture and purification platform supports reproducible, high‐yield production of MSC‐EVs with high purity in both 250 mL and 2 L STRs, compatible with clinical‐grade standards. The optimized STR‐based system and advanced purification strategies offer a scalable solution for MSC‐EV manufacturing, advancing their potential as cell‐free therapeutics in regenerative medicine.

Funding : This work was financed by national funds from FCT under projects UIDB/04565/2020 and UIDP/04565/2020 for iBB Research Unit, LA/P/0140/2020 for i4HB Associate Laboratory, and PTDC/EQU‐EQU/3708/2021 for NKboost.

Universal design of a macroporous material for the effective separating extracellular vesicles, viruses, and cells

Presenter: Takuya Kubo

Kyoto Prefectural University, Kyoto, Japan

Introduction : Previously, we developed a spongy‐like porous polymer consisting of poly(ethylene‐coglycidyl methacrylate) with continuous macropores that allowed efficient in situ reaction between the epoxy groups and proteins of interest. Immobilization of Protein A on spongy monolith enabled high‐yield collection of immunoglobulin G (IgG) from cell culture supernatant even at high flow rate. Additionally, this material can be moulded into any shape, enabling handling from tiny amounts of biological samples to large amounts of cell culture supernatant. In present studies, new spongy monoliths (SPMs) as separation media were developed for the separation of an extracellular vesicle, an exosome and a corona virus, SARS‐CoV‐2.

Methods : In former case, the specific lectins were immobilized onto a SPM and the selective separation of exosomes were successfully achieved. Furthermore, the SPM by hybridization with TiO2 nanoparticles effectively provided the concentration of small‐extracellular vesicles (SEVs) due to the interaction between phosphate in exosomes and TiO2. In case of the virus separation, an antibody was modified onto a SPM for the selective adsorption of a spike protein on SARS‐CoV‐2.

Results : In case of lectin immobilized SPMs, the separated exosomes included the different type proteins according to the proteome analysis. When we utilized TiO 2 containing SPMs, the yield of exosome was more than 100 times compared to the typical method, ultracentrifugation. This method could be applied to clinical samples, and efficient collecting of SEVs from 100 µL of plasma samples was achieved. We expect that our TiO2‐SPM will be a universal platform for collecting and purifying SEVs and significantly contribute to drug discovery research and biomarker exploration using SEVs. An anti‐spike protein (AS) based SPM (AS‐SPM) showed selective adsorption toward the spike protein in the protein mixture, and selective adsorption of SARS‐CoV‐2 was also confirmed. In contrast, the BSA‐SPM did not show adsorption of SARS‐CoV‐2 and adsorption of the rotavirus toward the AS‐SPM.

Summary/Conclusion : According to these applications, newly developed SPMs can be used for the rapid and effective separation of the bio‐related targets, such as proteins, glycoproteins, extracellular vesicles, viruses, and cells.

Rapid isolation and enrichment of EVs using solid phase separation

Gianna A. Slusher 1,2 , Andrei G. Fedorov 1,2

1 Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA; 2 The George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

Introduction : Extracellular vesicles (EVs) hold immense promise as diagnostic and therapeutic tools. However, their potential is constrained by the critical bottleneck of efficiently isolating pure EVs from other components in bodily fluids. Conventional isolation techniques, including ultracentrifugation (UC), size exclusion chromatography (SEC), and density gradient centrifugation (DG), face challenges related to enrichment, recovery, throughput, and reproducibility. While microfluidic devices enable rapid and precise processing, their small sample capacity and limited throughput restrict their suitability for clinical applications. This study aims to develop a precise, rapid, and selective EV isolation and enrichment method that can process large starting sample volumes by leverageing solid phase separation techniques.

Methods : Endothelial progenitor cell culture media is filtered through a pore vacuum filter and placed in different configurations atop a thermoelectric cooler. Different cooling rates, starting sample volumes, liquid film configurations, and spin rates are assessed for efficient separation and enrichment of EVs in the liquid phase. EV concentration is used as a metric for enrichment and evaluated with a tunable resistive pulse sensor. The entire setup is optimized for best recovery, throughput, and reproducibility.

Results : Preliminary results show an increase in EV concentration in the liquid phase following phase separation compared to the initial culture media.

Summary/Conclusion : This study presents a method for the isolation and enrichment of EVs from clinically relevant starting sample volumes. By enabling faster processing, improved handling, and more precise isolation compared to traditional methods like UC, SEC, and DG, this approach effectively overcomes critical limitations in the field. This advancement paves the way for significant progress in EV research and its clinical applications, ultimately contributing to more effective diagnostic and therapeutic tools.

Funding : The work described herein is supported by the National Science Foundation (NSF) Center for Cell Manufacturing Technologies (CMaT) Award No. 1648035. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NSF. The authors would like to acknowledge Ingrid Petersen of the Panitch Lab for technical assistance with EV concentration measurements.

Enhancing the therapeutic potential of mesenchymal stem cell‐derived extracellular vesicles through electrical stimulation

Sahba Mobini, Luna Pérez Troncoso, María Ujué González

Instituto de Micro y Nanotecnología, IMN‐CNM, CSIC (CEI UAM+CSIC), Madrid, Spain

Introduction : Stem cell transplantation shows promise for neurological conditions, but challenges like low cell survival and immune rejection limit its success. Extracellular vesicles (EV), key players in stem cell paracrine signalling, are emerging as an alternative treatment, though they struggle with low yield and variable cargo. Electrical stimulation (ES) has recently been suggested to boost stem cell regeneration capacity. We hypothesize and demonstrate that low voltage/low frequency ES could enhance the therapeutic potential of the mesenchymal stem cell (MSC) secretome, but the influence of ES parameters on MSC EV biogenesis and cargo remains unexplored.

Methods : We examined how frequency in voltage‐controlled pulsed ES affects EV production and their therapeutic content. EV from immortalized MSC lines were analysed under different ES conditions. Human adipose‐derived (ASC/TERT1) and bone marrow‐derived MSCs (BM‐MSC/TERT1) were cultured in a custom electro‐bioreactor and subjected to 1‐h daily ES for 3 days at 25 mV/mm, using 1 and 500 Hz frequencies with a 25% duty cycle. ES‐treated EVs (ES‐EV) from these regimes were then tested on MSCs and differentiated neurones to assess uptake and regenerative effects.

Results : EV were isolated using ultracentrifugation and characterized following ISEV guidelines, through nanoparticle tracking analysis and mass spectrometry‐based proteomics. We observed significant differences in yield and cargo of ES‐EV depending on the specific ES regimes and cell sources, up to 2 folds increase. our proteomics results show that ES‐EV enriched with distinct proteins compared control EV. Our bioassays confirmed that the effect of ES‐EV on cell proliferation in MSCs are more pronounced compared to control EV.

Summary/Conclusion : These preliminary results suggest that MSC‐derived EV are manipulable through ES and that ES parameters could potentially control EV properties. ES has the potential to be used as an effective tool for enrichment of EV from stem cells for regenerative medicine. Future experiments are required for defining mechanisms and fine‐teuning.

Funding : This study was supported by the Agencia Estatal de Investigación (AEI/Spain): CNS2023‐144736 funded by MCIN/AEI/10.13039/501100011033 and Unión Europea «Next Generation EU»/PRTR; PID2021‐128611OB‐I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe; “JAE Intro ICU” REF: IQM‐27. CSIC: I‐ILINK22025 funded by CSIC (I‐LINK2022); Connexion Nanomedicina CSIC

Optimization of blood and saliva EV isolation for the detection of the Parkinson´s disease‐related biomarker, aSyn

Fanni Annamária Boros 1 , Alexandra Gentschev 1 , Wei Xiang 1 , Philipp Arnold 2 , Martin Regensburger 1 , Friederike Zunke 1

1 Department of Molecular Neurology, University Hospital Erlangen, Friedrich‐Alexander‐University Erlangen‐Nuremberg (FAU), Erlangen, Germany; 2 Institute of Functional and Clinical Anatomy, Friedrich‐Alexander‐University Erlangen‐Nuremberg (FAU), Erlangen, Germany

Introduction : Parkinson´s disease (PD) is a progressive neurodegenerative disease caused by the loss of dopaminergic neurones in the central nervous system (CNS). The culprit of neurodegeneration is the accumulation of aggregates of aSyn in the CNS, and findings indicate that pathological forms of the protein are also present in extracellular vesicles (EVs) in peripheral tissues. Thus, EVs deserve particular interest in PD research as they can present a gateway to a better understanding of disease mechanism and inter‐organ spreading and can also serve as sources of potential disease biomarkers. The aim of this work is to establish an effective and ultrasensitive protein amplification assay (aSyn seed amplification assay, aSyn‐SAA), which enables detection of minimal amounts of pathological aSyn forms in EVs derived from peripheral body‐fluids. For this, we compare various EV‐enrichment methods and identify factors which influence aSyn‐SAA.

Methods : We isolated EVs from blood and saliva samples with polymer‐precipitation‐, membrane‐affinity‐, ultracentrifugation‐, and size‐exclusion chromatography‐based methods, and characterized the obtained samples for size and protein markers via nanoparticle tracking analysis, transmission electron microscopy, and western blots. For the optimization of pathological aSyn detection via aSyn‐SAA, we assessed the influence of monomer and seed concentrations, and reagents commonly used during sample preparation on the sensitivity and reliability of the assay.

Results : We show that different EV‐enrichment techniques result in differences in yield, integrity, and purity of vesicle preparations. We demonstrate that under optimal conditions aSyn‐SAA enables the detection of picograms of pathological aSyn conformers. However, the assay sensitivity and reproducibility heavily depend on the concentration of the monomer and the quality of seeds. Furthermore, the presence of reagents commonly used for sample preparation can negatively influence seed‐amplification.

Summary/Conclusion : The detection of pathological aSyn seeds in EVs obtained from peripheral tissue via minimally invasive route is a promising approach for PD diagnosis. However, due to the required very high sensitivity of the assay, the choice of EV‐enrichment method for obtaining seeding‐capable aSyn is of great importance, and the seed amplification assays need to be performed with great scrutiny.

A microfluidic device for on‐chip isolation and recovery of extracellular vesicle‐associated DNA

Teresa Lage, Hugo Silva, Lara Pierantoni, Sara Abalde‐Cela, Lorena Dieguez, Carlos Honrado, Alar Ainla

International Iberian Nanotechnology Laboratory, Braga, Portugal

Introduction : Current standard techniques for extracellular vesicles (EV) isolation based on ultracentrifugation are lengthy and burdensome, limiting broader clinical use. Microfluidics offers many benefits for miniaturization, cost reduction, faster processing, and automation. EV separation by physical features alone can be difficult due to the abundance of lipoproteins with overlapping sizes and densities. Thus, most microfluidic approaches utilize antibodies (ABs) against EV surface proteins, which, while highly specific, suffer from low stability and high cost. Alternatively, we propose an AB‐free microfluidic isolation approach targeting sulphated glycosaminoglycans (sGAGs) on the surface of EVs using chemical aggregation with a small molecule reagent. For biomarker selection, we have tailored our method for isolation of DNA cargo in EVs (evDNA). evDNA has only recently come into research focus, still having uncharted potential, but with some early findings indicating it could reveal the mutational landscape and methylation status of source cells, holding promise for liquid biopsy and diagnostics.

Methods : For evDNA isolation and recovery of we created a microfluidic device called ‘evDNA‐chip’ made of two parts: mixer and capture modules. The chemical aggregation of EVs is based on ExoGAG, utilizing cationic agent interacting with sGAGs. First, the mixer module combines ExoGAG with EV sample, followed by trapping in the capture module, where isolated EV aggregates are subsequently washed and lysed to recover the evDNA for downstream analysis. The evDNA‐chip performance was validated using conditioned medium from Hec‐1A cell line and human plasma. EV characterization was conducted using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Extracted evDNA was analysed by Qubit and droplet digital PCR (ddPCR).

Results : The evDNA chip successfully isolated and recovered evDNA from conditioned medium and human plasma samples. ddPCR analysis of isolated evDNA enabled the detection of clinically relevant DNA alterations.

Summary/Conclusion : The evDNA chip successfully isolates and recovers evDNA from complex body fluids, facilitating downstream analysis of their genetic cargo.

Funding : This study was supported by the Health from Portugal (C630926586‐00465198), through the NextGenerationEU Fund.

Rapid and efficient extracellular vesicle isolation using the EV‐osmoprocessor

Presenter: Casey Huang

National Taiwan University of Science and Technology, Taipei, Taiwan (Republic of China)

Introduction : Extracellular vesicles (EVs) are key mediators of intercellular communication with potential as biomarkers and therapeutics but isolating them from conditioned cell culture medium (CCM) remains challenging. Traditional methods, such as ultracentrifugation (UC), ultrafiltration, and size‐exclusion chromatography (SEC), have limitations in speed, purity, and ease of use. To address these challenges, we developed the EV‐Osmoprocessor (EVOs), a novel device leverageing osmosis‐driven filtration for rapid, efficient EV isolation with minimal user input.

Methods : The EVOs device was fabricated with a dual‐layer PVDF membrane system, housed between custom‐designed poly(methyl methacrylate) (PMMA) layers and immersed in a polyethylene oxide (PEO) solution to drive osmosis. The process reduced sample volume by 50‐fold in under two h, recovering concentrated EVs from a 200 µL reservoir. Characterization of the isolated EVs included cryo‐electron microscopy to confirm intact morphology, along with detection of key EV markers (CD81, CD63, TSG101, ALIX). Cardioprotective assays demonstrated that EVOs‐isolated EVs retained functional bioactivity comparable to those obtained by UC and ExoQuick (EQ).

Results : The EVOs device, composed of a dual‐layer PVDF membrane system, processes 10 mL of CCM in less than 2 h, concentrating it 50‐fold to 200 µL. Osmosis effectively removed small particles while retaining 75.6% of 100 nm and 81.5% of 200 nm EV‐sized particles. When used with CCM, EVOs reduced 99.7% of albumin and improved particle‐to‐protein ratios (3.3 × 10⁷ particles/µg). Cryo‐electron microscopy confirmed intact EV morphology, and key EV markers (CD81, CD63, TSG101, ALIX) were detected. Cardioprotective assays demonstrated that EVOs‐isolated EVs retained functional bioactivity comparable to those obtained by UC and EQ. While EVOs alone resulted in slightly lower purity than UC, combining it with SEC enhanced EV purity to 3.6 × 10⁹ particles/µg, exceeding UC performance.

Summary/Conclusion : Our findings suggest that EVOs is a promising new tool for rapid, high‐yield EV isolation with potential applications in biomarker discovery and therapeutic development. Further optimization of the filtration membrane could enhance its performance for broader applications.

Removal of chromatin by non‐woven fibre filtration and its effect on the biological activities of extracellular vesicles

Presenter: Nico Lingg

Austrian Centre of Industrial Biotechnology, Graz, Austria

Introduction : Advanced medicines based on extracellular vesicles (EVs) are showing promising results as therapeutic agents (both as delivery systems and intrinsic therapeutics) in several ongoing early‐stage clinical trials. To advance EV‐based medicines into late‐stage clinical trials and commercialization, scalable manufacturing processes are still needed. Currently available EV downstream processes require the combination of several unit operations, including ultracentrifugation, filtration, and/or chromatography, and suffer from poor scalability, low selectivity, and inadequate recovery. DNA in particular is a critical impurity that is challenging to remove, especially in the form of chromatin.

Methods : In this study, we developed a downstream process for the purification of EVs that combines the use of functionalized non‐woven fibres in flowthrough mode with tangential flow filtration. Non‐woven fibres are a type of convective chromatography media with an open‐pore architecture, offering a promising solution to the limited surface area and mass transfer limitations encountered with conventional chromatography resins, thereby improving process productivity. EV recovery and process yield were determined through nanoparticle tracking analysis, Western blotting, and bead‐based flow cytometry using different EV markers. Removal of chromatin was determined by Picogreen assay and anti‐histone Western blot.

Results : EV material that was purified using an efficient non‐woven fibre flow‐through step showed a strong decrease in histones and double stranded DNA. The concentration of EVs remained comparable in most cases. The biological activity after filtration were compared to an untreated control. A significant effect on the anti‐inflammatory activity was observed.

Summary/Conclusion : Our work demonstrates that EVs produced in different cell lines can be recovered, while critical impurities such as host cell DNA and chromatin are bound to the non‐woven fibre. The material resulting from the non‐woven fibre purification could then further processed using tangential flow filtration. The combination of these unit operations allowed the removal of host cell DNA, especially in the form of chromatin, increasing product purity and safety while maintaining its biological activity.

Funding : This project was funded by Austrian Research Promotion Agency FFG and Solventum Corporation.

Improved extracellular vesicle isolation by studying the hydrodynamics of micrometric particles within droplets confined in a microfluidic device

Beatrice Crestani 1 , Noemi Torriero 1 , Chiara Maceri 1 , Valentina Moccia 1,2 , Valentina Zappulli 2 , Davide Ferraro 1

1 Department of Physics and Astronomy, University of Padova, Padova, Italy; 2 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy.

Introduction : Microfluidics and droplet microfluidics permitted significant steps forward in biomedical diagnosis, allowing high throughput analysis and improved sensitivity. Droplet microfluidics, consisting of the generation of emulsions where aqueous droplets are suspended in oil, is currently applied in ddPCR and single‐cell sequencing. Here, we propose to use droplet microfluidics for the isolation of extracellular vesicles (EVs), mediated by immunocapturing on magnetic beads. Monophasic microfluidic has been employed for this scope with a low capture efficiency, mainly due to lack of mixing. Differently, droplets introduce recirculation zones promoting mixing between beads and EVs. In this work, we optimize the hydrodynamics of microparticles within droplets in microchannels to improve the EV isolation from plasma.

Methods : We analysed the behaviour of beads denser than the medium (2‐fold) under various experimental conditions (droplet speed, droplet volumes, channel diameters). We applied the best mixing conditions to optimize reaction time in a hydrogen peroxidase assay, and then to improve the EV isolation from frozen raw plasma. For the latter, experiments were carried out both by the microfluidic device and by an in‐batch protocol, varying the incubation time (1–4 h). Validation techniques were performed, including nanoparticle tracking analysis (NTA), BCA assay, and western blotting (WB), according to the MISEV‐2023 standards.

Results : From the microfluidic point of view, we observe that the redispersion of beads inside droplets is more homogeneous when the droplet aspect ratio is small (length‐width ratio between 1 and 3) and when they flow slowly (< 10 mm/s). Concerning the isolation of EV, results from NTA, BCA, and WB showed significant outcomes, with an isolation efficiency of about 70%. Microfluidic processing produced > 10‐fold higher WB signal intensities for EV markers CD9, CD81, and CD29 than in‐batch. The optimized droplet size and flow rates allowed to shorten the bead incubation time, achieving similar WB signals across 2‐, 3‐, 4‐h incubations.

Summary/Conclusion : This microfluidic platform improves EV isolation, addressing limitations of conventional approaches. Enhancing EV recovery from human plasma, droplet microfluidics offers a significant advance in liquid biopsy, showing a way for accessible, reproducible clinical diagnostics.

Funding : This research was funded by “Next Generation EU” Missione‐4 Componente‐1, for projects no. P2022J2ZC2 and 2022YYLSJ2.

Electrokinetic‐based micro‐nanofluidic device for EV isolation from urine for LPS recurrence detection

Premanshu K. Singh 1 , Ali F. Usmani 1 , Patricia Sarchet 2 , Federica Calore 2 , Debmalya Halder 1 , Raphael E. Pollock 2 , Shaurya Prakash 1,2

1 Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, Ohio, USA; 2 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA

Introduction : Liposarcoma (LPS), with 1500 US cases/year, has a 10‐year survival rate of only 10% due to late detection and high recurrence. Tissue biopsies and radiologic scans are costly and/or invasive, while liquid biopsy offers a minimally invasive alternative. We identified EV‐bound MDM2 DNA in serum as a biomarker for LPS recurrence, though limited sample volumes restrict analysis. Urine, offering larger volumes, could support broader analysis but requires improved isolation methods due to its variability and low EV concentrations, while conventional ultracentrifugation (UC) is labour‐intensive with low EV yield and purity. We developed a multi‐layered micro‐nanofluidic device with a sample injection channel (top) and a separation channel (bottom; each 3cm × 500µm × 60µm) integrated with a 200 nm nanocapillary membrane (NCAM) for selective capture of small EVs (< 200 nm) through immunocapture. We hypothesize that applying an electric field across the NCAM enhances the migration of negatively charged EVs into separation channel, improving capture efficiency.

Methods : Cells from the LPS cell line Lipo246 were cultured in serum‐free media for 48 h to produce conditioned media (LCCM), centrifuged at 2000 × g for 20 min to remove debris, and stored for EV isolation. Voltage biases (0–100 V) were tested on LCCM with anti‐CD81 for EV capture in the separation channel. EV isolation from pooled human urine was performed to assess device performance with clinical samples alongside UC for comparative EV isolation. Isolated EVs were characterized using Nanotracking Analysis, DNA quantification (Nanodrop), and MDM2 DNA analysis (qPCR). All experiments were conducted in triplicates.

Results : EV isolation from LCCM was optimal at 50 V, yielding 438 ± 59 ng/µL from a 450 µL sample, a 2.6× increase over 0 V. For urine samples (450 µL), 50 V resulted in a 1.5x increase in EVs ( p = 0.04, n = 2) and enhanced EV‐DNA yield to ∼98 ng, compared to ∼71 ng at 0 V. MDM2 DNA was detected in EVs from LCCM and urine. The device at 50 V isolated 98 ± 16 ng EV‐DNA from 450 µL of urine, offering 7.9× more EV‐DNA/mL than UC, which yielded 553 ± 111 ng from 20 mL.

Summary/Conclusion : We report an electrokinetic‐based microfluidic device capable of isolating 7.9x higher EV‐DNA/mL of urine than UC with a 75% reduction in time.

Funding : This work was supported by the US DoD through the PRCRP under Grant‐ CA210874 .

Enhanced dichotomic size‐exclusion chromatography to selectively remove lipoproteins for clinical isolation of plasma extracellular vesicles

Presenter: Xuhuang Xiao

Jinan University, Guangzhou, People's Republic of China

Introduction : The clinical translation of plasma‐derived extracellular vesicle (pEV)—relevant achievements was challenged by the abundant proteins and non‐vesicular extracellular particles (NVEPs) leftovers during isolation. Among which, the overwhelming of low‐density lipoproteins (LDL) were the primary contaminant in EVs acquired by size‐based isolation. We have previously developed the dichotomic size‐exclusion chromatography (dSEC) that could simply and efficiently remove abundant proteins and high‐density lipoproteins from pEVs. To provide a rapid method for the clinical isolation of high‐quality pEVs, we aimed to improve dSEC to selectively remove LDL.

Methods : Heparin‐conjugated agarose beads were employed to capture LDL and remove them from pEVs during dSEC isolations. The efficiency and specificity of the removal were parallelly estimated by nanoparticle tracking analysis (NTA), BCA, transmission electron microscopy (TEM), immunoblotting (IB), mass spectrometry (MS) and nanoflow cytometry (nFCM). Label‐free MS analysis was then performed to provide insights into the protein contents of the purified pEVs.

Results : Heparin beads could significantly remove 62% of particles and 60% of proteins in the EV fraction acquired by dSEC through overnight incubation, resulting an elimination of ApoB (a marker of LDL) without affecting the signal of EV markers in IB. EV‐like cup‐shaped illusion could only be observed in the TEM images of purified EV isolates, but none were found in the leftover on the beads. By integrating these heparin beads into our well‐characterized dSEC columns (dSEC_HP), we could remove over 90% of NVEPs including LDL‐particles by a single elution. Label‐free MS quantification reported that compared to the original dSEC, EV isolates from dSEC_HP showed increases in all identified transmembrane and cytosolic EV protein markers listed in MISEV2023, while various apolipoproteins including ApoB‐100 were found to be significantly decreased. Such high‐quality pEV isolates were proved to be capable for immunofluorescent nFCM analysis.

Summary/Conclusion : Lipoproteins in pEVs could be selectively removed or even eliminated by heparin‐conjugated agarose beads. Combining such beads with the dSEC, we have developed a simple and rapid isolation technique to acquire pEVs with low lipoprotein contaminants. Such a major‐equipment‐free method could be easily performed in clinical settings, thus could potentially facilitate the clinical translation of EV‐relevant achievements.

V‐Disk: automated multimodal extracellular vesicle purification from one mililitre of human plasma sample

Presenter: Ehsan Mahmodiarjmand

Hahn‐Schickard, Freiburg, Germany

Introduction : The clinical application of extracellular vesicle (EV) research, such as in liquid biopsies, is limited by the lack of reproducible, automated, and affordable EV purification methods. An ideal method would standardize the entire workflow from blood collection and plasma separation to EV purification for reliable and comparable results. Our microfluidic cartridge (V‐Disk) aims to achieve this. The current version of V‐Disk processes 1 mL of pretreated plasma, and the next version will automate plasma separation from 3 mL of blood, completing the purification workflow.

Methods : Using disposable centrifugal microfluidic cartridges manufactured by injection molding, we automate a multimodal EV purification process consisting of cation exchange chromatography (CEX), two filtration steps to remove particles > 800 nm and < 20 nm and concentrate the EVs, and a final treatment with a mixed‐mode chromatography (MMC) resin to remove remaining protein contaminants.

Results : After establishing the layout and manufacturing processes of the V‐Disk, we conducted an initial study comparing it with size‐exclusion chromatography (SEC), using three melanoma patient samples. ELISA results of EV surface markers reveal that the V‐Disk isolates exhibit a higher EV yield than those obtained via SEC. Measuring proteins, HDLs, and (V)LDLs, we demonstrate that the V‐Disk can remove impurities from plasma samples to achieve purity comparable to that of SEC. Nanoparticle‐tracking analysis reveals that isolated particles' size distribution is around 100 nm.

Summary/Conclusion : Starting from CEX‐treated plasma, the V‐Disk version already demonstrates significant potential, competing with SEC in terms of EV purity and yield. In the future, the V‐Disk will offer a fully automated, standardized label‐free isolation of EVs from whole blood in a highly reproducible manner in a small point‐of‐care compatible device.

Funding : Financial support by the Federal Ministry of Education and Research, Germany within the project KI‐VesD‐2 (No. 16LW0339) is gratefully acknowledged.

Process intensification by replacing centrifugation with chromatographic filtration in the production of therapeutic extracellular vesicles

Presenter: Tomas Mesurado

acib GmbH, Graz, Austria

Introduction : Extracellular vesicle (EV)‐based advanced medicines are demonstrating promising therapeutic potential in early‐stage clinical trials, both as delivery systems and intrinsic therapeutics. However, to transition EV‐based therapies to late‐stage trials and commercialization, scalable manufacturing processes are essential. Current EV downstream processes, which involve multiple unit operations such as ultracentrifugation, filtration, and chromatography, face challenges including poor scalability, low selectivity, and inadequate recovery. Chromatographic filtration utilizing non‐woven fibres offers an attractive method to intensify the process.

Methods : In this study, we evaluated the impact on EV quality if the traditional clarification steps of differential centrifugation and sterile filtration were replaced by a single chromatographic unit operation. Functionalized non‐woven fibres have been used to intensify and streamline manufacturing processes for monoclonal antibodies. Here, we tested their applicability to a typical EV production process. EV recovery and process yield were determined through nanoparticle tracking analysis, Western blotting, and bead‐based flow cytometry using different EV markers. We compared the biological activity of EVs produced in a conventional process to those produced with the intensified process.

Results : Replacing two centrifugation and one filtration step with a single chromatographic filtration step, resulted in drastically reduced process times while not affecting the recovery of EVs. The impurity profile was comparable, with the chromatographically clarified material showing lower concentrations of dsDNA. The biological activity after filtration were compared to an untreated control and a significant effect on the anti‐inflammatory activity was observed.

Summary/Conclusion : In this study, we developed the effective EV preparation method that enable a large quantity at high purity without decreasing the bioactivity of EVs. Furthermore, this method can also remove critical DNA impurities alongside of cellular debris in a single step. Therefore, non‐woven fibre filtration method, which overcomes all issues of current EV preparation method, must be gold standard for the therapeutic EV preparation in the future.

Funding : This work was supported by Solventum Corporation.

Validation of an immunoprecipitation‐based protocol to enrich brain, lung, liver, and placenta‐derived extracellular vesicles from human plasma

Biancamaria Pierri 1 , Gabriela L. Jackson 1 , Fang Wang 1 , Erez Eitan 2 , Olga Volpert 2 , Vrinda Kalia 1 , Andrea Baccarelli 3 , Haotian Wu 1

1 Columbia University, New York, New York, USA; 2 NeuroDex Inc., Cambridge, Massachusetts, USA; 3 Harvard University, Cambridge, Massachusetts, USA

Introduction : Extracellular vesicles (EVs) in circulation are informative tissue‐specific biomarkers that potentially reflect the phenotypic state of the parental cells, but their high heterogeneity poses a challenge for the enrichment of tissue‐specific EVs, limiting their utility. The absence of standardized, validated methods undermines the reliability of isolated materials and data reproducibility, causing significant obstacles to the translation of EVs as biomarkers. We propose a general workflow to enrich tissue‐specific EVs from human plasma, targeting tissue/cell‐specific surface proteins and validating EV subpopulations through multiple cytosolic targets.

Methods : Total EVs were purified by ultracentrifugation, size exclusion chromatography, or polyethylene glycol isolation reagent. An immunoprecipitation (IP) protocol followed by immunoassay validations was performed to enrich tissue‐specific EV subpopulations derived from brain, lung, liver, and placenta cells. In accordance with MISEV guidelines, we visualized the resulting EVs, measured particle concentrations and sizes, and verified the presence of canonical EV tetraspanins markers. The enrichment of target fractions was validated using ELISA assays for two tissue‐specific cytosolic proteins per tissue. Total EVs, IP unbound fractions, and IgG were considered as controls to compare the relative enrichment obtained for each EV subpopulation.

Results : ELISA assays showed >10× enrichment for all tissue‐specific cytosolic protein markers in IP‐purified subpopulations compared to total EVs (all p values < 0.05). General EV characterization confirmed the successful enrichment of the targeted EVs, demonstrating the colocalization of tetraspanins alongside tissue‐specific markers.

Summary/Conclusion : Our results show the effectiveness of an immunoprecipitation‐based protocol targeting tissue/cell‐specific surface antigens to enrich subpopulations of EVs from human plasma. The use of multiple cytosolic cell‐specific markers to validate the enrichment of the targeted EVs strengthens the reliability of our findings. This approach can be replicated to enrich other EV subpopulations, providing further insights into the physiology or pathology of the source tissue, and enhancing the application of EVs through a liquid biopsy perspective.

Funding : This research is supported by the National Institute of Environmental Health Sciences grant 1R35ES031688‐01A1.

Synergistic purity enhancement in extracellular vesicle isolation by integrating electrokinetic and tangential flow filtration: ExoTFF

Presenter: KIM YongWoo

Korea University, Seoul, South Korea

Introduction : As the importance of extracellular vesicles (EVs) grows in diagnostics and therapeutics, there is a pressing need for technologies that enable both large‐scale EV extraction and high‐purity isolation. Conventional methods struggle to effectively separate EVs from other particles and soluble proteins, creating challenges in clinical and research applications. The ExoTFF system is introduced as a solution, combining electrokinetic mesh filtration (ExoFilter) with size‐exclusion tangential flow filtration (TFF) to enhance EV isolation efficiency.

Methods : ExoTFF leverages two complementary filtration techniques. First, TFF removes particles smaller than 30 nm, effectively filtering out impurities. Second, the ExoFilter applies electrokinetic filtration to capture negatively charged EVs, resulting in an optimized separation process. This hybrid system was tested to evaluate improvements in yield and purity over traditional TFF methods.

Results : In proof‐of‐concept experiments, ExoTFF successfully processed 10 mL of plasma within 10 min, achieving near‐total removal of albumin and HDL. The hybrid system demonstrated nearly 100% yield and an 800% increase in purity compared to conventional TFF. Further tests with an automated ExoTFF system confirmed these results, maintaining the same yield and purity levels when processing 500 mL of plasma in 50 min.

Summary/Conclusion : small‐scale research and industrial EV production. The platform's ability to produce high‐quality EVs efficiently supports the growing demands in precision medicine and therapeutic applications, positioning it as a valuable tool for advancing EV‐based diagnostics and treatments.

Funding : This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government, MISP (2016RIA5A1010148).

Biomimetic culture system constructed with chorionic villi nanofibres enhanced the secretion of mesenchymal stem cell‐derived extracellular vesicles and its repair of intrauterine adhesions

Presenter: Xiaomei Liang

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Intrauterine adhesion (IUA) is one of the main causes of female infertility. Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs) rich in multiple factors and miRNAs related to tissue regeneration, which are the main effective paracrine components of MSCs to treat IUA. However, the poor secretion and bioactivity of EVs have been obstacles to their widespread use in clinical practice. Here, we developed human chorionic villi (CV)‐derived nanofibres and then constructed the CV‐nanofibre bionic culture system for MSCs. The mechanisms of the culture system were explored in maintaining the stemness of MSCs over a long period and enhancing EV secretion and bioactivity. Finally, we formed EVs‐ECM aggregate for treatment of rat IUA model and explored its efficacy and mechanisms to provide experimental evidence for IUA clinical therapy.

Methods : CV tissue was collected and separated from human placenta. CV‐nanofibre was prepared through decellularization and processing. The bionic culture system for MSCs based on CV‐nanofibre was constructed to collect high‐concentration EVs enrichment supernatant. EVs enrichment degree was detected by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot. Finally, EVs‐ECM aggregate was formed for treatment of rat IUA model.

Results : We reconstructed CV‐ECM into CV‐nanofibre, which retained the ECM protein component completely and was rich in proteins of enhancing EV formation, release and transport. We constructed the bionic culture system for MSCs based on CV‐nanofibre, cultured in which MSCs stemness maintained for up to 30 days and EVs production efficiency was enhanced greatly. These EVs meeting international standards contained proteins of promoting tissue regeneration. Finally, we formed EVs‐ECM aggregate. Experimental results showed that EVs‐ECM could effectively repair endometrium, treat IUA model and support healthy live birth.

Summary/Conclusion : In summary, we constructed the CV‐nanofibre biomimetic culture system to achieve large‐sale culture of MSCs and enhance EV secretion and bioactivity. EVs obtained by this system were encapsulated with CV‐nanofibres to form EVs‐ECM aggregate, which can effectively promote endometrium regeneration toward supporting foetal development and live births and overcomes the main obstacle preventing EV.

Funding : This work was supported by the National Natural Science Foundation of China (82371725 and 32301204).

Acoustofluidic chromatography for extracellular vesicle enrichment from microlitre blood plasma samples

Michael Gerlt and Thomas Laurell

Lund University, Lund, Sweden

Introduction : Blood plasma is a key medium for Extracellular vesicles (EV) research due to its accessibility but presents challenges for EV isolation, including high lipoprotein levels and other abundant proteins. Traditional ultracentrifugation methods are limited by low purity and high sample requirements, prompting interest in microfluidic technologies. This manuscript introduces ‘acoustofluidic chromatography,’ a method leveraging acoustic waves and micro‐capillaries for high‐throughput EV trapping with enhanced capacity and minimal sample volumes.

Methods : The trapping device comprised a glass capillary (20 mm length, 4 mm width, 0.4 mm height, 0.25 mm wall thickness) attached to a piezoelectric element. The capillary was filled with polystyrene beads (100 µm diameter). Upon actuation of the piezoelectric element in the MHz range using a wave generator, ultrasonic waves are generated and scattered at the polystyrene beads enabling the trapping of nanometre sized particles (seed particle trapping). The setup consists of two syringe pumps and a fluorescent microscope enabling the analysis of trapping efficiency in flow, which we used for thorough device characterisation. The blood plasma samples were analysed using nanoparticle tracking analysis and micro BCA.

Results : The device effectively traps particles over a broad frequency range (0.45–4 MHz) and supports high flow rates up to 100 µL/min, achieving trapping efficiencies of 82% ± 4% with electric power as low as 55 mW. Following comprehensive characterization, we performed blood plasma experiments using just 4 µL of triple‐centrifuged plasma, successfully isolating up to 2 × 10 9 particles/mL with the highest concentration of Particles with 50–60 nm diameter in only 8 min with a PBS wash of 360 µL. Protein contamination was minimal, with micro BCA measurements showing levels below detection (< 2 µg/mL). Our recent discovery of a frequency‐switching mode enhances trapping capacity by 100 times and throughput up to 4 times. Current efforts focus on isolating distinct EV subpopulations by size and density and confirming low lipoprotein contamination due to a repulsive acoustic force on these particles.

Summary/Conclusion : Acoustofluidic chromatography is a promising method to enrich different EV subpopulations with high purity from minute plasma samples within min.

Funding : This project is funded by the European Union—EIC transition.

Analysis of the efficacy and integrity of extracellular vesicles from concentrated BeWo cell culture media and placental explant supernatant

Presenter: Natalie Kaplanyan

University of California, Berkeley, California, USA

Introduction : Concentration of EVs is frequently used to increase sensitivity of downstream analyses and optimize functional studies. Ultracentrifugation is considered the gold standard and provides an unbiased approach for EV isolation. However, this method may cause EV aggregation which compromises further characterization. Amicon concentration of BeWo cell culture media (isolated from choriocarcinoma) and placental explant supernatant (PES), which are widely used in vitro models to study placental function, explores the efficacy and integrity of concentrating EVs in both cell culture and explant medias.

Methods : BeWo cell culture media (CCM) and PES were concentrated to 2×, 3×, 4×, 5×, and 10× using a 100 kDa molecular weight cut‐off Amicon filter. Amicon columns were blocked with 0.05% BSA prior to adding neat sample, then centrifuged at 3000 × g for various times to achieve the desired concentration. Vesicle flow cytometry (vFC) using antibodies raised against canonical EV markers (CD9, CD81, CD63) and a trophoblast‐specific marker (PLAP) was performed to quantify and characterize the concentrated EVs. Transmission Electron Microscopy (TEM) allowed for high‐resolution imaging to analywe EV size and morphology.

Results : vFC analysis shows loss in the total number of EVs recovered and hence decrease in concentration efficiency following Amicon concentration for both BeWo CCM and PES. According to TEM data, as the samples are concentrated, their biophysical properties change in terms of number, size, and circularity. The size increases and the circularity decreases, suggesting the formation of EV‐aggregates during the process of Amicon concentration.

Summary/Conclusion : Both vFC and TEM data provide evidence that Amicon concentration leads to decreased particle number, increased size, and decreased circulatory, consistent with EV aggregation, for the two sample types that were tested. We therefore conclude that Amicon concentration should be avoided for downstream analytical and functional studies of EVs, as concentrating EVs might change the cargo composition and/or biological properties of EVs.

Funding : UH3CA241687.

Miniaturized electrical fractionation of extracellular vesicles from placental samples by depletion‐zone isotachophoresis

Presenter: Andrea Capuano

Leiden Universiteit, Leiden, The Netherlands

Introduction : Placenta‐derived extracellular vesicles (EVs) play key roles in fetomaternal communication, modulating immune responses and cellular functions. Efficient isolation of EVs is crucial to enable accurate analysis for downstream applications. Using depletion zone isotachophoresis (dzITP), we achieve high‐purity EV separation and enrichment, enhancing analytical sensitivity and reproducibility. This method supports EV characterization, advancing research on pregnancy‐related pathologies.

Methods : Miniaturized silicon‐glass devices based on dzITP were used to concentrate EVs and separate them from contaminants. Optimal electrical parameters were established with JEG‐3‐derived EVs (from cell cultures), followed by the analysis of EVs from human placenta perfusate—both pre‐purified (clean) and unprocessed (raw). These samples were collected and characterized (NTA, dot blot) by the Placenta Lab at Jena University Hospital (Germany). Sample collection from the microfluidic chip was optimized for downstream analysis.

Results : Experiments showed how different pre‐treatments of JEG‐3‐derived EVs affected dzITP fractionation. Small EVs, isolated via serial ultracentrifugation steps, and larger, more contaminated EVs obtained with less refined ultracentrifugation processing showed distinct patterns—more complex samples yielded more fractions. Mixed EV samples displayed cumulative electropherograms and retained both profiles. Under the same dzITP conditions, EVs from raw and clean placenta perfusate were fractionated, reflecting their complexities. An electroosmotic flow‐based method enabled selective retrieval of fractions for downstream analysis.

Summary/Conclusion : dzITP enables efficient, precise electrical‐based fractionation of placenta‐derived EVs, reflecting sample complexity. Its ability to concentrate EVs, separate them from contaminants, and retrieve the different fractions will potentially enhance downstream analyses, supporting research on EV roles in pregnancy‐related conditions.

Funding : This study was supported by HORIZON EUROPE Marie Skłodowska‐Curie Actions (Grant No. 101111215).

Scalable GMP‐compatible process solution for MSC‐EV purification with 10× yield improvements

Stephen Lenzini, Jae Jung, Madeline Cramer, Elie Zakhem, Jon A. Rowley

RoosterBio, Inc., Frederick, Maryland, USA

Introduction : Scalable GMP‐compatible production of MSC‐EVs requires bioreactor expansion and downstream processing (DSP) for purification. We have published MSC‐EV bioreactor processes from 3‐50L with DSP operations including clarification, tangential flow filtration (TFF), chromatography, and formulation. Total cumulative yields of MSC‐EVs are often < 10%, representing a major challenge by limiting lot sizes and increasing cost of goods. Recently, we discovered a novel reagent (Agent V, patent pending) for addition during cell culture or to harvested conditioned medium (CM) that streamLines MSC‐EV DSP by greatly increasing yield. Our goal in this study was to determine the Agent V concentration in bioreactor‐harvested conditioned medium (CM) that maximizes post‐DSP yield while maintaining quality attributes.

Methods : Human bone marrow MSC‐EVs were produced in a 3L bioreactor (Eppendorf) using RoosterCollect‐EV for a 5‐day collection. Agent V was added to harvested CM at 0.1×, 1× and 5× for at least 15 min and MSC‐EV yield though clarification filters (Sartorius) was quantified by NTA (Particle Metrix). The optimal Agent V dose (1X) was chosen to treat remaining CM. MSC‐EV yield and purity (particles/mg protein) for each additional unit operation (TFF, Repligen; chromatography, Cytiva) were measured +/‐ Agent V treatment. Purified MSC‐EVs were tested for MSC‐EV identity (CD63/CD9/CD81 via ProteinSimple Jess) and function (CD73 activity).

Results : Treating CM with Agent V led to a dose‐dependent yield improvement through a 5 µm clarification filter. Without Agent V, pressure in the filter rises, leading to filter fouling and particle loss. With Agent V, pressure rise during filtration is minimized. Treated and untreated CM were processed further through each DSP unit operation, demonstrating that treatment leads to significant cumulative yield improvements (∼5% vs. ∼50%, a 10‐fold increase). Treatment and filtration steps did not affect particle size distribution. Furthermore, MSC‐EV markers (ProteinSimple Jess) and MSC‐EV potency (CD73 activity) in the purified EVs from the Agent V treated group were maintained.

Summary/Conclusion : In conclusion, Agent V is a novel, GMP compatible reagent that simplifies and streamLines EV purification by increasing MSC‐EV DSP yield 10‐fold while maintaining their quality attributes.

Assessing commercial size exclusion chromatography columns for isolation of extracellular vesicles from malignant ascites

Šárka Hrachovinová 1 , Stanislava Sladeček 1 , Janka Rakytová 1 , Martina Hýžďalová 2 , Nikol Straková 2 , Michal Felsinger 3 , Markéta Bednaříková 4 , Jitka Hausnerová 5 , Daniel Pinkas 6 , Václav Pustka 7 , Zbyněk Zdráhal 7 , Vít Weinberger 3 , Vendula Pospíchalová 1

1 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; 2 Department of Pharmacology and Toxicology, Veterinary Research Institute, Brno, Czech Republic; 3 Department of Obstetrics and Gynecology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic; 4 Department of Internal Medicine—Hematology and Oncology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic; 5 Department of Pathology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic; 6 Cryo‐EM and Tomography Core Facility, Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 7 Proteomics Core facility, Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Introduction : Malignant ascites is a common feature in advanced ovarian cancer, particularly high‐grade serous carcinoma (HGSC) of the ovaries, fallopian tubes, and peritoneum. Ascitic fluid, often collected during cytoreductive surgery, contains abundant components from the tumour microenvironment, including extracellular vesicles (EVs). Isolating EVs from ascites via size exclusion chromatography (SEC) may improve diagnostic accuracy and offer prognostic insights by enabling detailed EV profiling. This approach provides a valuable, accessible resource for translational cancer research.

Methods : EVs were isolated from five malignant ascites samples using two SEC column types: IZON qEV1 70 nm (IZON) and Immunostep EVs SEC 70 nm (Immunostep). Parameters evaluated included sample volume requirements, separation times, and EV recovery efficiency. EV fractions were analysed using Western blotting, cryo‐electron microscopy (cryo‐EM), multi‐angle dynamic light scattering (MADLS), and proteomics to assess purity, size distribution, and molecular profiles.

Results : Results indicated that EVs isolated with the Immunostep column exhibited higher levels of lipoprotein contamination, as confirmed through proteomic analysis. Proteomic profiling revealed distinctive protein enrichment patterns between the two columns: IZON‐isolated EVs predominantly showed MISEV2023 protein categories 1 and 2, while category 3 proteins were more abundant in Immunostep isolates. Categories 4 and 5 showed differential enrichment across both columns, suggesting isolation of distinct EV subtypes. Size distribution analysis demonstrated that IZON columns isolated larger EVs, while Immunostep columns captured smaller particles. Notably, both columns preserved the EV corona, ensuring robust isolation of intact EVs across a broad size range.

Summary/Conclusion : Optimizing EV isolation from malignant ascites could improve HGSC diagnostics and prognostics. This study outlines the unique strengths of IZON and Immunostep columns, helping researchers choose the best option for their needs. StreamLined EV isolation may boost the reliability and impact of biomarker research.

Funding : This work was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102)—funded by the European Union—Next Generation EU. CIISB, Instruct‐CZ Centre of Instruct‐ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127, is gratefully acknowledged for the financial support of the measurements at the CEITEC Proteomics Core Facility.

Magnetic bead‐assisted enrichment of extracellular vesicles for proteomic analysis of cardiometabolic disorders

Daniela Dzubinska, Jana Frydlova, Martin Vokurka, Petr Prikryl

Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Prague, Czech Republic

Introduction : Extracellular vesicles (EVs) are crucial for intercellular communication, with emerging diagnostic and therapeutic potential in cardiovascular and metabolic disorders. However, EV isolation remains challenging due to their small size and the complexity of biological samples. Traditional isolation methods like density gradient ultracentrifugation (DGUC) and size‐exclusion chromatography (SEC) often require post‐isolation washing and concentration steps that risk EV sample loss and their functional protein corona or can disrupt EV integrity. We adapted and optimized the single‐pot solid‐phase‐enhanced sample preparation (SP3) method using paramagnetic bead‐assisted precipitation to simplify and accelerate EV isolation for proteomic analysis by mass spectrometry.

Methods : EV proteins were enriched and lysed on beads and solubilized proteins were recaptured, trypsin digested on the same beads and UHPLC‐MS were used for detection and identification EV proteome. The SP3‐EV method applicability was tested on various biological samples, including human plasma, urine, pericardial fluid, cell culture media, and explant culture media of adipose and pancreatic tissue of cardiac/diabetic patients and prediabetic cell culture or mouse models. Ethical approval was obtained, and informed consent was received from all participants.

Results : The SP3‐EV method efficiently concentrated and purified EV proteins from density gradient ultracentrifugation fractions and is capable of eliminating contaminating iodixanol so that it would not interfere with proteomic mass spectrometry. This was documented by gel electrophoresis, western blot analysis, NTA, MALDI‐TOF etc. SP3‐EV method was compared to standard DGUC approach using qualitative mass spectrometry analysis. This approach can replace the washing and concentration steps by directly preconcentrating EV proteins from diluted samples, such as DGUC iodixanol or SEC fractions.

Summary/Conclusion : Our SP3‐EV approach offers a versatile, rapid, and efficient possibility of EV protein enrichment for proteomic analysis, with broad‐ranging potential for biomedical and clinical research. In combination with DGUC, SP3‐EV preserves protein corona and EV integrity and facilitates high‐purity EV isolation from complex samples, so SP3‐EV enables precise profiling of EV subpopulations, which is crucial for understanding functional heterogeneity. Its compatibility with automation and high‐throughput workflows aligns with emerging trends in clinical EV research, enhancing reproducibility and reducing hands‐on time.

Funding : The project Programme EXCELES LX22NPO5104‐Funded by the European Union‐Next Generation EU.

Development of a method for large‐scale purification of extracellular vesicles using the PS affinity method

Takamasa Ishidome, Masayuki Yamane, Ryo Ukekawa, Takahiro Nishibu

FUJIFILM Corporation, Tokyo, Japan

Introduction : Extracellular vesicles (EVs) are lipid bilayer membrane structures secreted by cells and contain various bioactive substances such as lipids, nucleic acids, and proteins. Since EVs derived from some stem cells, such as mesenchymal stem cells (MSCs), have therapeutic potential on various diseases, the use of EVs as therapeutic agents in medicine has been thought to be promising. Consequently, multiple methods have been developed for a larger scale EV purification. Here, we have sought to develop a scalable and reproducible method for affinity purification of EVs by using Tim4 protein, which specifically binds phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+‐dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. This method is capable of purifying EVs derived from cell culture supernatants with high recovery and purity.

Methods : Recombinant Tim4 protein was covalently immobilized on the surface of agarose resin, and the Tim4‐resin was packed into a 1 mL column. For EV isolation, 200 mL of MSC cell culture supernatant was directly applied into the Tim4 column. After washing the column, 4 mL of EDTA solution was applied to elute the captured EVs. The resulted EVs were analysed by using NTA, ELISA, BCA methods, and cell‐based functional assay. In parallel, EVs were also purified from the 200 mL of the same culture supernatant by using TFF, TFF + SEC, or TFF + AEX, and the resulted EVs were analysed for comparison.

Results : It was confirmed that EVs can be purified with higher yield and higher purity than other commonly used methods for large‐scale purification of EVs such as tangential flow filtration (TFF) combined with SEC or anion exchange chromatography (AEX) method. Furthermore, we observed Tim4 column purified EVs showed higher anti‐inflammatory effect than other conventional method.

Summary/Conclusion : Our affinity purification method using Tim4‐immobilized resin can purify EVs with higher recovery, purity and biological activity in fewer steps than competing methods and is also scalable. Therefore, we propose that our affinity purification method has potential for use in manufacturing process of therapeutic EVs.

Construction and application in colorectal cancer of isolation method for bacterial extracellular vesicles from human faeces

Presenter: Yicong Xue

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Colorectal cancer (CRC) is a major health challenge, ranking third in incidence and second in cancer‐related mortality. Evidence highlights the interplay between CRC and the gut microbiome. Bacterial extracellular vesicles (BEVs), originating from bacteria, contribute to inter‐bacterial and bacteria‐host communication by transporting proteins, lipids, and nucleic acids. BEVs from gut bacteria may play a critical role in CRC pathology. Investigating BEVs from human faeces is essential for understanding gut BEV heterogeneity in CRC, emphasizing the need for improved isolation and purification methods. This study optimized BEV isolation and applied it to assess BEVs’ diagnostic potential in CRC.

Methods : BEVs from gram‐positive and gram‐negative bacteria, as well as from human faeces, were analysed to evaluate their distribution in density gradient centrifugation (DGC) using both top‐down and bottom‐up methods, along with size exclusion chromatography (SEC). These methods were optimized individually and in combination to establish the most effective separation strategy. Evaluation metrics included particle morphology, size, concentration, and specific markers (LPS, OmpA, LTA for BEVs). These were analysed using TEM, NTA, NanoFCM, and WB. DNA from fecal bacteria and fecal BEVs in both healthy individuals and CRC patients was amplified (16S rRNA gene v3‐v4) and sequenced using Illumina MiSeq. A random forest model was developed to assess diagnostic performance through ROC analysis.

Results : A concentration of BEVs was observed in DGC fractions 6‐8 (F6‐8), with F5 being rich in eukaryotic EVs, and F4‐7 for SEC. The combination of top‐down DGC and SEC was identified as the optimal approach for isolating fecal BEVs from clinical samples, yielding high purity and more protein types regarding faecal BEVs, with a faecal BEVs concentration of 10 8 /mg. Sequencing results showed marked compositional differences between gut bacteria and fecal BEVs, notably between healthy and CRC individuals, both at the phylum and genus levels. Compared to whole bacteria, faecal BEVs demonstrated equally important diagnostic potential with fewer genus‐level types.

Summary/Conclusion : The optimized method enables standardized BEV isolation, providing a robust foundation for multi‐omics and functional analyses. Significant changes in the types and abundance of fecal BEVs in CRC underscore their potential as diagnostic markers.

Funding : This study was supported by the National Science Fund for Distinguished Young Scholars (82025024).

A novel hydrogel‐based method for efficient extracellular vesicles isolation from various biological samples

Ryota Kajihara, Riho Tanigaki, Kenji Yoneoka, Kyohei Tobinaga, Ryosuke Suzuki

Sanyo Chemical Industries, Ltd., Osaka, Japan

Introduction : The isolation of extracellular vesicles (EVs) from biofluids is a fundamental step in EV research. The most common protocol is differential ultracentrifugation (dUC) which utilizes size and density to separate EVs from sample solutions. However, dUC requires extremely high centrifugal force and has limitations regarding the volume and the number of samples that can be processed simultaneously. We developed a novel EV isolation method called hydrogel adsorption separation (HAS) using cross‐linked polyacrylate hydrogels. By modifying the chemical properties of the hydrogel surface, we achieved EV adsorption and desorption on the hydrogel. Here, we report the application of HAS for isolating EVs from various biofluids and the features of EVs which are purified through this process.

Methods : Cross‐linked polyacrylate hydrogel was manufactured by Sanyo Chemical Industries, Ltd. Human body fluid samples were purchased or collected under ethical approval. The conditioned medium was prepared by culturing HEK293 cells in an FBS‐free medium.

Results : First, we evaluated the recovery rate of urinary EVs purified by HAS in comparison with dUC. The isolated EV from urine sample was quantified by CD9/CD63 sandwich ELISA, with HAS achieving approximately a 10‐fold higher recovery rate than dUC. EVs isolated from other biofluids (serum, plasma) and conditioned medium were also examined by HAS. The obtained EVs showed sharp size distribution between 50 and 200 nm, with mode diameters around 80 nm. The EV markers CD9, CD63, and CD81 were detected by ELISA or western blotting. Additionally, RT‐PCR analysis of miRNAs in EVs isolated by HAS revealed the amplification of miRNAs (miR‐15a, miR‐22, let‐7a). Proteomic analysis of purified EVs from a conditioned medium was also performed, and EV‐specific proteins were predominantly identified from the isolated EVs.

Summary/Conclusion : Cross‐linked polyacrylate hydrogel serves as an effective scaffold for EV isolation from various solutions. EVs isolated by HAS exhibit uniform size distribution and contain distinct molecular markers. This method is scalable for large sample volumes and supports the simultaneous isolation of multiple samples, offering a powerful tool for EV research.

Platelet‐derived CD61+ extracellular vesicle depletion and isolation in less than 20 min

Presenter: Mandy Chan

STEMCELL Technologies, Vancouver, Canada

Introduction : Platelet‐derived extracellular vesicles (PLT‐EVs) are the most abundant EV subtype in blood and are often further elevated during illnesses. The increased level of PLT‐EVs is a double‐edged sword as PLT‐EVs may contain valuable cargoes for immunological functions (e.g., coagulation or inflammation), but their high abundance can mask the signals from other EV subtypes and hinders biomarker discovery. In this study, we developed new immunomagnetic isolation methods (EasySep) to either positively select or deplete CD61+ PLT‐EVs.

Methods : Plasma was obtained from blood collected in acid‐citrate‐dextrose tubes. Bulk plasma EVs were obtained using differential ultracentrifugation (dUC‐EVs). Starting with either untreated plasma or dUC‐EVs, PLT‐EVs were labelled with anti‐CD61 antibody complexes and magnetic particles for positive selection or depletion. The tube containing the labelled sample was then placed in an EasySep magnet. Magnetically labelled PLT‐EVs were retained on the tube wall, while the fraction containing PLT‐EV depleted plasma or dUC‐EVs was poured off into a new tube. To assess depletion efficiency, pan‐EVs were isolated from PLT‐EV depleted or untreated plasma using immunomagnetic isolation targeting CD9, CD63, and CD81 or size exclusion chromatography (SEC). PLT‐EV recovery and depletion were assessed by western blotting.

Results : CD61+ EVs that were isolated by positive selection had the expected PLT‐EV phenotype: CD61+ (platelet marker), CD63+ and CD9+ (common EV markers), and CD81‐ (non‐PLT EV marker). The depletion method efficiently removed CD61+ EVs from plasma (94.7% ± 3.5%, N = 17) and dUC‐EVs (95.7% ± 4.9%, N = 10). All PLT‐EV‐depleted and control samples had similar non‐PLT EV recoveries. In preliminary experiments, the signal‐to‐noise ratio (non‐PLT‐EV:PLT‐EV) increased from 0.2 to 4.4 after CD61 depletion when 1.3 × 10 10 depleted or control EVs were analysed. Pilot data demonstrated the feasibility of performing both depletion and positive selection from the same plasma sample.

Summary/Conclusion : Immunomagnetic isolation or depletion of PLT‐EVs can easily fit into common EV isolation workflows. PLT‐EV positive selection allows researchers to specifically analyse PLT‐EVs in diagnostic applications, while PLT‐EV depletion improves signal‐to‐noise ratio for biomarker discovery and functional characterization of EVs.

Improving hypoxic‐ischaemic encephalopathy (HIE) in children with umbilical cord mesenchymal stem cells enriched in extracellular matrix protein CD49e and extracellular vesicles

Presenter: Kuan‐Der Lee

Taichung Veterans General Hospital, Taichung, Taiwan (Republic of China)

Introduction : Recent advances in mesenchymal stem cells (MSC)‐secreted extracellular vesicles (EVs) provide another innovative treatment option for various diseases. Hypoxia significantly affects many properties of MSCs, including cell viability, proliferation, differentiation, and cell migration. Changes in cell culture conditions can affect the release of different exosomes. Therefore, this study explores the effect and therapeutic effect of hypoxia on clinical‐grade mesenchymal stem cell and exosomes.

Methods : hUC‐MSC‐EVs were isolated and expanded using animal‐free/serum‐free medium from Wharton's jelly from healthy human umbilical cord. EVs isolated from normoxic (21% O 2 , 5% CO 2 ) or hypoxic (2% O 2 , 5% CO 2 ) cultured MSCs were sorted and concentrated by membrane‐based tangential flow filtration. We evaluate the size and distribution of nanoparticles through nanoparticle tracking analysis (NTA) and evaluate morphology through transmission electron microscopy. Immunophenotyping of two EV populations was assayed by flow cytometry (Miltenyi Biotec, Germany).

Results : hUC‐MSC‐EVs were enriched by hypoxia in mean expression values for CD9 (EV‐NormO 2 89.12% ± 4.04%; EV‐2% O 2 96.76% ± 1.02%), CD63 (EV‐NormO 2 97.63% ± 1.49%; EV‐2% O 2 99.04% ± 1.23%), CD81 (EV‐NormO 2 84.88% ± 19.77%; EV‐2% O 2 94.75% ± 4.31%), and particles concentration (EV‐NormO 2 6.41 × 10 8 ± 3.47 × 10 8 /10 5 cells/mL; EV‐2% O 2 1.75 × 10 9 ± 3.34 × 10 8 /10 5 cells/mL). hUC‐MSC‐EVs cultured in hypoxia expressed much higher levels of the surface markers CD49e than those cultured in normoxia. MicroRNA‐seq showed EVs had high expression of miR‐151‐3p and miR‐21‐5p. Herein, we will present these hUC‐MSCs have demonstrated clinical improvement in children with HIE.

Summary/Conclusion : hUC‐MSCs cultured under hypoxic conditions secrete EVs enriched in the extracellular matrix protein CD49e and miRNAs and may improve children with HIE.

Funding : This study was supported by the Intramural research grant from Taichung Veterans General Hospital, Taichung, Taiwan.

Translation of combined density gradient ultracentrifugation and size exclusion chromatography‐based EV isolation from rat to human plasma for metabolomic and proteomic analyses

Presenter: Dóra Kapui

Semmelweis University, Budapest, Hungary

Introduction : To implement extracellular vesicle (EV)‐based biomarker analyses for diagnostics and prognostics, isolation protocol resulting in high purity EV samples is needed. An established method based on iodixanol density gradient ultracentrifugation (DGUC) followed by CaptoCore700 bind‐elute size exclusion chromatography (BE‐SEC) was successfully applied to identify EV‐based metabolic alterations correlating with hypercholesteremia‐induced cardiotoxicity in rats. However, this workflow has not been tested for human samples. Thus, our aim was to apply this technology for human blood plasma samples.

Methods : EVs were isolated from human platelet‐free plasma by iodixanol DGUC, qEV and Exo‐spin SEC columns, and iodixanol DGUC followed by Sepharose CL‐2B SEC column. EV isolates were characterized by Western blot (WB), nanoparticle tracking analysis, immune electron microscopy, and bicinchoninic acid protein assay.

Results : WB analysis of human plasma DGUC isolates showed a significant amount of contaminants previously not detected in rat plasma, which made EV markers undetectable by WB. These results made it impossible to directly assess which DGUC fractions contain human EVs. By isolating EVs with qEV or Exo‐spin column, EV markers could be detected by WB, however, significant lipoprotein contaminants were present. Therefore, to eliminate the lipoprotein contaminants, DGUC was performed, and lipoprotein‐poor, particle‐rich fractions were further purified with a large‐scale Sepharose CL‐2B SEC column, which has similar separation characteristics to qEV columns. This resulted in the isolation of EVs positive for CD81, Tsg101 and HSP70 with lipoprotein contaminants below detection limits.

Summary/Conclusion : Using Sepharose CL‐2B SEC column, a methodology similar to earlier used technology, resulted in EVs with lipoprotein contamination below detection limit. The applied method could be used to test the diagnostic potential of circulating EV metabolites in cardiovascular diseases. Further studies to test the effect of the difference in Sepharose CL‐2B versus CaptoCore700 SEC is needed.

Funding : The Ministry for Innovation and Technology of Hungary from the source of National Research, Development and Innovation Fund supported CK (EKÖP‐2024‐226 New National Excellence Program), ZG (K139105) and the project (2020‐1.1.5‐GYORSÍTÓSÁV‐2021‐00011). The project was further supported by the European Union (RRF‐2.3.1‐21‐2022‐00003).

Testing of high‐throughput methods for urinary extracellular vesicle isolation for biomarker discovery studies

K. Barreiro 1 , R. Hartman 1 , I. Quintero 1 , F. Roessler 1 , P. H. Groop 2,3,4 , M. Puhka 1

1 HiPREP core, Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland; 2 Folkhälsan Research Center, Biomedicum Helsinki, Helsinki, Finland; 3 Department of Nephrology, University of Helsinki, Helsinki University Hospital, Helsinki, Finland; 4 Faculty of Medicine, Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, Finland

Introduction : Urinary extracellular vesicles (uEV) provide valuable non‐invasive biomarkers for urogenital diseases. However, progress in biomarker discovery and validation is slow because of lack of uEV isolation methods suitable for robust high‐throughput processing. For this purpose, we tested methods based on precipitation, ultrafiltration and immunomagnetic bead capture in comparison to ultracentrifugation. Methods were evaluated based on processing format and time, RNA or protein yield and quality and uEV sample morphological analysis.

Methods : uEVs were isolated from urine samples with or without a preconcentration step. The urine donors included healthy donors or donors with type 1 diabetes with or without kidney disease. uEV samples were viewed with transmission electron microscopy, RNAs profiled with the Bioanalyzer Pico kit and proteins quantified with micro BCA or similar methods. For a deeper insight, proteins were run on SDS‐PAGE gels and EV‐enriched markers and contaminants detected by Western blotting, or samples were subjected to RNA sequencing.

Results : EVs, EV‐like structures, small RNA and proteins were detected using all methods. The yield, quality and purity assessments showed small differences between the methods. EV markers, contaminants and sequencing results were at an acceptable level in comparison to ultracentrifugation. Processing time and sample format varied the most between the methods.

Summary/Conclusion : All methods showed initial promise for scalable EV isolations. However, the need for sample preprocessing and output sample format impact the throughput or the selection of usable downstream techniques. Further optimization may allow a wide variety of large‐scale studies for biomarker discovery.

Funding : This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115974. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA and JDRF.

Separation of small extracellular vesicles (sEV) from human blood by superose 6 size exclusion chromatography

Gonzalo Bustos‐Quevedo 1,2 , Jerome Nouvel 1,2,3 , Tony Prinz 1,2 , Ramsha Masood 1,2 , George Daaboul 4 , Tanja Gainey‐Schleicher 1,2 , Uwe Wittel 2,5 , Sophia Chikhladze 2,5 , Bence Melykuti 1,2 , Martin Helmstaedter 2,6 , Gerhard Pütz 2,3 , Irina Nazarenko 1,2,7,8

1 Institute for Infection Prevention and Hospital Epidemiology, Freiburg, Germany; 2 Faculty of Medicine, Medical Center, University of Freiburg, Freiburg, Germany; 3 Institute of Clinical Chemistry and Laboratory Medicine, Freiburg, Germany; 4 NanoView Biosciences, Boston, Massachusetts, USA; 5 Department of General and Visceral Surgery, Freiburg, Germany; 6 IMITATE EM Core Facility, Freiburg, Germany; 7 Hahn‐Schikard, Freiburg, Germany. 8 German Cancer Consortium (DKTK), Partner Site Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany

Introduction : EVs are particles released from all types of cells sharing similarities with the cells from where they were released. Many isolation techniques are being developed for EV isolation because of their applicability as liquid biopsies, and specifically as a way of understanding their role in the progression of different diseases. Blood is one of the biofluid more used, where other EV‐like particles are found, such as lipoproteins. To obtain EV samples without lipoproteins is quite used in the research community consecutively EV isolation based on different approaches, having a first step of size exclusion isolation followed by a step of ultracentrifugation. Resulting in a high purity of EVs but a low EV recovery. We addressed this problem using fast protein liquid chromatography (FPLC) as an EV isolation method.

Methods : NTA, TEM, MACSPLEX, TECAN, cell culture, lipoproteins isolation, spiking blood samples with EVs derived cell culture, western blot.

Results : FPLC was validated using pre‐isolated EVs from cell culture and lipoproteins from blood. EVs were visualized in fraction 16, and lipoproteins in fraction 25. TEM and western blot were used to corroborate integrity from fraction 16 and the presence of CD63 as a surface marker and ALIX, TSG101, and HSP70 as associated EV markers. Those markers were missing in the rest of the fractions. Then, plasma samples from healthy donors were run using antibodies using CD9 and CD63 for EVs, and ApoB100 and ApoA1 for lipoproteins. Fraction 16 being the highest in EV particles and fraction 25 for lipoproteins. Then, EVs from four blood samples with Pancreas adenocarcinoma (PDAC) were isolated by FPLC to validate that the EV‐rich fractions are useful for detecting EV‐derived cancer cells. Isolated particles were measured by NTA and MACSPLEX. Fraction 16 did not contain a high particle number, but EV biomarkers such as CD63 and EV cancer biomarkers such as TSPAN8, and EpCAM were detected with the maximum signal in fractions 16 and 17.

Summary/Conclusion : The applicability of the FPLC for EV isolation technique was corroborated having a higher resolution of EV rich fractions with a lower number of lipoproteins co‐isolated, and also isolating EVs carrying EV cancer biomarkers.

Comparative efficiency of ultracentrifugation and size‐exclusion chromatography in exosome isolation from limited clinical sample

Yeliz Altınok 1 , Gülşah Çeçener 1 , Şenay Vural Korkut 2 , Salih Haldun Bal 1 , Gözde Arslan 1

1 Bursa Uludag University, Bursa, Turkey; 2 Yildiz Technical University, Istanbul, Turkey

Introduction : Exosomes are small extracellular vesicles (30–150 nm) released by most cell types into fluids like blood plasma and urine, carrying biomolecules that indicate the physiological state of their origin cells. Their heterogeneity in size and composition complicates isolation and characterization. Traditional methods, such as ultracentrifugation, often result in plasma protein contamination and yield variability, underscoring the need for standardized protocols to enhance exosome research.

Methods : Blood samples were collected from three healthy individuals with ethics‐approved informed consent. Plasma was separated by centrifugation at 4000 rpm for 30 min within 1 h of collection. Exosomes were isolated from 2 mL of plasma via ultracentrifugation at 100,000 × g , 125,000 × g , and 150,000 × g to determine the optimal speed for limited sample volumes. Additionally, exosomes were isolated from 1 mL plasma using size‐exclusion chromatography (SEC). Western Blot was used to detect CD9, CD63, and CD81, with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) providing morphological and concentration data.

Results : CD9, CD63, and CD81 were present in all samples via Western Blot. Exosomes isolated by SEC had a mode size of 111.5 nm and an average of 145.67 nm, indicating a more homogeneous size range. Ultracentrifugation yielded mode sizes of 104.17, 114.17, and 122.17 nm at 100,000 × g , 125,000 × g , and 150,000 × g , with average sizes of 189.67, 211, and 186.67 nm, respectively. The difference between mode and average in ultracentrifugation suggests potential plasma protein co‐precipitation. SEC's narrower size distribution indicates higher purity, suitable for sensitive applications. In terms of particle concentration, 100,000 × g produced the highest average concentration (2.48 × 10 10 particles/mL), followed by 150,000 × g (1.42 × 10 10 ) and 125,000 × g (1.30 × 10 10 ). Two samples had the highest concentration at 100,000 × g , while one sample peaked at 150,000 × g , suggesting optimal speed varies between individuals.

Summary/Conclusion : This study evaluated ultracentrifugation and SEC for exosome isolation. SEC yielded exosomes with a narrower size distribution, indicating higher purity, making it suitable for biomarker research with limited clinical samples. While ultracentrifugation can produce higher concentrations in some cases, it requires more sample volume and poses a contamination risk, limiting its utility for high‐purity applications.

Optimization and scale‐up of Human Milk Extracellular Vesicle isolation for the prevention of Necrotizing Enterocolitis in preterm infants

Jose Luis Moreno‐Casillas 1 ,2 , Laura Ripoll‐Seguer 1,2 , Eduardo López Briz 3 , Ana María Padilla‐ López 3 , María Gormaz 1,4 , Julia Kuligowski 1,2,5

1 Neonatal Research Group, Health Research Institute Hospital La Fe, Valencia, Spain; 2 Servicio de Análisis de Vesículas Extracelulares (SAVE), Health Research Institute Hospital La Fe, Valencia, Spain; 3 Hospital pharmacy department, University and Polytechnic Hospital La Fe, Valencia, Spain; 4 Division of Neonatology, University and Polytechnic Hospital La Fe, Valencia, Spain; 5 Primary Care Interventions to Prevent Maternal and Child Chronic Diseases of Perinatal and Developmental Origin Network (RICORS‐SAMID) (RD21/0012/0001), Instituto de Salud Carlos III, Madrid, Spain María Gormaz and Julia Kuligowski are co‐senior authors .

Introduction : Human milk (HM) is a complex biofluid enriched with extracellular vesicles (EVs), which contribute to the mitigation of intestinal damage associated with necrotizing enterocolitis (NEC). This study aims to develop an optimized ultracentrifugation (UC) protocol for the isolation of HMEVs, facilitating their use as a nutritional supplement to evaluate their potential in NEC prevention.

Methods : Five different isolation protocols were assessed, each processing 25 mL of HM and differing in the number and intensity of centrifugation steps for lipid and protein removal, as well as for HMEV pelleting. Isolated HMEVs were analysed by tunable resistive pulse sensing (TRPS) for particle size and concentration determination, and immunoassays via Leprechaun platform. HMEV isolation was performed in triplicate, with samples resuspended in PBS (control), saline (0.9% NaCl), and 5% glucose solutions as candidate carriers for stability and osmolarity assessment.

Results : While the same size distribution (170–450 nm) was observed across protocols 1–5, the particle concentrations increased from 3 to 6 × 10 1 1 , with a significant rise when two UC steps were omitted (protocol 4), as well as when one lipid and protein removal step were skipped in addition to two UC steps (protocol 5). The Leprechaun platform, employing antibodies against CD9, CD81, and CD63, yielded comparable HMEV concentrations across methods (one‐way ANOVA, p < 0.05). No significant variation in particle concentration was observed over a 1‐month period across different solvents (one‐way ANOVA, p < 0.05). Osmolarity values (287–307 mosmol/kg) were below 380 mosmol/kg, being similar to those of HM and suitable for supplementation.

Summary/Conclusion : Although the elimination of purification steps compromises the purity of isolated HMEVs, the shortened protocol 3 allows for the efficient retrieval of EVs from HM for the preparation of nutritional supplements. This streamlined approach reduced the isolation time from > 12 to ∼4 h, minimizing manipulation and enabling the processing of the necessary volume of HM for one week of patient treatment in only two isolation cycles.

Funding : This work was supported by the Instituto de Salud Carlos III (ISCIII), grant number CD22/00081 Spain, and co‐funded by the European Union [PI23/00202]; and grant CNS2022‐135398 funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “European Union NextGenerationEU/PRTR.”

Efficient isolation and characterization of extracellular vesicles and insights into their role in prostate cancer progression and treatment response

Presenter: Carlotta Schudy

University Medical Center Hamburg‐Eppendorf, Hamburg, Germany

Introduction : Prostate cancer (PCa) is the most prevalent malignant tumour among men. Despite early detection, diagnosis and treatment, PCa remains among the most frequent causes of cancer‐related deaths. Extracellular vesicles (EVs), with their cargo that reflects the characteristics of their parent cells, offer a potential means of communication with other cells and present various opportunities for therapeutic and diagnostic applications. A primary aim of this study is to identify an efficient, time‐saving method for exosome isolation, as the current gold‐standard method, ultracentrifugation, is time‐intensive, unsuitable for small volumes, and risks damaging exosomes. Additionally, this study explores the potential role of exosomes in the prognosis of PCa.

Methods : Hormone sensitive (LNCaP) and hormone‐resistant LNCaP sublines were used. EVs were isolated by ultracentrifugation, precipitation, and a combined method. Nanoparticle tracking analysis assessed EV size and concentration, and Western blotting and PKH67 staining characterized CD9/CD63 expression and exosome uptake, respectively. LC‐MS/MS compared EV proteomics to parent cells, with validation through patient‐derived tissue slice cultures. Informed consents were obtained from all patients.

Results : Our findings indicate that the combined concentration‐precipitation method achieves the highest EV yield and concentration. EVs were characterized by identifying specific markers, and confocal microscopy confirmed robust cellular uptake of PKH67‐labelled exosomes. Proteomic analysis revealed significant differences between EVs and their parent cells, with distinct proteomic profiles noted between EVs from LNCaP cells and their hormone‐resistant sublines. Several pathways were uniquely enriched in EVs. These results were further validated using EVs isolated from patient‐derived tumour slice cultures.

Summary/Conclusion : Our study highlights the effectiveness of a newly established isolation method using combined concentration‐precipitation approach, revealing unique cargo and functional differences between EVs and their parental cells. These distinctions suggest that EVs may play key roles in prostate cancer progression. Ongoing mechanistic studies are now focused on uncovering how EVs influence the response to anti‐hormone therapy in PCa.

Automated, medium and high‐throughput immunomagnetic isolation of extracellular vesicles from biofluids

Presenter: Jason Dixon

STEMCELL Technologies, Vancouver, Canada

Introduction : Traditional extracellular vesicle (EV) isolation methods such as differential ultracentrifugation (UC) are time consuming, difficult to implement in clinical settings, and cannot be automated. We have recently developed immunomagnetic methods (EasySep) to isolate EVs using tetraspanin surface markers. We have now fully automated these methods using RoboSep‐S (up to 4 samples) and RoboSep‐16 (up to 16 samples) platforms, allowing for the scalable isolation of EVs for sample volumes of 0.5 to 8.0 mL.

Methods : EVs from human plasma were labelled directly with a tetraspanin (CD9, CD63, and/or CD81)‐specific antibody cocktail. The labelled EVs were bound to magnetic particles and separated from unwanted EVs using a specialized magnet. Automated isolation using RoboSep‐S or RoboSep‐16 was performed as follows: guided by the on‐screen prompts, plasma, reagents, tips, tubes, and wash solutions were loaded onto the instrument; the instrument automatically labelled EVs with antibody cocktails and particles and transferred the sample to the magnets on the instrument deck for magnetic separations. EVs isolated manually or via the automated platforms were analysed by western blot to assess EV recovery and albumin contamination.

Results : The fully automated RoboSep‐16 pan‐EV protocols recovered 89%–98% EVs ( n = 6) while the RoboSep‐S protocols recovered 93%–111% ( n = 6) EVs compared to the manual separation using EasySep magnets. The automated platforms consistently demonstrated lower albumin contamination compared to manual isolation. Both platforms were able to isolate EVs in less than 60 min.

Summary/Conclusion : EasySep allows for fast and easy immunomagnetic enrichment of EVs. Fully automated EV isolation on RoboSep‐S and RoboSep‐16 platforms further reduces impurities and user variability, offering scalable EV isolation solutions for labouratories using EVs as biomarkers of disease.

Subtopic EV sizing and counting 2

A novel method of size measurement in super‐resolution microscopy for extracellular vesicles characterization

Nicolas Kuszla 1 , Lucile Alexandre 1 , Laura Fouassier 2 , Florence Gazeau 1 , Stéphanie Mangenot 1

1 Université Paris Cité, NABI Laboratory, CNRS UMR 8175, INSERM U1334, Paris, France; 2 Sorbonne Université, Centre de Recherche Saint‐Antoine, INSERM UMR S938, Paris, France

Abstract unavailable

Quantification of extracellular vesicles by flow cytometry: fluorescence versus light‐scatter detection

Liam Barry‐Carroll 1 , Céline Gounou 2 , Jean‐Christophe Delpech 1 , Stéphane Mornet 3 , Alain R. Brisson 2

1 INRAE UMR‐1286, Bordeaux, France; 2 University of Bordeaux, Bordeaux, France; 3 CNRS UMR‐ICMCB, Pessac, France

Introduction : The quantification of extracellular vesicles (EV) constitutes a major challenge in the EV field. In this study, we compared two approaches of EV quantification by flow cytometry, namely the conventional approach of light‐scatter detection using a new‐generation flow cytometer dedicated to small particle detection (NanoAnalyzer, NanoFCM), and the alternative approach of fluorescence‐based detection, using a previous‐generation instrument (Gallios, Beckman‐Coulter). In addition, immuno‐gold cryo‐electron microscopy (immuno‐cryo‐EM) was used to determine the EV size and phenotype distributions.

Methods : EV derived from red blood cell (RBC) and platelets (PLT) were prepared from blood bags, by osmotic lysis and activation with A23187 ionophore, respectively. EV derived from reticulocytes (RET) were isolated from blood samples obtained from sickle cell disease (SCD) patients, after written informed consent. EV samples were labelled with Annexin‐5 (A5) for identifying phosphatidylserine‐exposing EV, or with antibodies (Ab) specific of RBC (CD235), PLT (CD41) or RET (CD71), either fluorescently labelled for flow cytometry or conjugated to gold particles for immuno‐cryo‐EM.

Results : Two types of EV samples were selected for this study: (1) EV derived from RBC and PLT, which are large EV (∼200 nm average size, as determined by cryo‐EM) and present a high density of membrane surface receptors, around 7000 CD235 (RBC) and 3000 CD41 (PLT) per µm 2 , respectively, and (2) RET‐EV expressing CD71, which are prototypical exosomes of small size (∼100 nm diameter). For RBC‐ and PLT‐EV, essentially identical numbers of EV were detected by the two flow cytometry approaches compared here, with all the markers used ‐A5, CD235 and CD41‐, over 20 independent experiments. As an example, the ratio between the numbers of A5‐labelled EV detected by light‐scattering vs. fluorescence was 1.4 ± 0.3 ( n = 11) for RBC‐ and PLT‐EV. In contrast, in the case of CD71‐expressing RET‐EV, we found that only a small amount (< 10%) of EV was detected by fluorescence triggering. This latter point is under further investigation.

Summary/Conclusion : This study demonstrates, for the first time to our knowledge, that the fluorescence triggering approach allows an accurate and reliable quantification of EV, at least in the case of large EV expressing a high surface density of antigens.

Funding : ANR grant

Characterizing EV preparations via gas‐phase electrophoresis on a nES GEMMA instrumentation

Presenter: Victor U. Weiss

TU Wien, Vienna, Austria

Introduction : As recommended by the European Commission, nanoparticle characterization should be based on particle number concentrations (2011/696/EU from October 18th, 2011, updated in 2022). Gas‐phase electrophoresis on a nano electrospray gas‐phase electrophoretic mobility molecular analyser (nES GEMMA) allows such an approach. Single‐charged (bio‐)nanoparticles obtained after a nES process with subsequent surface‐drying and charge equilibration are separated based on their electrophoretic mobility diameter (EMD) corresponding to the particle diameter for spherical analytes. By voltage variation, particles of different EMD successively reach the instruments’ detector unit and are counted, resulting in a spectrum in the size range of several few up to several hundred nm EMD.

Methods : EVs were either purified via ultracentrifugation or a combination of precipitation and centrifugation. Subsequently, the sample buffer was exchanged for ammonium acetate. EV monitoring was via nanoparticle tracking analysis (NTA, Particle Metrix, Inning am Ammersee, Germany), gas‐phase electrophoresis was on nES GEMMA instrumentation (TSI Inc., Shoreview, MN, USA).

Results : In previous work, we succeeded in demonstrating the applicability of nES GEMMA for the analysis of liposomes, vesicles consisting of a lipid bilayer encapsulating an aqueous lumen. nES GEMMA enabled the characterization of vesicles in terms of size distribution, particle number concentration and the occurrence of smaller‐sized building blocks next to large vesicles. Also, offline hyphenation of gas‐phase electrophoresis with orthogonal analysis methods, for instance atomic force microscopy, MALDI mass spectrometry or spectroscopy was shown. In vivo, cell‐derived EVs, being part of cell/cell communication, are comparable to liposomes. They are envisioned as pharmaceutical cargo transporters, explaining the need for their characterization. We succeeded in porting gas‐phase electrophoresis of liposomes to the characterization of EVs, demonstrating protein co‐purification and loss of EV stability upon further polishing of vesicle preparations. Our nES GEMMA results were compared to data from NTA.

Summary/Conclusion : We demonstrate gas‐phase electrophoresis relating additional characteristics for EV containing samples in comparison to prevailing techniques like, for example, NTA.

Standardization of data analysis for investigating extracellular vesicles in plasma utilizing high‐resolution flow cytometry with medium angle light scatter detection

Rikke W. Rasmussen 1 , Rikke B. Eschen 1 , Morten H. Nielsen 1 , Rikke Bæk 2 , Malene M. Jørgensen 2,3 , Aase Handberg 1,3 , Maiken Mellergaard 1,3

1 Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark; 2 Department Of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark; 3 Department Of Clinical Medicine, Aalborg University, Aalborg, Denmark

Introduction : High‐resolution flow cytometry (hFCM) is highly efficient for characterization of phenotype on a single‐EV level. Detailed protocols are required from the collection of samples to data analysis and publication of reliable and translatable results. To document the credibility of data, relevant controls are required (MISEV 2023). However, hFCM analysis is complex and results are affected by the optical and physical configuration of the applied instrument as well as scatter characteristics of a given sample. To be able to reproduce and compare hFCM data between laboratories, calibration and conversion of signals such as arbitrary scatter signals to particle diameter are crucial. However, this is challenged by the variability in hFCM instrument properties and currently, no tools for size scatter calibration of medium angle light scatter (MALS) data are available. Finally, the risk of introducing bias in data analysis and gating strategy even within the same research group challenge the outcome.

Methods : We have optimized the analysis of EVs detected directly in platelet poor plasma samples by our hFCM instrument (Apogee A60) designed to include a MALS detector to increase sensitivity. We have used Rosetta calibration of data to convert particle scatter signals to size in nm to enable us to specifically report the concentration within a certain size range of particles (MIFlowCyt‐EV, 2020,). Moreover, we tested the robustness of our gating strategy and subsequent reporting of our findings by comparison of data analysis from the same study performed independently by two different researchers.

Results : We found that the limit of detection (LoD) is significantly lowered using MALS (∼110 nm) over LALS (∼250 nm) in hFCM and report how to perform size scatter calibration using Rosetta beads for data acquired by MALS analysis. Moreover, our data analysis test revealed coefficient of variations of 2.8%, 13%, and 10% for the gating and analysis of CD9+ EVs, CD81+ EVs, and CD9+CD81+ EVs, respectively.

Summary/Conclusion : Our data demonstrate the advantage of using MALS for EV studies and a need for proper tools for reporting calibration of LoD. Moreover, we report a robust data analysis strategy especially for the CD9+‐positive EVs.

Funding : This study was supported by the Novo Nordisk Foundation: NNF22OC0080036 and Research Cube AAUH 2022.

Precision measurements of size, refractive index and concentration of nanoparticles in polydispersions

Presenter: Anna Kashkanova

Max Planck Institute for the Science of Light, Erlangen, Germany

Introduction : Characterization of the size, material properties and concentration of particles in liquid suspensions is in high demand, for example, for the analysis of extracellular vesicles (EVs) in body fluids. However, existing methods are often limited in sensitivity and cannot handle a high degree of polydispersion.

Methods : We present interferometric nanoparticle tracking analysis (iNTA), which combines nanoparticle tracking analysis (NTA) with interferometric scattering (iSCAT) microscopy to achieve higher sensitivity than traditional NTA instruments. We show that iNTA can measure the size and refractive index of individual nanoparticles with unprecedented precision. Furthermore, we demonstrate that the concentrations of different subpopulations in polydispersions can be determined from the trajectory numbers.

Results : We benchmarked iNTA with synthetic nanoparticles and showed that gold nanoparticles as small as 10 nm can be measured. Afterwards we show that iNTA can extract the size and internal structure of liposomes and EVs. In addition, we demonstrate that iNTA can differentiate EVs from lipoproteins in blood plasma‐derived samples, which is not possible with conventional NTA. Finally, we show that iNTA can differentiate viruses from EVs in SARS‐CoV2 infected cell supernatants.

Summary/Conclusion : We have shown that iNTA is a useful tool not only for characterization of particle size and refractive index, but also for quantitative assessment of particle concentration, even when sizes overlap. This is very useful for the quantification of EVs where co‐isolates are often present in the samples.

Funding : This study was supported by the Max Planck Society, Bundesministerium für Bildung und Forschung, Alexander von Humboldt Foundation, Christiane Nüsslein‐Volhard Foundation.

PHoNUPS—plotting the histogram of non‐uniform particles' sizes, a program for the analysis of extracellular vesicles

Bence Mélykúti, Gonzalo Bustos‐Quevedo, Tony Prinz, Irina Nazarenko

Institute for Infection Prevention and Control, Medical Center—University of Freiburg, Freiburg, Germany

Introduction : The processing of size and zeta potential measurements of extracellular vesicles is plagued by the lack of an output standard shared by the measuring instruments. We envision software which can process measurement data in a high‐throughput fashion. It should compute basic statistics and should plot histograms in a standard but also adjustable way. It should be extensible with the data formats of additional measuring instruments.

Methods : We release the free/libre and open‐source software (FLOSS) PHoNUPS which can read multiple measurement data formats, which computes simple statistics of the input data and plots their histograms. The program PHoNUPS is in the programming language R.

Results : PHoNUPS is currently able to read data from the nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) instruments of a certain German manufacturer. PHoNUPS displays the statistics that the instruments do: total concentration, and mean, median, mode/peak of the particle size distribution. It processes multiple measurements in one run, potentially from different instruments. It plots their histograms both in one chart and in separate subcharts in a joint plot. The plots can be shown on screen, or they can be saved in a range of vector and raster graphics formats, such as .svg and .png. The vector graphics output enables further graphical editing in batch for the user, even for disparate input instruments.

Summary/Conclusion : PHoNUPS is made with extensibility in mind, and we call on the community to contribute the capability to parse further input formats.

Funding : This development was funded by the German Federal Ministry of Education and Research.

Standardizing extracellular vesicle analysis with CytoFLEX nano flow cytometer and FCMPASS software

Presenter: Jen Bon Lui

Beckman Coulter, Brea, California, USA

Introduction : Traditional flow cytometry often falls short in sensitivity and resolution for extracellular vesicle (EV) analysis. By converting scatter parameters from arbitrary to standardized units, researchers can better compare results across different cytometers, enhancing study interpretation and reproducibility.

Methods : The CytoFLEX nano flow cytometer paired with FCMPASS software offers a solution for the analysis EVs which are difficult to analyse due to their small size, heterogeneity, and low abundance. The CytoFLEX nano flow cytometer is equipped with a Violet Side Scatter (VSSC1) detector capable of detecting particles as small as 40 nm polystyrene NIST (National Institute of Standards and Technology) reference particles, and up to 1000 nm using the VSSC2 channel. This advanced detection capability is crucial for accurately analysing small particles like EVs. To facilitate cross‐platform comparisons, scatter data is calibrated to nanometres using Mie‐modelling software.

Results : Here we show a workflow where VSSC data is converted from arbitrary units into standardized units (nanometres) for particle analysis, using the CytoFLEX nano flow cytometer and FCMPASS software. Standardizing this data into nanometres using a ready‐to‐use, NIST‐traceable size standard mixture, the nanoViS Low and High Sizing Standards, significantly enhances the value and comparability of small particle research data.

Summary/Conclusion : This approach aligns with MISEV and MIFlowCyt‐EV guidelines, ensuring that research involving EVs and other submicron particles is repeatable, verifiable, and traceable.

EV shape classification using deep learning analysis based on scattered light intensity information obtained from nanoparticle tracking method

Takanori Ichiki 1,2 , Hiromi Kuramochi 1 , Yasushi Shibuta 1 and Naohiro Seo 1

1 The University of Tokyo, Tokyo, Japan; 2 Innovation Center of NanoMedicine (iCONM), Kawasaki, Japan

Introduction : Since EVs are a heterogeneous population in which each particle differs in size and zeta potential, it is necessary to analyse each particle separately when evaluating their properties. The nanoparticle tracking (NTA) method, which is currently widely used for single‐particle analysis of nanoparticles in liquids, is a method in which a laser beam is irradiated into a microfluidic channel, scattered light images from the nanoparticles are continuously recorded, and the particle size is calculated for each individual particle. We have further developed the NTA method to simultaneously measure particle size and zeta potential. Furthermore, we have attempted to construct a new analytical method that can determine the presence of non‐spherical particles by using machine learning for analysing Brownian motion. In a previous study, it was shown that particle shape classification is possible by deep learning analysis of the Brownian motion trajectory of particles. In this study, we focused on scattered light intensity information as a new feature to replace the Brownian motion trajectory and examined shape classification using deep learning analysis.

Methods : Four types of gold nanoparticles (two spherical: d = 40 nm, 80 nm; two rod‐shaped: d = 40 nm × L = 180 nm, d = 50 nm × L = 150 nm) were prepared and each was measured with an NTA system to obtain continuous images (100 frames/s). Based on this, the Brownian motion of each particle was tracked, the scattered light intensity around the obtained centre‐of‐gravity coordinates (100 × 100 pix 2 ) was integrated, and the rate of change in the time series was calculated to create a dataset for deep learning.

Results : Using a one‐dimensional convolutional (1D‐CNN) model, the “sphere‐bar” classification achieved a correct response rate of approximately 88% for the test data. The results also indicated that increasing the variation of particles to be trained may approach a more general shape classification model with less size dependence.

Summary/Conclusion : These results are expected to contribute to the development of a technological platform for the simple and high‐quality mass preparation of exosomes and promote the possibility of their clinical application.

Funding : The open innovation platform for industry‐academia co‐creation (COI‐NEXT) program supported by Japan Science and Technology Agency (Grant No. JPMJPF2202).

Multi‐parametric surface plasmon resonance—a label‐free new technology for EV corona and cell interaction studies

Abhishek Sharma, Justas Svirelis, Matias Jokinen, Annika Järvinen

BioNavis Ltd., Tampere, Tampere, Finland.

Introduction : Multi‐Parametric Surface Plasmon Resonance (MP‐SPR) is a label‐free technique for detecting and quantifying biochemical interactions, such as drug‐protein binding, in real time without labels like fluorophores or radiolabels. MP‐SPR collects the complete SPR angular spectrum, enabling analysis of parameters like thickness, refractive index, and optical dispersion. It is compatible with crude samples, including serum.

Methods : MP‐SPR instruments can be used to study extracellular vesicles (EVs) and their interactions with plasma or serum. When EVs interact with plasma, they can form a protein layer known as the EV protein corona, which might affect EV function [1]. MP‐SPR has been used to measure the thickness of this protein layer on nanoparticles [2]. Otto et al., for example, measured the ‘soft’ and ‘hard’ corona layers formed on EVs by plasma [2]. Lipophilic dyes (used to label EVs), can interfere with EV measurements and uptake studies [3,4], MP‐SPR enables label‐free study of EV interactions with mammalian cells [5–7].

Results : MP‐SPR, is also a valuable tool in EV diagnostic and therapeutic research, allowing precise measurement of binding kinetics between EVs and capture agents like antibodies or aptamers [8].

Summary/Conclusion : BioNavis's multi‐wavelength instruments enhance MP‐SPR's utility by enabling EV size and concentration measurements, especially for small EVs (< 100 nm) [9‐11]. These measurements leverage different evanescent fields produced by multiple wavelengths. BioNavis's regenerable avidin for fully reversible ligand immobilization tool for EV capture [12, 13], making it a powerful, versatile tool for EV and therapeutic research.

Funding : 1. Dietz et al. https://doi.org/10.1002/jev2.12399.2. Otto et al. https://doi.org/10.1007/s13346‐016‐0320‐0.3. Dominkus et al. https://doi.org/10.1016/j.bbamem.2018.03.013.4. Wan et al. https://doi.org/10.2147/IJN.S475060.5. Koponen et al. https://doi.org/10.1016/j.bios.2020.112510.6. Suutari et al. https://doi.org/10.1002/smLl.201601815.7. Scurti et al. https://doi.org/10.1021/acs.langmuir.3c00728.8. Carney et al. https://doi.org/10.1002/adbi.201600038.9. Parkkila et al. https://doi.org/10.1016/j.colsurfa.2022.130015.10. Norling et al. https://doi.org/10.1021/acs.langmuir.1c03096.11. Rupert et al. https://doi.org/10.1021/acs.analchem.6b01860.12. https://www.bionavis.com/en/publications/application‐notes/166‐regenerable‐avidin‐kit‐assay‐working‐principle‐1/13. https://www.bionavis.com/en/publications/application‐notes/168‐regenerable‐avidin‐kit‐assay‐concentration‐kinetics/

Role of the protein corona in modulating the mineralization capabilities of matrix vesicles

Juçara Gastaldi Cominal 1 , Larwsk Hayann 1 , Maryanne Trafanni Mello 1 , Luiz Henrique da Silva Andrilli 1,2 , Ana Paula Ramos 1 , Saida Mebarek 3 , Massimo Bottini 2,4 , José Luís Millán 2 , Pietro Ciancaglini 1

1 University of Sao Paulo—Faculty of Philosophy, Sciences and Letters at Ribeirao Preto, Chemistry Department, Ribeirao Preto, Brazil; 2 Sanford Burnham Prebys Medical Discovery Institute—Sanford Children's Health Research Center, La Jolla, California, USA; 3 Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR CNRS 5246, Université Claude Bernard Lyon 1, Villeurbanne, France; 4 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy

Introduction : Mineralizing cells release a special class of extracellular vesicles, known as matrix vesicles (MVs), with the unique property to bind to collagen fibres and contribute to mineralization. Following their release, MVs bind to the extracellular matrix (ECM), where their highly specialized enzymatic machinery facilitates the formation of seed minerals within the vesicles’ lumen, subsequently releasing the minerals onto the ECM. However, how MVs propagate minerals onto the collagenous ECM remains unclear. We address these questions by exploring the “protein corona” paradigm whereby nanoparticles entering a biological milieu become cloaked by a corona of soluble proteins modifying their biological functions.

Methods : Here, we isolated native MVs from the growth plates of chicken embryos. After removing the protein corona from the native MVs using high ionic strength buffer, we obtained shaved MVs. Reconstituted MVs were produced by incubating shaved MVs with the removed protein corona constituents. MVs were obtained by different ultracentrifugation steps and characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), scanning electron microscopy (SEM). The protein content was assessed by electrophoresis and proteomic analyses.

Results : Our results showed that both the removal and reconstitution of the protein corona significantly affect the biochemical and physicochemical properties of MVs, resulting in three well‐defined groups. Shaved MVs exhibited an increase in the activity of tissue non‐specific alkaline phosphatase (TNAP) and a decrease in mineral deposition compared to native MVs. Reconstituted MVs partially recovered these functions, showing a reduction of TNAP activity and mineral deposition compared to native MVs. Furthermore, changes in the protein corona affect MV ability to anchor to the collagenous ECM, which is crucial for initiating the propagation of the mineral phase within this organic matrix. Proteomic analyses revealed changes in the protein profile of the MVs resulting from the removal of the protein corona, indicating that shaved proteins were primarily related to external structural and ECM organization and catabolism.

Summary/Conclusion : These findings underscore the role of the protein corona in modulating the mineralization capabilities of MVs. Understanding these interactions could lead to new therapeutic strategies for enhancing bone repair and regeneration (https://academic.oup.com/jbmrplus/advance‐article/doi/10.1093/jbmrpl/ziae168/7931791).

Funding : FAPESP 2019/08568‐2; CNPq 305426/2021‐4.

Multi‐parametric surface plasmon resonance‐based sensing platforms for platelet derived extracellular vesicles characterization

Elena Scurti 1 , Johanna Puutio 1 , Martina Hanzlikova 1 , Iida Kähärä 2 , Elina Vuorimaa‐Laukkanen 2 , Saara Laitinen 3 , Pia Siljander 1 , Tapani Viitala 1,4

1 University of Helsinki, Helsinki, Finland, 2 Tampere University, Tampere, Finland; 3 Finnish Red Cross Blood Service, Helsinki, Finland; 4 Åbo Akademi University, Turku, Finland

Introduction : The multi‐parametric surface plasmon resonance (MP‐SPR) technique is a versatile optical real‐time label‐free detection technique that can be adapted into several different sensing platforms for characterizing the size and amount of extracellular vesicles (EVs), as well as for monitoring the interaction kinetics of EVs with cells and induction kinetics of EVs. Here we use platelet derived EVs (PLT EVs) to demonstrate these unique analysis platforms.

Methods : For the determination of particle size and concentration of differently prepared PLT EVs the MP‐SPR sensors were functionalized with biotinylated CD 41 for specific capture of PLT EVs. A two‐wavelength MP‐SPR analysis approach was used to determine the particle size and concentration of the captured EVs. Real‐time cell interactions of fluorescently labelled and non‐labelled PLT EVs were performed by seeding HeLa cells on fibronectin coated MP‐SPR sensors. Monitoring the induction of EVs from PLTs was achieved by immobilizing PLTs on MP‐SPR sensors and injecting an ionophore to stimulate PLT EV formation while simultaneously monitoring the MP‐SPR signal response in real time.

Results : Ionophore induced PLT EVs were efficiently captured by CD 41, while PLT EVs prepared by ultracentrifuging the supernatant of PLT preparations contained impurities that did not allow to efficiently capture EVs for further MP‐SPR analysis. The size of the captured ionophore induced PLT EVs determined through the MP‐SPR analysis were 76 ± 16 nm and the concentration ranged between 4 and 40 µg/mL depending on the particle amount used in the measurements. Cell interactions of non‐labelled PLT EVs with HeLa cells showed concentration dependent MP‐SPR responses, and labelling of the EVs did not significantly affect their interaction with HeLa cells. Stimulation of PLTs immobilized on MP‐SPR sensors with an ionophore induced clear MP‐SPR signal changes that suggest that MP‐SPR could be used to monitor EV formation kinetics in real‐time without using labels.

Summary/Conclusion : We envision that the MP‐SPR‐based analysis platforms presented here have a great potential to provide unprecedented information about EVs that is not accessible by any of the currently widely used methodologies in EV research.

Funding : Business Finland, Academy of Finland, The Finnish Research Impact Foundation and Blood Service Research Fund.

Back to basics: how should we measure EV protein concentration?

Heikki Saari 1,2 , Jacopo Zini 2 , Julia Monola 2 , Riina Harjumäki 2 , Saara Laitinen 1

1 Finnish Red Cross Blood Service, Helsinki/Vantaa, Finland; 2 University of Helsinki, Faculty of Pharmacy, Helsinki, Finland

Introduction : Protein quantification is an extremely basic part of biochemical research, including those regarding extracellular vesicles (EVs). Total protein concentration is a useful characteristic of EV samples, which can be used for normalizing samples for functional assays or molecular characterizations such as western blotting. However, since EVs are a complex, heterogeneous nanoparticles containing a mixture of proteins and other biomolecules that vary between different cell types, accurate protein quantification can be less straightforward than expected. As the exact protein composition of EVs cannot be provided, only nonspecific assays are applicable, with their inherent estimations and errors. In this study, selected colourimetric and spectroscopic methods were compared for assessing protein concentration from platelet‐ and PC‐3 prostate cancer EVs.

Methods : EVs were purified from expiring platelet concentrates from donated blood or PC‐3 conditioned cell culture medium by ultracentrifugation and size exclusion chromatography. The EVs were characterized by Nanoparticle Tracking Analysis (NTA), cryo‐TEM and western blotting. Compared protein concentration methods were UV absorbance (Nanodrop) with 205 nm, 280 nm or the Scopes method, and BCA assay with and without SDS. Purified egg ovalbumin was used as a known concentration reference protein.

Results : Based on the egg ovalbumin reference, the Scopes method gave the most accurate estimate for protein concentration. It is based on peptide bond absorbance at 205 nm, correcting for aromatic amino acid overlap. Compared to Scopes method, uncorrected A205 was naturally very close, but A280 significantly overestimated the EV protein concentration about 150% for platelet EVs and 240% for PC‐3 EVs. BCA assay on the other hand underestimated the protein concentrations to about 50% of the Scopes method, and addition of SDS slightly increased the estimate to about 60%.

Summary/Conclusion : In our study, we found that there is significant variation between EV protein quantification methods, which may either over‐ or underestimate the protein concentration in an EV source‐dependent fashion. We suggest that the Scopes method is the most reliable protein quantification method for EVs and recommend it for the EV community.

Funding : This study was supported by the Instruct‐ERIC R&R pilot funding #2729.

Exploring ATP as a mediator of chemoresistance in cancer: single‐particle detection of ATP‐EVs via nano‐flow cytometry

Xu Xiao, Cheng Lu, Chenxi Liu, Yunyun Hu, Xiaomei Yan

Xiamen University, Xiamen, China

Introduction : Chemotherapy remains the cornerstone in cancer treatment. However, its efficacy is often compromised by the development of drug resistance. A central mechanism of chemoresistance involves overexpression of ATP‐binding cassette (ABC) transporters that utilize ATP to actively expel chemotherapeutic drugs from cancer cells. Intriguingly, extracellular vesicles (EVs), key mediators of intercellular communication, have been found to contain ATP, potentially linked to the mechanism of chemoresistance. Yet, detecting ATP in single EVs and understanding the heterogeneity of ATP in EVs have not been reported. This study aims to develop a high‐throughput single‐particle technique using nano‐flow cytometry (nFCM) to detect ATP‐containing EVs (ATP‐EVs) and unveil their role in chemoresistance.

Methods : EVs were isolated from both drug‐sensitive and resistant cancer cell lines via differential ultracentrifugation and stained with ATP‐Red 1, a fluorescent probe specific to ATP. A laboratory‐built nFCM equipped with a 532‐nm laser was employed to analyse these ATP‐EVs. The effects of glycolysis and mitochondrial function inhibitors, as well as knockdown of ABC transporters, were assessed to determine their impact on the secretion and profile of ATP‐EVs. EVs were further fractioned through high‐resolution iodixanol density gradient centrifugation. Comprehensive molecular characterization (ATP, ABC transporters, and classic EV protein markers) was performed for EVs in each fraction using either the 532 nm laser equipped nFCM or a dual‐laser equipped nFCM (488 and 647 nm).

Results : Our approach successfully identified ATP within individual EVs and revealed that drug‐resistant cells secreted higher levels of ATP‐EVs compared to their sensitive counterparts. Mitochondrial inhibitors were more effective than glycolytic inhibitors in reducing ATP‐EV secretion. The suppression of ABC transporter expression significantly decreased the release of ATP‐EVs. Furthermore, ATP‐EVs exhibited similar density profiles to ABC transporter‐containing EVs, highlighting their potential role in drug efflux mechanisms.

Summary/Conclusion : This research introduces an advanced single‐particle detection method for ATP‐EVs, elucidating their augmented role in drug‐resistant cancer cells. Our findings suggest a novel mechanism by which ATP‐EVs may be associated with chemoresistance, providing insights that could lead to targeted therapeutic strategies against drug‐resistant cells.

Funding : This research was supported by the National Natural Science Foundation of China (Grants 21934004 and 32450337).

Comparative analysis of in‐house and commercial CD9/CD81 Fab‐streps for extracellular vesicle isolation

Syeda Menahil Fatima 1 , Jens Guðmundur Hjörleifsson 1 , Berglind Eva Benediktsdóttir 2

1 Faculty of Life‐and Environmental Sciences, University of Iceland, Iceland, Reykjavík, Iceland; 2 Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavík, Iceland

Introduction : Efficient extracellular vesicle (EV) isolation for downstream nanoparticle therapeutic applications requires a method that preserves EV size and activity. Immunoaffinity capture using commercial CD9 or CD81 Fab‐streps has previously been shown by our group to yield EVs with minimal protein contamination and preserved integrity. However, the commercial CD9 and CD81 Fab‐streps were discontinued by IBA as off January 2025. By developing in‐house Fab‐streps, costs could able further optimization of the isolation process for academic use.

Methods : Bl21(DE3) E. coli cells were transformed with the pET11a plasmid encoding codon optimized for CD9 or CD81 Fab‐strep ORFs, gifted by IBA‐Life Sciences, and cultured at either 18°C or 25°C in shake flasks. Cells were harvested, lysed and clarified for affinity purification using ÄKTA Pure 25 FPLC, with a Strep‐Tactin 4Flow column and desthiobiotin for elution. MEXi HEK293E cells were cultured at 37°C in serum‐free Mexi‐CM medium. For each experiment, 25 mL of conditioned medium (viability at least 90%) was collected for each EV isolation and clarified. EVs were isolated via immunoaffinity chromatography using Strep‐TactinTACS agarose with either commercial (IBA) or newly generated CD9 or CD81 Fab‐strep (0.05 mg/mL). After biotin elution and concentration (100 kDa), EVs were analysed using NanoSight, CL 15, pump speed 100.

Results : In‐house CD9 and CD81 Fab‐strep were achieved in concentrations of 5–20 mg/L LB culture, with > 95% purity, MW of ∼48 kDa as measured with SEC. CD9 expression was 4–5× greater than CD81. Preliminary EV isolation using in‐house CD81 Fab‐strep yielded 1.76 ± 0.08 × 10 11 particles/mL while commercial Fab‐strep yielded 4.44 ± 0.4×10 11 particles/mL (2.5× higher). EVs from in‐house CD9 Fab‐strep reached 1.48 ± 0.05 × 10 11 particles/mL compared to 2.23 ± 0.05 × 10 11 particles/mL using commercial Fab‐strep (1.5× higher). EVs isolated with commercial Fab‐Strep had a mean size of 128.5 ± 1.0 nm (CD81) and 137.5 ± 0.8 nm (CD9), while those obtained with in‐house Fab‐Steps were somewhat smaller or 103.6 ± 1.2 nm (CD81) and 133.3 ± 1.2 nm (CD9).

Summary/Conclusion : While in‐house CD9 or CD81 Fab‐streps effectively isolated EVs, initial tests yielded lower values than commercial alternatives. Further optimization and characterization of the purity and activity of the Fab‐streps is needed to improve efficiency.

Funding : This study was supported by the Icelandic Cancer Institute Science Foundation, the National Breast Cancer Awareness Fund (Göngum Saman), and STAFN Grant.

Exploring cell extrusion as a scalable method for large‐scale production of functional cell‐derived vesicles (CDVs)

Negar Sedghi Aminabad 1 , Antri Stefani 1,4 , Pauline Ley 1 , Nicole Maeding 1,4 , Rodolphe Poupardin 1 , Thomas Heuser 2 , Samir El Andaloussi 3 , Martin Wolf 1,4 , Dirk Strunk 1,4

1 Institute of Experimental and Clinical Cell Therapy, Paracelsus Medical University (PMU), Salzburg, Austria; 2 Vienna Biocenter Core Facilities, Vienna, Austria; 3 Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden; 4 Austrian Red Cross Research, Vienna, Austria

Introduction : Extracellular vesicles (EVs) hold great promise in therapeutic and diagnostic applications. Extrusion is well‐established for creating liposomes and offers certain advantages for artificial cell‐derived vesicle (ACDV) manufacture. Here, we explored cell extrusion as an alternative time‐saving large‐scale ACDV production method.

Methods : We extruded fluorescent HEK CD63‐mNeon or HEK GFP, at 0.25 and 1 × 10⁶ cells/mL, through 0.8 and 5 µm pore size membranes, using syringe pumps at varying flow rates to investigate effects of variable parameters on nano‐vesicle quantity and quality. ACDVs were characterized using tunable resistance pulse‐sensing (TRPS) to measure size distribution and concentration. In parallel, we isolated EVs from culture supernatants of HEK‐GFP cells to compare natural EVs with ACDVs.

Results : We validated presence of typical EV markers via dot blot. ACDVs produced from HEK‐GFP had larger mode size compared to EVs (mean 180 ± 4.5 vs. 140 ± 1.6 nm) but production yield was six‐times higher for ACDVs compared to EVs (1488 vs. 236 particles/cell). Size distribution of ACDVs from HEK‐GFP was more heterogeneous compared to EVs with bigger mean mode‐size (242 ± 4.1 vs. 161 ± 1.7 nm). Extrusion cycles (1–3–9) did not change ACDV size and concentration. Increasing flow rate from 10 to 20 mL/min significantly increased ACDV mode size from 111 ± 4.5 to 134 ± 2.6 nm. Sequential extrusion of HEK CD63‐mNeon with reducing membrane pore size from 5 to 0.8 µm, with extrusion cycles 3‐9, respectively, further decreased increased ACDV mode size (190 to 110 ± 4.96 to 190 ± 3.4 nm). ACDV production by cell extrusion resulted in > 6× vesicle quantity compared to EV production via collecting conditioned medium from the same cell lines. Further data on EV/ACDV quality will be reported. The most influential parameter for ACDV production output was initial cell input concentration.

Summary/Conclusion : Increase in yield positions cell extrusion as a candidate for large‐scale vesicle manufacturing. Remaining challenges like polycarbonate membrane clogging and loading quantity need to be further optimized for individual donor cell types.

Comparative analysis of techniques for quantifying EV release from hydrogel scaffolds

Hannah Aris 1 , Pamina Contreras Kallens 1 , Yeyu Shen 1 , Andrew Daly 1 , Xandra O. Breakefield 2 , Garry D. Duffy 1 , Meadhbh Á. Brennan 1

1 University of Galway, Galway, Ireland; 2 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) offer tremendous potential in tissue repair as acellular alternatives to MSC therapies due to their regenerative properties. Numerous studies show EVs administered to injury sites via biomaterials are retained longer than those delivered via saline solution, facilitating greater therapeutic impact. As such, researchers are implementing intrinsic and modified hydrogel properties to generate scaffolds with sustained EV release. Various methods are employed during hydrogel development to quantify EVs as they are released from scaffolds to understand the EV‐retention capacity of the gels. However, no work has been conducted to assess if hydrogels interfere with EV quantification, potentially limiting the accuracy of hydrogel characterization. Here, we have assessed the interference of three hydrogels commonly used for EV delivery on multiple EV quantification techniques.

Methods : Hydrogels were cross‐linked to the bottom of well‐plates and bathed in double‐filtered PBS. At various time points, the hydrogel‐conditioned PBS (HG‐CPBS) was collected. Naïve, fluorescent, or bioluminescent/fluorescent EVs were obtained from murine bone marrow‐derived MSCs, the latter two EV types from cells transduced with genes encoding for palmitoylated tandem dimer Tomato (PalmtdTomato) or palmitoylated GFP‐nanoluciferase bioluminescence resonance energy transfer reporter (PalmGRET) for EV membrane labelling. EVs were isolated via differential centrifugation followed by size exclusion chromatography. EVs were serially diluted in HG‐CPBS and PBS for quantitative assessment by Nanoparticle Tracking Analysis (NTA), Bicinchoninic Acid (protein) assay (BCA), phospholipid assay, RNA quantification, CD9 ExoELISA, and fluorescence/bioluminescence measurements of PalmtdTomato or PalmGRET EVs.

Results : Results suggest hydrogels interfere considerably with NTA, BCA, ExoELISA, and RNA measurements, and to a lesser but noteworthy extent with phospholipid quantification, making these methods unreliable in this context. Fluorescence measurements of PalmtdTomato EVs offer accurate quantification. Bioluminescent and fluorescent measurements of PalmGRET EVs are being investigated.

Summary/Conclusion : Based on these results, it is recommended that fluorescent EVs are used for accurate EV quantification in the presence of hydrogels. It is essential to develop an accurate depiction of EV release in the development of EV carriers with optimized release profiles for tissue regeneration, and precise EV quantification is central to achieving this goal.

Funding : This study was supported by the Science Foundation Ireland Starting Investigator Research Grant.

Swarm detection can be your friend: rapid estimation of fluorescent fraction of stained exosome samples via flow cytometry

Presenter: Alan Chin

Kinetic River Corp., Boston, Massachusetts, USA

Introduction : Determining the fluorescent fraction of stained exosomes can be a relatively cumbersome task, requiring measurements of the total exosome concentration, the fluorescently tagged exosome concentration, and the background concentration along with processing of the data involving subtracting the background concentration from the total exosome concentration to accurately determine the fluorescent fraction of stained exosomes to total exosome population. Although swarm detection is now widely regarded as a situation to avoid when using flow cytometry to study exosomes, it is only necessary to avoid swarm detection if one requires single‐particle information. Because the fluorescent fraction (while obtainable via single‐particle statistics) is a bulk sample measurement (e.g., using nanoparticle tracking analysis), it is possible to use swarm detection to increase the scatter and fluorescence signals and obtain a rapid, accurate estimate of the fluorescent fraction.

Methods : We utilized a custom‐built flow cytometer to perform measurements on samples with concentrations up into the swarm regime. We also utilized a nanoparticle tracking analyser to characterize each sample to obtain a baseline concentration as well as a measurement of the fluorescent fraction for each sample. Titrations were prepared ranging from the single‐particle regime to the swarm regime.

Results : We examined fluorescent bead samples (40‐nm yellow‐green, 75‐nm MD655) with size and/or fluorescence below the detection limit of a flow cytometer. By increasing the concentration of the bead samples, we were able to confirm that both the scatter and fluorescence signals increase in concert to yield a rapid fluorescent fraction estimation without background subtraction. A fluorescent fraction of 100% can be achieved if an additional step with background subtraction is performed. We utilized the same procedure to measure the fluorescent fraction of fluorescently labelled exosomes (HEK293‐derived CD63‐EGFP) and were able to achieve a fluorescent fraction that matches the specified value.

Summary/Conclusion : Our results demonstrate that swarm detection in flow cytometry can be a useful tool for rapidly and accurately determining the fluorescent fraction of exosomes.

Funding : This work was made possible in part by US government support under award number 1R43GM155492‐01 from the NIH.

Quantifying EV‐based communication in the TME: a novel flow cytometry approach to track extracellular vesicle dynamics

Timea Boroczky 1,2,3 , Maria Harmati 1 , Matyas Bukva 1,2 , Gabriella Dobra 1,2 , Edina Sebestyen‐Gyukity 1,2 , Edit Kotogany 4 , Gabor Szebeni 4 , Peter Horvath 1 , Krisztina Buzas 1,2

1 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, HUN‐REN Biological Research Centre, Szeged, Hungary; 2 Department of Immunology, University of Szeged, Szeged, Hungary; 3 Doctoral School of Interdisciplinary Medicine, University of Szeged, Szeged, Hungary; 4 Laboratory of Functional Genomics, HUN‐REN Biological Research Centre, Szeged, Hungary

Introduction : Tumour progression and metastasis are strongly influenced by the intercellular communication between tumour cells and the surrounding tumour microenvironment (TME), with extracellular vesicles (EVs) as key players. EVs carry bioactive molecules that can alter the recipient cells’ behaviour, supporting tumour development. However, a comprehensive understanding of EV dynamics—including both release and uptake by various cell types—remains challenging due to EV heterogeneity and the complex interactions within the TME. Therefore, our goal was to develop a robust, flow cytometry‐based method to quantitatively measure EV dynamics across diverse cell populations in the TME.

Methods : To track vesicle release and uptake with precision, EVs were labelled within the donor cells rather than isolating and staining them, preventing artificially high EV concentrations. Tumour and stromal cells were stained with different DiO and DiD dyes respectively, causing each cell to produce vesicles in its corresponding colour. After one day, media were swapped allowing DiO vesicle‐producing cells to take up DiD vesicles, and vice versa. Using flow cytometry, EV dynamics were analysed measuring the uptake and release of dyed vesicles. Vesicle size, concentration and surface markers were also assessed by nanoparticle tracking analysis and flow cytometry. To quantify the EV uptake‐release balance over time, ‘EV‐rate’ was developed. This reproducible and quantifiable measure is suitable for comparing EV dynamics across different cell types, including fibroblasts, endothelial cells, and cancer cells. Moreover, to mimic chemotherapeutic stress, low‐dose doxorubicin was used, and the samples were compared based on EV‐rate.

Results : Our data show that the EV‐rate was similar across cell types, indicating balanced uptake and release, although different cell types maintained different EV concentrations. Furthermore, doxorubicin treatment induced a notable shift in EV‐rate, with treated tumour cells showing increased EV production. These findings suggest that chemotherapy may modulate EV‐mediated communication, potentially impacting therapeutic resistance.

Summary/Conclusion : This study presents a novel, quantifiable approach, the EV‐rate, to assess EV uptake and release in the TME through flow cytometry, providing a valuable tool for investigating intercellular communication in cancer. Our approach holds promise for future research on targeted EV‐based cancer therapies.

Funding : This study was supported by the TKP‐EGA‐09; OTKA‐K143255, EKÖP‐24‐3; MEC_R_149867.

Optimizing isolation of gingival fibroblast‐derived extracellular vesicles for enhanced wound healing

Presenter: Paula Horta Hugues

King's College London, London, UK

Introduction : The skin and oral cavity both undergo the same wound healing process, yet the oral mucosa heals without scarring, with reduced inflammation and faster re‐epithelialization. These differences may arise from cell‐intrinsic fibroblast properties. Extracellular vesicles (EVs), which transport biomolecules like DNA, RNA, lipids, and proteins, have been increasingly studied for their regenerative potential. While mesenchymal stem cell‐derived EVs have been shown to promote wound healing, invasive isolation methods and the wound‐healing relevance of fibroblasts suggest the need to investigate fibroblast‐derived EVs. We aim to explore the role of gingival fibroblast‐derived EVs in wound healing and compare transcriptomic and proteomic profiles with dermal fibroblast‐derived EVs. Here, we optimize isolation conditions and techniques to develop protocols for both cell types.

Methods : Primary gingival fibroblasts from patients were cultured under three conditions (serum‐free, 10% ultralow endotoxin FBS, and 2% Ultroser G) for 24 h following a 10% FBS culture period. Proliferation and morphology were monitored for 72 h using CellCytex. EVs were isolated by ultracentrifugation and ExoQuick‐TC, following the manufacturer's protocol. EVs were characterized by Exoview R200 and NanoSight LM10.

Results : Serum‐free conditions showed no growth after 72 hs, while cells in 10% FBS, 2% Ultroser G, and ultralow endotoxin FBS reached 35%, 28%, and 30% confluency, respectively, with no morphological changes. ExoQuick‐TC yielded 4.4 times higher protein concentration than ultracentrifugation. Exoview R200 results indicated a low particle range, suggesting subpopulations may be lost with these purification methods. In unpurified serum‐starved samples, CD81 was the most abundant exosome marker (44%), followed by CD63 (33.7%) and CD9 (22.3%). Using NanoSight, the mean particle size from ExoQuick‐TC was 202.3 nm with a concentration of 4.942 × 10 9 particles/mL, whereas ultracentrifugation yielded 361 nm particles at 3.4 × 10 8 particles/mL.

Summary/Conclusion : Low endotoxin FBS and Ultroser G conditions promoted greater cell growth for EV isolation. ExoQuick‐TC provided faster, higher‐concentration particle yields compared to ultracentrifugation, with more precise purification. These optimized conditions will be applied to purify EVs from dermal fibroblasts in future stages.

Disease and therapy

Subtopic Biologic Activity and therapeutic potency of EVs 2

Optimization of extracellular vesicles‐based therapy in the treatment of ischaemic stroke

Presenter: Ons Ben Hadj Hassen

Université de Caen‐Normandie, Caen, France

Introduction : Ischaemic stroke remains a major health problem. Extracellular vesicles (EV), derived from mesenchymal stem cells (MSC), have been shown to mitigate brain damage and improve neurorepair and neurological outcome following ischaemic stroke. Though promising, EV production and their route of delivery remain a major challenge. Here, the effects of EV generated by an innovative turbulence approach were characterized on stroke‐induced brain lesion and neurological deficits in the rat.

Methods : EV were isolated from human adipocyte tissue stromal cells (hASC) using a patented high‐yield turbulence bioproduction process. EV were quantified and characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), ExoView and proteomic analyses. Rats were subjected to a transient occlusion (1 h) of the middle cerebral artery (tMCAO) and treated with EV at reperfusion time (1010 EV/rat; I.A.) or 24 h later (1011 EV/rat; I.V.). MRI were performed at 1 and 14 days after tMCAO to quantify brain lesion volume. Behavioural tests were carried out up to 40 days post‐tMCAO.

Results : NTA, TEM and proteomic analyses confirmed EV characteristics (size, shape, tetraspanin expressions). While I.V. EV administration showed limited therapeutic effects. I.A. EV delivery at reperfusion time reduced the lesion volume 24 h post‐tMCAo compared to the control group ( p < 0.05). Moreover, 7 days after tMCAO, rats treated with EV immediately after reperfusion tend to improve their neurological score ( p = 0.07) and showed a significantly better limb placing test performance ( p < 0.05). In addition, these rats were less lateralized compared to the control ones in the corner test and performed better in the adhesive test 30 days after tMCAO ( p < 0.01). In contrast, I.V. EV administration 24 h post‐tMCAO showed only a significant effect on spatial memory ( p < 0.05, t ‐test). Immuno‐histological analyses of brain tissues are ongoing to investigate post‐ischaemic cellular effects.

Summary/Conclusion : These results suggest that (1) MSC‐derived EV produced by an original turbulence method are efficacious on stroke‐induced brain lesion and neurological deficits and (2) optimizing the administration route and timing enhances the efficacy of EV after stroke. Overall, the results showed that MSC‐derived EV produced by an original turbulence method are promising as an efficient therapeutic intervention in ischaemic stroke.

Small extracellular vesicles released by cardiac fibroblasts from hypertrophic cardiomyopathy patients alter calcium transients and contribute to hypertrophy of human cardiomyocytes

Presenter: Rahul Sanwlani

University of Surrey, Guildford, UK

Introduction : Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic cardiomyopathy. However, the role of cardiac fibroblasts (CFs) in HCM is still not completely understood. Previously, we reported paracrine signalling mediated regulation of cardiomyocyte electrophysiology by CFs. The role of extracellular vesicles (EVs) has been increasingly highlighted in cardiovascular pathophysiology. In this study, we investigated the role of HCM CF EVs in regulation of cardiomyocyte electrophysiology.

Methods : Primary CFs were isolated from HCM patient biopsies. HCM and healthy donor CFs were treated with TGFβ receptor inhibitor SD‐208 and characterised with RT‐qPCR and flow cytometry. CF EVs were isolated with size exclusion chromatography (SEC) and characterised using MISEV 2023 guidelines. EVs were used to treat human iPSC derived cardiomyocytes. Effects of EVs on cardiomyocyte electrophysiology were characterised by measuring changes in contractility, calcium transients and action potential. Proteomics and RNA sequencing were performed to understand the molecular effects of EV treatment.

Results : SD‐208 exhibited anti‐fibrotic effects in HCM CFs as suggested by significant reduction in mRNA and protein levels of fibroblast activation markers ACTA2, POSTN, FAP, COL1A1, IL‐6 and IL‐11 ( p < 0.0001), significant reduction in α‐SMA positive cells ( p = 0.0043) in flow cytometry and reduction in collagen secretion ( p = 0.00062) followed by treatment. CF EVs collected using SEC were observed to be in the small EV range. Interestingly, SD‐208 treatment led to significant (6.5‐fold) reduction in EV particles secreted by HCM CFs ( p < 0.0001). HCM CF EVs altered cardiomyocyte electrophysiology, leading to increased spontaneous beating frequency and metabolic potential but shorter calcium time to peak and decay. EV treatment also imparted hypertrophic phenotype to cardiomyocytes as suggested by significantly higher cell size and nucleation. Molecular characterisation with RT‐qPCR, RNA sequencing and proteomics revealed an increased expression of hypertrophy markers and calcium ion channels in cardiomyocytes following HCM CF EV treatment.

Summary/Conclusion : Overall, these findings demonstrate antifibrotic activity of SD‐208 in HCM CFs and its potential role as regulator of EV biogenesis and secretion. Furthermore, the results in this investigation highlight the novel role and regulatory mechanism of HCM CF EVs in cardiomyocytes with potential implications in disease prognosis and potential therapeutic utility in HCM treatment approaches.

Subtopic Cancer progression and metastasis 2

Multiple myeloma plasma EV protein signatures for monitoring patient response to daratumumab

Kieran Brennan 1 , Katrine F Iversen 2 , Alfonso Blanco‐Fernández 3 , Thomas Lund 4 , Torben Plesner 2 , Margaret M Mc Gee 1

1 School of Biomolecular & Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Dublin, Ireland; 2 Department of Internal Medicine, Section of Hematology, Lillebaelt Hospital, and Institute of Regional Health Science, University Hospital of Southern Denmark, Vejle, Denmark; 3 Flow Cytometry Core Technology, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Belfield, Dublin, Ireland; 4 Department of Hematology, Odense University Hospital, Odense, Denmark

Introduction : Our previously published data revealed that peripheral blood plasma (PB) EVs, isolated by differential centrifugation and density gradient ultracentrifugation, from 57 multiple myeloma (MM) patients treated with daratumumab (DARA) contain elevated CD55, CD59 and CD147 relative to healthy PB EVs ( n = 12), with elevated PD‐L1 associated with patient response to DARA (Brennan et al. 2022, PMID: 36359760). The aim of this study was to determine if a mass spectrometry could identify additional proteins altered in these MM patients and if these proteins could be combined with the proteins from our previous study in order to generate two multiple myeloma plasma EV protein signatures for 1, detecting Myeloma, and 2 monitoring patient response to Daratumumab.

Methods : A shortlist of 8 additional proteins upregulated in MM patients or associated with patient response to DARA were identified by mass spectrometry and validated by flow cytometry analysis.

Results : Flow cytometry analysis revealed that all 8 proteins were present in all EV samples with 3 of the 8 proteins being significantly elevated in MM PB EVs relative to healthy PB EVs, while 2 of the 8 proteins were significantly decreased. Since the original study was performed, there is now 2+ years additional follow‐up on these patients, which allowed these patients to be subdivided into long‐term responders (> 2 years), short‐term responders (6–24 months) and non‐responder (< 6 months). Three proteins were significantly higher in PB EVs of patients with a long‐term response to DARA compared to the PB EVs of non‐responders and short‐term responders. PCA analysis revealed a distinct separation between healthy PB EVs from MM PB EVs, as well as separating long‐term responders from both short‐term responders and non‐responders. Multivariate ROC curves revealed that the MM PB EV signature has an area under curve (AUC) of 0.92 (Sens: 0.86/Spec: 0.92), while a long‐term response signature has an AUC of 0.733 (Sens: 0.80/Spec: 0.91).

Summary/Conclusion : In conclusion we identified two EV signatures that may have potential as a non‐invasive liquid biopsy to complement or replace invasive Bone Marrow sampling for monitoring patient response to DARA.

Funding : This study was supported by Research Ireland RD&I Fellowship (23/IRDIFB/12112) and the TwinFlag consortium (HORIZON‐WIDERA‐2021‐ACCESS‐03‐01).

EV total RNA offers a dual perspective on prostate cancer patients’ prognosis through tumour and immune system cell profiles

Federico Vannuccini 1 , Yari Ciani 1 , Vera Mugoni 1 , Orsetta Quaini 1 , Caterina Nardella 1 , Francesco Orlando 1 , Veronica Weber 1 , Alessia Marinelli 1 , Irene Casanova‐Salas 2,3 , Fabio Pessina 4 , Alessandro Vannini 4 , Ugo De Giorgi 5 , Orazio Caffo 6 , Consuelo Buttigliero 7 , Umberto Basso 8 , Joaquin Mateo 2,3 , Gerhardt Attard 9,10 , Francesca Demichelis 1

1 Computational and Functional Oncology Laboratory; University of Trento, Trento, Italy; 2 Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain; 3 Vall d'Hebron University Hospital, Barcelona, Spain; 4 Structural biology research centre, Human Technopole, Milan, Italy; 5 Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori”‐ IRST, Meldola, Italy; 6 Department of Medical Oncology, Santa Chiara Hospital Trento, Trento, Italy; 7 Department of Oncology, University of Torino, Torino, Italy; 8 IOV‐Istituto Oncologico Veneto, Padova, Italy; 9 Department of Medical Oncology, University College London Hospitals, London, UK; 10 UCL Cancer Institute, University College London, London, UK

Introduction : Liquid biopsies offer a minimally invasive complement to tissue biopsies for monitoring cancer by capturing multiple molecular components circulating in body fluids, including cell‐free (cfDNA) and extracellular vesicles (EVs). Plasma‐derived coding and non‐coding EV‐ associated RNAs (EV‐RNA) offer insights into EV cellular origin, informing about disease status and ongoing cellular processes.

Methods : In the framework of the multi‐institutional research program PRIME for the development of multi‐modal liquid‐biopsy assays for prostate cancer, we characterized plasma‐derived EVs and the EV‐total RNA cargo from longitudinal samples from 54 metastatic castration‐resistant prostate cancer (mCRPC) patients treated with an Androgen Receptor signalling inhibitor. Plasma samples were processed using ONCE, an in‐house developed approach for isolating EVs and cfDNA from a single plasma aliquot. EVs were isolated through ultracentrifugation, RNA libraries were obtained using the Takara Bio SMARTer smRNA‐Seq kit and then sequenced on NovaSeq 6000 with an SR150 protocol. cfDNA was profiled using the targeted PCF_SELECT assay.

Results : We identified differences in EV physical properties and RNA cargo when stratifying patients based on prognosis. Worse prognosis patient plasmas contain larger EVs than better prognosis patients and healthy donors (HD; median diameter of 189, 158, and 119 nm, respectively) and exhibit stronger evidence of prostate‐related transcriptional signals. EV‐RNA cargo deconvolution indicates i) enrichment for epithelial cells and prostate luminal KLK3+ cells signal in patients versus HDs and ii) marked differences in immune cells contribution based on patients’ prognosis. Specifically, polarized Macrophages and prostate‐related EV components are associated with prognosis ( p value < 0.0001 for both) and are independent prognostic factors when accounting for circulating tumour DNA signal. Coherent trends are observed in a second mCRPC cohort ( n = 21).

Summary/Conclusion : EV‐RNA profiling has the potential to contribute to mCRPC prognostic assessments by revealing tumour and immune cell components associated with patient prognosis.

Funding : Accelerator Award 20218 funded by Cancer Research UK (A26822) and Fondazione AIRC per la ricerca sul cancro ETS (22792).

Small extracellular vesicles from triple‐negative breast cancer cells increase angiogenesis onto chicken and quail chorioallantoic membrane (CAM) assay

Leticia A. Fernandes 1 , Ana Claudia O. Carreira 1,2

1 University of São Paulo, São Paulo, Brazil; 2 Federal University of ABC, Santo André, Brazil

Introduction : Angiogenesis is a crucial physiological process for tissue homeostasis and is directly involved in tumourigenesis and progression. Different molecules are involved in the angiogenesis process, such as small extracellular vesicles (sEVs) secreted by cells. Animal models are essential for uncovering their regulatory mechanisms and answering key biological questions when investigating angiogenesis. However, aiming to replace animal models, the chick embryo chorioallantoic membrane (CAM) assay surges as one alternative method for studying angiogenesis and the action of EVs.

Methods : sEVs were collected from malignant triple‐negative breast cancer cells (Hs578T) in 2D (106 cells) or 3D (1.6 × 10 5 ) cultures in DMEM media sEV without foetal bovine serum. sEVs were isolated from 24 h of conditioned media using PEG precipitation technique. Total protein was isolated with RIPA lysis buffer plus protease inhibitor and quantified using the Bradford Protein Assay. Chicken and Quail embryonated eggs were incubated at 37.5°C under a humid atmosphere for 3 days. Then, the chorioallantoic membranes were exposed under sterile conditions. CAM was stimulated with sEVs total protein (15 µg) using a space‐temporal assay at 7 days and investigated following 24 and 48 h. It was further analysed for 72 and 96 h to complement the data. CAMs were collected and analysed through histology (H&E, Picrosirius Red, Fuchsin‐Resorcin staining), immunohistochemistry (Collagens I, III, V, Laminin, CD31), and scanning electron microscopy (SEM). The experiments were performed in triplicate.

Results : Over the time series, CAM membranes stimulated with sEVs showed larger caliber vessels, sprouting, and increased blood vessels compared to the unstimulated control. Additionally, higher labelling of collagen I, III, and CD31 was detected.

Summary/Conclusion : These results demonstrate that the Chicken and Quail CAM assays are viable and efficient alternative methods for studying angiogenesis and tumour progression. Compared to other animal models, it involves lower costs and less labour.

Funding : This work was supported by the Coordination for Improvement of Higher Education Personnel (CAPES).

Subtopic EV biomarker development 2

Small extracellular vesicles carry differential miRNA cargo in progressive myotonic dystrophy type 1 patients

Presenter: Andrie Koutsoulidou

The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

Introduction : Myotonic Dystrophy Type 1 (DM1) is a hereditary disorder and the most common adult‐onset muscular dystrophy worldwide. DM1 hallmark symptoms are myotonia, muscle weakness, and wasting which steadily worsen over time. We previously reported that small extracellular vesicles (EVs) isolated from serum samples of DM1 patients contain elevated levels of muscle‐specific miRNAs, miR‐1, miR‐133a, miR‐133b, and miR‐206, compared to healthy participants. Importantly, the levels of the four muscle‐specific miRNAs encapsulated within small EVs were found to be associated with the progression of muscle wasting in DM1 patients. Moreover, we showed that these miRNAs were increased in EVs circulating in the serum of a DM1 animal model. In this study, we aimed to investigate miRNAs in small EVs that could potentially differentiate between stable and progressive DM1 patients.

Methods : Serum samples from DM1 patients were obtained from the ‘PhenoDM1’ study. Patients were categorized as progressive or stable at the time of blood collection based on their outcome measures. Small EVs were isolated using size‐exclusion chromatography method. EV characterisation was confirmed with TEM imaging and small‐particle flow cytometry. Total RNA, including small RNA, was extracted from EV samples followed by transcriptomics using small RNA next‐generation sequencing (NGS). Bioinformatics analysis allowed for the identification of deregulated miRNA cargo encapsulated in EVs. The best candidate miRNAs were selected for validation using Real‐Time PCR.

Results : A total of 69 progressive and stable DM1 and healthy serum samples were used for small EV isolation followed by small RNA‐NGS. The total miRNA population was identified and analysed with bioinformatics tools. Heatmaps between progressive and stable DM1 patients and healthy controls were used to assess the expression signatures of miRNAs within small EVs. Several novel EV‐miRNAs were detected showing altered levels and were associated with the disease progression. Validation of the top miRNAs was performed with RT‐qPCR.

Summary/Conclusion : This study suggests that circulating EVs encapsulate differential miRNA profile between stable and progressive DM1 patients and healthy individuals. This strongly highlights the promising use of EVs and their miRNA content as biomarkers for disease progression in patients with DM1.

Funding : This work was funded by AFM Téléthon France and A. G. Leventis Foundation.

Evaluation of plasma‐ and CSF‐derived extracellular vesicles as biomarkers for post‐COVID

Hanna Reßin 1 , Shreyans Chatterjee 1 , Deborah Erhart 2 , Hayrettin Tumani 2,3 , Jan Münch 1 , Rüdiger Groß 1

1 Ulm University Medical Centre, Ulm, Germany; 2 University and Rehabilitation Clinics Ulm, Ulm, Germany; 3 German Centre for Neurodegenerative Diseases (DZNE) Site Ulm, Ulm, Germany

Introduction : Extracellular vesicles (EVs) are produced by all cells and contain biomolecules that give clues on the state of the producer cell. Acute infections with SARS‐CoV‐2 are associated with increased EV release in plasma and increased pro‐coagulative properties of these EVs. We here investigated whether patients with persistent fatigue (range 1.0–102.0 months) after infection (post‐COVID, PC) also show a characteristic EV profile in serum or CSF, which could be used for diagnostics and give clues on disease aetiology.

Methods : Plasma and cerebrospinal fluid (CSF) samples were collected from patients presenting to the Ulm University Hospital, Dept. of Neurology, Post‐COVID out‐patient clinic, pre‐cleared and stored at −80°C. Reconvalescent (RC) patients with a history of COVID‐19 but no persistent symptoms were used as a control cohort. For all samples, nanoparticle tracking analysis (NTA) was used to determine particle concentration and particle diameter. Bead‐assisted flow cytometry was used to analyse EV surface epitopes using detection of tetraspanin (TSPN) (αCD9, αCD63, αCD81) and phosphatidylserine (PS) (lactadherin).

Results : Biophysical parameters (particle concentration and size) were not significantly altered between the PC and RC cohorts for plasma or CSF. Bead‐based flow cytometry showed significant elevation of CD142+ and CD105+ vesicles in CSF of PC patients, hinting at endothelial damage. In plasma, PS‐based detection of vesicles revealed upregulation of several proteins involved in immune activation, including CD25, CD29, CD56 and HLA‐DRDPDQ. Upregulated HLA‐DRDPDQ was also detectable on EVs in the CSF of the PC cohort, suggesting an overall immune‐activated state.

Summary/Conclusion : Several EV populations appeared altered in plasma and CSF of PC patients, overall suggesting endothelial damage and/or persistent immune activation. The underlying mechanisms driving these changes, as well as their potential utility as biomarkers for PC is subject to further investigation.

Funding : This study was supported by the Ministerium für Wissenschaft, Forschung und Kunst Baden‐Württemberg, COVID Sonderforschung

Exosomal proteins as potential stress‐related biomarkers in inflammatory bowel disease animal model

Min‐Jung Ma

Kyungpook National University, Daegu, Republic of Korea

Introduction : Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal disorder characterized by persistent inflammation of the digestive tract, significantly affecting patients' quality of life. IBD is also associated with psychological stress, creating a bidirectional relationship between disease progression and stress responses. Despite advances in understanding its pathogenesis, the exact aetiology remains unclear, and current diagnostic methods rely on invasive procedures such as endoscopy and biopsy, necessitating the development of alternative biomarker‐based approaches. Exosomes, known as small extracellular vesicles carrying bioactive molecules, have gained attention as potential biomarker sources for various diseases.

Methods : In this study, serum‐derived exosomes were analysed to identify IBD related stress biomarkers using a mouse model. Exosomes were isolated from serum samples obtained from a mouse model induced with IBD, and exosome characterization was performed using NTA, TEM, and flow cytometry analysis. For flow cytometry, exosomes were labelled with antibodies targeting CD9, CD63, and CD81. Protein profiling of exosomes from IBD and control mice was subsequently performed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS).

Results : Our findings revealed distinct protein expression patterns in the exosomes of IBD mice model compared to controls. Notably, proteins associated with inflammation, gut barrier integrity, and stress pathways were significantly altered in IBD samples. These results suggest that exosome analysis could provide valuable insights into both the physiological and psychological dimensions of IBD.

Summary/Conclusion : This study highlights the potential of exosome‐based biomarkers discovery in advancing the understanding of IBD pathogenesis and stress‐related mechanisms, offering new avenues for diagnosis and disease management.

Analysis of plasma‐derived extracellular vesicles to determine the HER2 status in breast cancer patients

Presenter: Alexis Wilhelm

Technical University Munich, Munich, Germany

Introduction : Breast cancer, one of the most prevalent cancers among women, is characterised by the expression of hormone receptors and the growth factor receptor HER2. This information is crucial for the development of personalised tumour treatment using HER2‐targeted therapies. Tumour biopsies are necessary for the histopathological diagnosis of HER2 expression by breast cancer cells, but they are subject to sampling error.

Methods : In this study, we present a successful method for identifying and analysing cancer‐derived extracellular vesicles (EVs) from plasma for the detection of HER2 expression in breast cancer without the need for additional processing steps. We have optimised the detection of nano‐sized particles by flow cytometry and employed these settings to detect HER2‐expressing EVs and quantify the expression level of HER2 on these EVs.

Results : In a clinical study of 115 patients with breast cancer, optimised flow cytometric analysis detected between 50 and 1.3 × 103 HER2+EVs per µL in the plasma of patients. While the numbers of HER2+EVs did not directly correlate with HER2 expression of tumour tissue, tumour size, grade, or metastasis, the integration of the data deriving from the enumeration of HER2‐positive EVs and the determination of their HER2 expression levels enabled the reliable identification of HER2‐expressing tumours, while preventing the misclassification of false positive results.

Summary/Conclusion : Our findings demonstrate the viability of analysing cancer‐derived EVs from plasma by integrating the count of HER2+ EVs and the expression level of HER2 on these EVs for the diagnosis and personalised therapy of breast cancer patients.

Funding : This project was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) (ZIM) to P.A.K. and O.W. B.H. was supported by funding from the School of Medicine and Health. P.K., T.B. and J.K. are supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project‐ID 210592381 ‐ SFB 1054 (TP B03; TP Z02), CRC‐TRR179.

The role of phosphatidylserine‐positive extracellular vesicles in the communication between head and neck cancer‐associated fibroblasts and tumour cells

Presenter: Jan Balvan

Masaryk University, Brno, Czech Republic

Introduction : Phosphatidylserine‐positive extracellular vesicles (PS‐EVs) are involved in cell signalling in the tumour microenvironment, have immunomodulatory properties and are secreted not only by tumour cells but also by cancer‐associated fibroblasts (CAFs). The aim of this study was to investigate the effect of PS‐EVs released from head and neck tumour‐associated fibroblasts on recipient cells of the primary tumour cell lines.

Methods : Collection of tumour samples: The study was approved by the ethical committee of Motol University Hospital, Prague, Czech Republic. Ex vivo cell cultures and cell line cultivation and characterisation: All cell lines were characterised by LC‐MS, cytokine arrays, western blot, senescence essay, atomic force microscopy and metabolic essay. EV isolation and characterisation: Conditioned media were pre‐cleared by differential centrifugation and filtration and then concentrated. Phosphatidylserine‐positive EVs were isolated using TIM4 receptor coated magnetic beads and characterised by NTA and DLS, TEM and cryo‐EM, and the presence of EV markers was assessed by western blotting. The proteomic composition of EVs was determined by LC‐MS.

Results : We identified 113/39 proteins with higher/lower abundance in PS‐EVs derived from CAFs compared to PS‐EVs derived from non‐transformed human gingival fibroblasts (hGFb). Initial findings indicate that the proteomic profiles of individual patients' PS‐EVs correlate with the progression of HNSCC to more advanced stages. We investigated the impact of PS‐EVs isolated from patients' ex vivo cultures and hGFb on the viability of FaDu cells. PS‐EVs derived from tumour samples significantly increased the viability of the FaDu cell line compared to those derived from hGFb.

Summary/Conclusion : Our results indicate that PS‐EVs derived from HNSCC patients influence the viability of recipient cells. The proteomic profiles of PS‐EVs from patient‐derived CAF samples reveal fibroblast activation and the advancement of HNSCC to more advanced stages. Thus, these PS‐EVs could serve as a source for discovering new diagnostic and prognostic biomarkers.

Funding : This study was supported by the Ministry of Health of the Czech Republic NU22‐03–00202.

Extracellular vesicles as a source of biomarkers for Fragile X syndrome therapeutic assessment

G. Venturini 1,2 , F. Fratini 3 , P. Piscopo 3 , L. Bertuccini 4 , F. Iosi 4 , A. Ferrante 1 , N. Di Carlo 1 , P. Popoli 1 , A. Martire 1 , Z. Boussadia 1

1 National Centre for Drug Research and Evaluation, Italian National Institute of Health, Rome, Italy; 2 Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy; 3 Department of Neurosciences, Italian National Institute of Health, Rome, Italy; 4 Core Facilities, Italian National Institute of Health, Rome, Italy

Introduction : Fragile X syndrome (FXS) is the leading genetic cause of intellectual disability, characterized by autism spectrum disorder traits such as social deficits, hyperexcitability, and anxiety. Despite extensive research, effective therapeutic strategies remain elusive, partly due to lack of reliable biochemical markers to evaluate their efficacy. Extracellular vesicles found in body fluids could represent a promising source of biomarkers as they reflect intracellular processes being enriched in proteins and nucleic acids coming from the cells of origin. This study aimed to characterize small EVs (sEVs) derived from a FXS mouse model to identify biomarkers potentially useful to assess therapies efficacy.

Methods : Blood‐derived sEVs were isolated from FXS and wild‐type (WT) mice by using both ultracentrifugation and size‐exclusion chromatography (SEC). Then, vesicles were characterized by nanoparticle tracking analysis (NTA), western blot for main EV markers, and transmission electron microscopy (TEM). Proteomic analyses and miRNA evaluation by Polymerase Chain Reaction (PCR) were performed to identify molecular differences between FXS and WT genotypes. Selected proteins and miRNAs were finally validated via WB and Reverse Transcription Polymerase Chain Reaction) RT‐PCR.

Results : SEC proved superior to ultracentrifugation for sEV isolation, yielding higher purity and greater identification of proteins and EV markers. Comparable number of sEVs are found in the plasma of WT and FXS mouse model, with overlapping size distribution and morphology. Proteomic analysis comparing WT and FXS blood‐ derived sEVs revealed differentially expressed proteins, including proteins related to mitochondrial function and linked to pathways deregulated in FXS. miRNA profiling identified seven differentially expressed miRNAs, with few promising biomarkers which will be object of further validation studies.

Summary/Conclusion : This study demonstrates the potential of sEVs as a source of novel measurable biochemical parameters for FXS. These biomarkers could serve as an essential tool for the preclinical and clinical evaluation of therapies, as well as providing new insights into the cell signalling alterations underlying FXS.

Extracellular vesicle‐derived biomarkers surpass cfDNA performance in a next‐generation early detection strategy for colorectal cancer

Devin F. Perez, Sudipto K. Chakrabortty, J. Christian J. Ray, Joe Johnson, Julia Doo, Yang Yang, Sonia Kumar, Vinita Verma, T. Jeffrey Cole, Christopher J. Benway, Shuran Xing, Yiyuan Yao, Sinead Nguyen, Allan George, Pawan Upadhyay, Kyle Manning, Dulaney Miller, Brian Haynes, Sivakumar Gowrisankar, Johan Skog

Exosome Diagnostics, a Bio‐Techne brand, Cambridge, Massachusetts, USA.

Introduction : Colorectal cancer (CRC) increasingly impacts younger individuals, emphasizing the need for earlier, more accurate and minimally invasive screening methods. Current plasma‐based liquid biopsy tests, typically relying on cell‐free DNA (cfDNA) and/or free proteins, effectively detect CRC but fall short in identifying early pre‐cancerous lesions such as advanced adenomas (AAs). We hypothesized that a multi‐omic approach combining cfDNA and extracellular vesicle (EV)‐derived biomarkers could enhance early detection of CRC, including AAs.

Methods : A statistical analysis plan guided the enrolment of ∼230 individuals undergoing colonoscopy across three independent clinical sites. Plasma cfDNA methylation levels were analysed utilizing enzymatic conversion and enrichment of a panel covering over 4 million CpG sites. Comprehensive profiling of EV‐derived long RNAs to measure gene expression and splice variants were performed using a hybrid capture panel covering 20,000 protein‐coding RNAs and 20,000 long non‐coding RNAs (lncRNAs). Unbiased EV small RNA profiling was performed to measure small non‐coding RNAs between 18 and 200 nts. Levels for up to 552 cancer associated EV proteins were measured using the Olink platform. Stringent quality control ensured reproducibility and no significant batch effects across sites. Feature selection and model development employed a stratified, 5‐fold cross‐validation approach with a support vector machine model. Sensitivities were evaluated using a Clopper‐Pearson estimator at a target specificity of 91% and prevalence‐adjusted.

Results : Among the omics channels, EV‐derived RNA demonstrated the highest sensitivity for detecting both CRC and pre‐cancerous AAs at 87% (±6%) and 34% (±7%), respectively, significantly outperforming cfDNA methylation (76% ± 7% and 10% ± 4%, respectively), EV‐derived protein (85% ± 7% and 24% ± 7%, respectively), and EV‐derived miRNA (49% ± 9% and 21% ± 7%, respectively). Cross‐validation identified features relevant to CRC biology, with pathway enrichment analysis of EV‐derived gene expression revealing significant activation of immune and inflammatory pathways.

Summary/Conclusion : While most EV‐derived analytes surpassed cfDNA methylation performance, EV long RNAs emerged as the most sensitive analyte for early detection of CRC and AAs. This study highlights the potential of EV‐derived multi‐omic biomarkers to address critical gaps in CRC screening, offering a transformative approach to early detection and improving patient outcomes. Combining EV‐RNA with cfDNA may give synergistic performance improvements when applied in larger cohorts.

Cell‐free EV‐associated proteomic and transcriptomic biomarker signature candidates for off‐label use of vandetanib in anaplastic thyroid cancer

Christian Grätz 1 , Prashant Changoer 2 , Dapi Menglin Chiang 1 , Johannes Kersting 1 , Martin Jaeger 2 , Romana Netea‐Maier 2 , Markus List 1 , Benedikt Kirchner 1,3 , Marlene Reithmair 4 , Michael W. Pfaffl 1

1 Technical University of Munich, Munich, Germany; 2 Radboud University Medical Center, Nijmegen, The Netherlands; 3 German Cancer Research Center, Heidelberg, Germany; 4 Ludwig‐Maximilians‐University Munich, Munich, Germany

Introduction : Anaplastic thyroid cancer (ATC) is a rare but very aggressive and undifferentiated form of thyroid cancer with a high metastatic tendency and poor prognosis. It lacks targeted therapy options, but studies have shown therapeutic potential in off‐label use of drugs like vandetanib. Since off‐label cancer therapy monitoring is not yet established in clinical practice, we assessed whether EVs are suitable sources of protein and transcriptional biomarkers for this purpose in an in vitro approach, evaluating vandetanib treatment response of the ATC cell line Cal62.

Methods : Cal62 cells were grown in EV‐depleted medium and treated with three doses of vandetanib (EC10, EC50 and EC80, as previously determined) besides the DMSO control. EVs were enriched from the supernatant using PEG precipitation to include as much cell‐free transcriptional and translational information as possible. The EV preparation was characterized by nanoparticle tracking analysis (NTA) for size and particle concentration and flow cytometry for EV marker expression. RNA and proteins from cells and the EV preparation were isolated and analysed by total RNA sequencing and LC‐MS/MS proteomics to identify biomarker signature candidates.

Results : Treatment with the EC80 dose of vandetanib led to significantly increased particle concentrations in the culture supernatant, while the treatment did not influence the particle size. The percentages of membranous EV‐marker positive particles (CFSE+, CD9/CD63/CD81+) were significantly increased through vandetanib treatment (EC50 and EC80 doses). Through differential gene expression analysis, dimensional reduction (sPLS‐DA) and pathway analysis, we identified sets of 4–8 protein and RNA biomarker candidates each from the proteomics and RNA‐Seq datasets that could be detected both intracellular and in the cell culture supernatant. The transcriptional biomarker signature was further validated by RT‐qPCR.

Summary/Conclusion : The identified cell‐free biomarker signatures enable the assessment of Cal62 cell response to vandetanib treatment in vitro. They may serve as a basis for follow‐up clinical studies with blood samples from ATC patients receiving vandetanib therapy. Given the limited number of such patients, our pre‐clinical findings address a significant knowledge gap in the use of vandetanib for ATC treatment. Further, these results underscore the potential of cell‐free EV‐associated RNA and proteins as promising targets for non‐invasive monitoring of cancer therapy.

Extracellular vesicles and intensive aerobic exercise in Parkinson's disease: preliminary insights

D. Di Martino 1,2 , R. Stiuso 2 , C. Fazio 1,2 , P. Grillo 1,2 , A. Calculli 1,2 , D. Comolli 1,2 , G. Di Lazzaro 3 , P. Calabresi 3,4 , S. Cerri 2 and A. Pisani 1,2

1 University of Pavia, Pavia, Italy; 2 IRCCS Mondino Foundation, Pavia, Italy 3 Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy; 4 Università Cattolica del Sacro Cuore, Rome, Italy

Introduction : Preclinical studies suggest a beneficial effect of physical activity in Parkinson's disease (PD) through an increase in BDNF levels and stimulation of anti‐inflammatory pathways, although the underlying mechanisms are not fully understood. Exercise induces the release of circulating extracellular vesicles (EVs), potentially mediating health benefits. Beyond altering EVs quantity, exercise can influence their cargo. This study aims to explore the role of EVs in the therapeutic effects of physical activity and their potential as biomarkers in PD.

Methods : A total of 59 PD patients were recruited in the study. A subgroup of patients were selected for a longitudinal study, and randomly assigned to a PD‐active group ( n = 22), undergoing 3 months of high‐intensity treadmill training (T1), or a PD‐sedentary group ( n = 19), receiving 3 months standard care. To optimize EVs recovery while minimizing sample volume, a PEG‐based kit was used for isolation, followed by characterization steps to ensure vesicle purity and integrity. EVs were characterized using NTA for particle size and concentration, and western blot (WB) analysis for EVs markers (CD63, Alix, TSG101) and the negative marker calnexin. Inflammatory cargo (IL‐1β, TNFα, IFN‐γ, IL‐17A, IL‐6, and IL‐10), as well as α‐synuclein and BDNF levels, were assessed by WB and ELISA assay. Plasma EVs subpopulations were analysed by flow cytometry.

Results : Preliminary analysis showed that at baseline, EVs levels correlated with UPDRS III ( r = −0.37, p = 0.017), MOCA ( r = 0.4, p = 0.0087), and age ( r = −0.31, p = 0.045). EVs concentration was similar between baseline and T1 in both groups. In the PD‐active group, a negative correlation was observed between ∆EVs levels and both disease duration ( r = −0.78, p = 0.0043) and ∆UPDRS III ( r = −0.73, p = 0.01).

Summary/Conclusion : Preliminary results suggest a correlation between EVs and motor and non‐motor parameters, with higher EVs levels observed in younger and less severe patients. Physical exercise did not affect EVs levels, but significant improvements in motor function were associated with slight increases in EVs. Further analysis of vesicle load and characterization is ongoing.

Funding : This project was supported by the European Union—Next Generation EU—PNRR M6C2 ‐ 2.1 (PNRR‐MAD‐2022‐12375804).

Exploring Phosphatidylserine as a potential tumour EV marker

Srinidhi Rangarajan, Oscar P.B. Wiklander

Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden

Introduction : Considering precision medicine, antibody‐ and small molecule‐based drugs have been developed, but resistance limits their effectiveness. Liquid biopsies, such as extracellular vesicles (EVs) offer advantages over conventional tissue biopsies, including minimal invasiveness, and real‐time tumour monitoring. EVs, which reflect the cellular content, are promising biomarkers. Phosphatidylserine (PS), normally found on the inner leaflet of cell membranes, is often exposed on the outer surface of cancer cells and corresponding tumour‐EVs (tEVs). This PS exposure is thought to be contacted by Annexin V (ANXV), a protein that binds PS with high affinity. Both PS as a target, and Annexin V as a therapeutic agent, are explored as a novel cancer therapy. This project aims to evaluate PS on tEVs to identify patients who may benefit from the ANXA5 treatment and to optimize dosing strategies.

Methods : Bead‐based capture of EVs is explored using general and specific markers, such as tetraspanins and Annexin V, and detected via flow cytometry. EVs were isolated from cell‐conditioned media using size exclusion chromatography and coupled to polymethyl methacrylate (PMMA) beads, which were functionalized with carbodiimide chemistry. Utilization of these beads demonstrate an improved PS quantification, enhanced EV capture and a minimized nonspecific binding.

Results : Our results demonstrate variability in PS expression across EVs derived from cancer cell lines and highlight differential expression of tetraspanins, essential markers for EV isolation and analysis. Key findings include the optimization of Annexin V as a primary binder for detecting PS+ EVs, and the successful development of a competitive binding assay to assess concentration‐dependent binding dynamics. The study also investigated strategies to enhance assay sensitivity, such as pre‐incubating EVs with labelled Annexin V to mimic physiological conditions, improving translational relevance.

Summary/Conclusion : This study highlights PS on tumour‐EVs as a potential biomarker for cancer diagnostics and treatment stratification. Optimizing Annexin V‐based detection and competitive binding assays improves assay sensitivity and translational relevance. These findings advance liquid biopsy tools for real‐time tumour monitoring and personalized Annexin V‐based therapies.

Funding : This study was supported by the Swedish Research Council (VR); the Swedish Cancer Society (Cancerfonden); the Center for Innovative Medicine (CIMED); the Cancer Research Funds of Radiumhemmet (Radiumhemmets Forskningsfonder); and Annexin Pharmaceuticals.

Profiling bronchoalveolar lavage extracellular vesicle miRNA for detection of lung cancer

Miks Priedols 1 , Edgars Dauss 2 , Reinis Vangravs 2 , Dārta Pūpola 2,3 , Rihards Mikilps‐Mikglebs 2,3 , Arnis Kiršners 2,4 , Ilmārs Stonāns 2 , Artūrs Ābols 1 , Alvils Krams 2,3

1 Latvian Biomedical Research and Study Centre, Riga, Latvia; 2 University of Latvia, Institute of Clinical and Preventive Medicine, Riga, Latvia; 3 Riga East University Hospital, Riga, Latvia; 4 Riga Technical University, Institute of Information Technology, Riga,, Latvia

Introduction : Lung cancer remains the leading cause of cancer‐related mortality globally, with early detection crucial for improving patient outcomes. Despite advancements in diagnostics, peripheral lung cancers are often identified at advanced stages due to non‐specific symptoms and the absence of effective screening programs. Extracellular vesicle (EV)‐associated microRNAs (miRNAs) represent a promising biomarker for minimally invasive diagnostics, reflecting the molecular landscape of lung tumours. We evaluated the diagnostic utility of EV‐associated miRNA profiles from bronchoalveolar lavage fluid (BALF) for detecting peripheral lung cancer.

Methods : EVs were isolated from BALF using differential ultracentrifugation. RNA isolation and cDNA synthesis was performed using commercially available kits. Seven hundred fifty‐four miRNAs profile analysis was performed on 10 malignant cancer and 10 benign tumour patient BALF samples using the TaqMan Advanced miRNA assay panel. The most promising miRNA targets identified were subsequently validated in an additional cohort of 50 patients with peripheral lung lesions.

Results : On average, 232 target genes were detected in the malignant cancer patient group, compared to 209 in the benign group. Notably, hsa‐let‐7a‐5p was detected approximately 22 times higher in the malignant cancer group compared to the benign group, while hsa‐miR‐576‐3p was lower by approximately 27 times.

Summary/Conclusion : Our findings underscore the potential of BALF‐derived EV‐associated miRNAs as minimally invasive biomarkers for enhancing early peripheral lung cancer detection.

Funding : This study was supported by the Latvian Council of Science, Project No: lzp‐2022/1‐0410

Searching the diagnostic potential of extracellular vesicles with distinct surface molecule signatures

Klára Hánělová 1 , Jan Balvan 1 , Monika Kratochvílová 2 , Martina Raudenská 2 , Jiří Navrátil 1 , Tomáš Vičar 2 ’⁵, Michal Masařík 1 ’ 3 ’⁴

1 Faculty of Medicine, Department of Pathological Physiology, Masaryk University, Brno, Czech Republic; 2 Faculty of Medicine, Department of Physiology, Masaryk University, Brno, Czech Republic; 3 BIOCEV, First Faculty of Medicine, Charles University, Prague, Czech Republic; 4 International Clinical Research Centre (ICRC), St. Anne's University Hospital, Brno, Czech Republic; 5 Faculty of Electrical Engineering and Communication, Department of Biomedical Engineering, VUT, Brno, Czech Republic

Introduction : Various populations of small extracellular vesicles (sEVs) are widely heterogeneous, even within a single cell type. These populations are marked by different surface molecules, such as EpCAM, CD63, or phosphatidylserine (PS). This implies that EV subpopulations may carry diverse proteomes, which could be essential for their wide range of biological functions. These variations allow sEVs to be involved in processes, such as the immune response, disease progression, and fibroblast activation into CAFs. This work aimed to explore, how distinct surface markers shape the functional roles of sEVs in the context of head and neck cancer (HNSCC).

Methods : EVs were isolated from conditioned media (obtained from HNSCC cell line FaDu) that were subjected to differential centrifugation, filtration, and concentration and EVs were isolated using affinity‐based methods targeting EpCAM, CD63, and PS on EV surface. Then, isolated EVs were characterised using NTA or DLS, cryo‐EM, TEM, WB for EV surface markers, and LC‐MS for their proteomic composition. Furthermore, the effect of various groups of EVs on recipient cells was assessed using WB for CAF activation, LC‐MS, and measurement of mitochondrial energetics in recipient cells.

Results : So far, we have been mainly focused on the first subgroup, PS‐positive sEVs, as PS is of high importance in the tumour microenvironment. By inhibition of autophagy, specifically lysosomal degradation, we observed the accumulation of proteins in PS‐EVs as an alternative pathway for autophagic degradation. This led us to the assumption that isolated EVs are of endosomal origin rather than microvesicles. Accumulation of lysosomal membrane marker, LAMP1, and decrease in the plasma membrane marker, basigin, in PS‐EVs after bafilomycin treatment supported our previous finding.

Summary/Conclusion : Our results indicate that the clinical use of autophagy modulators in cancer must be taken cautiously, since they also affect the PS‐EVs content. We also observed that PS‐EVs derived from cancer cells were taken up by normal fibroblasts, activating them into CAFs. Thus, molecules carried in PS‐EVs can be critical for cancer development, progression, and potential treatment resistance. Further research will be focused on other aforementioned subgroups of EVs.

Funding : This study was supported by the Ministry of Health, Czech Rep. NU22‐03–00202 Internal grant MUNI/LF‐SUp/1405/2024

Subtopic Cancer biomarkers 2

Extracellular vesicle‐derived circRNAs as biomarkers for brain metastasis

Ruo Lin 1 , Jingyu Li 2 , Boya Peng 1

1 The Eighth Affiliated Hospital, Sun Yat‐sen University, Guangzhou, China; 2 Beijing Tiantan Hospital, Capital Medical University, Beijing, China

Introduction : Brain metastasis (BrM) is a leading cause of cancer‐related mortality, with non‐small cell lung cancer (NSCLC) being a primary contributor. Despite advances in understanding metastatic mechanisms, the molecular factors that drive cancer cell entry and adaptation to the brain remain poorly understood. The brain microenvironment presents unique challenges for metastatic cells, and the development of targeted therapies for BrM is hindered by the complexity of this interaction. Identifying tumour‐intrinsic properties that influence this crosstalk is crucial for the prevention and treatment of BrM. Extracellular vesicles (EVs), which mediate cell‐to‐cell communication, have been implicated in reshaping distant microenvironments, including pre‐metastatic niches. Circular RNAs (circRNAs), a novel class of non‐coding RNAs, are emerging as important regulators of metastasis. Here, we identify differentially expressed circRNAs in EVs from NSCLC cells with distinct metastatic potential, focusing on brain‐specific tropism, to assess their potential as therapeutic biomarkers for BrM.

Methods : A brain‐tropic metastatic NSCLC cell line was established by performing two cycles of intracardiac injection of H2030 cells. Differential circRNA expression between H2030 and the brain‐tropic metastatic H2030‐BrM cells was assessed using circRNA sequencing. GO and KEGG pathway analyses were performed to identify enriched biological processes and molecular pathways. Cancer cell‐derived EVs were purified by ultracentrifugation and characterized by western blot, nanoparticle tracking analysis, and transmission electron microscopy. The expression of circRNA candidates in EVs was validated by RT‐qPCR.

Results : CircRNA sequencing revealed a significant difference in expression profiles between H2030 and H2030‐BrM cells (|log2FC| ≥ 2, p < 0.05). Heatmap analysis displayed distinct circRNA expression patterns in both cell lines. GO and KEGG pathway analyses highlighted the enrichment of molecular pathways associated with cancer, the blood‐brain barrier, and neurodegenerative diseases. RT‐qPCR validation confirmed the upregulation and downregulation of the top twenty differentially expressed circRNAs in EVs derived from H2030 and H2030‐BrM cells.

Summary/Conclusion : This study identifies circRNA‐associated EV biomarkers that may play a role in the adaption of cancer cells to the brain microenvironment. Further functional investigations are required to elucidate their biological roles in BrM. Exploring their potential as therapeutic targets could offer valuable insights into the development of novel strategies to prevent and treat brain metastasis in NSCLC.

Subtopic EV biomarker clinical translation 2

Lipidomic profiling of plasma small extracellular vesicles via FTIR spectroscopy for early breast cancer detection

Presenter: Abhay Mishra

All India Institute of Medical Sciences, New Delhi, India

Introduction : Breast cancer remains a leading cause of female mortality, primarily due to delayed detection. Current diagnostic methods lack specificity for early‐stage identification, necessitating novel approaches. Small extracellular vesicles (sEVs) present in plasma exhibit lipidomic signatures with potential as biomarkers. Fourier‐transform infrared (FTIR) spectroscopy provides a label‐free method for analysing these lipid profiles.

Methods : Plasma samples from breast cancer patients (BrCa, n = 74) and healthy controls (HC, n = 30) were used for sEV isolation. Characterization employed transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting for markers (CD9, CD81, CD63, TSG101). FTIR spectra of sEVs were analysed to identify lipid and other biomolecular changes, employing spectral ratio calculations (e.g., I3015/I2929). Principal component analysis (PCA) and receiver operating characteristic (ROC) curve analyses assessed the diagnostic potential.

Results : Significant lipid alterations were identified in BrCa sEVs, including elevated I3015/I2929 and I2960/I2929 ratios. PCA differentiated BrCa from HC with 94.5% explained variance, supported by ROC analysis where I2960/I2929 achieved 100% sensitivity and specificity (AUC = 1.000). Additionally, spectral markers of proteins, nucleic acids, and carbohydrates revealed distinct biomolecular signatures in BrCa sEVs compared to controls.

Summary/Conclusion : This study demonstrates that FTIR spectroscopy of sEVs can discern breast cancer‐associated lipidomic changes with high precision. Spectroscopic lipid‐to‐lipid ratios, particularly I2960/I2929, emerged as robust markers for early detection. These findings highlight FTIR‐based sEV profiling as a promising non‐invasive diagnostic tool, paving the way for enhanced clinical outcomes through early breast cancer detection.

Funding : This work was supported by the Department of Bio‐technology, India, through JRF funding‐ DBT/2021‐22/THSTI/1601.

Circulating extracellular vesicles contribute to type 1 diabetes pathogenicity

Presenter: Nagesha Guthalu Kondegowda

City of Hope, Duarte, California, USA

Introduction : Type 1 diabetes (T1D) results from diminished functional β‐cell mass due to autoimmunity. Plasma‐derived circulating extracellular vesicles (cEVs) contribute to multiple diseases. However, their impact on T1D remains unknown. Our finding that serum from T1D subjects is cytotoxic to human β‐cells led to our hypothesis that cEVs mediate β‐cell cytotoxicity through their distinct molecular cargo and thus contribute to T1D pathogenesis.

Methods : cEVs isolated from T1D (< 5 years since diagnosis), multiple autoantibody‐positive (Aab+), age, sex, and ethnicity‐matched non‐diabetic healthy donor (HD), and T1D subjects pre‐ and post‐islet transplantation, were characterized physically, and protein cargo identified. Human islet cells cultured with T1D or HD plasma (10% vol/vol) or cEVs (≥ 50 µg/mL) were examined for cell death by TUNEL staining. Glucose and β‐cell homeostasis was assessed in NOD/ShiLtJj female mice, an autoimmune T1D mouse model, treated with GW4869, an EV secretion inhibitor. PBMC‐derived EVs from T1D and HD subjects were evaluated for β‐cell cytotoxicity.

Results : Plasma and cEVs from T1D but not HD subjects ( n = 10/group) significantly increased human β‐cell but not α‐cell death, mimicking disease pathology. Proteomic analysis ( n = 5/group) identified differential cargo in T1D vs HD cEVs. Proinflammatory cytokine, IFN‐gamma, was increased in T1D cEVs, mediating its β‐cell cytotoxic effect ( n = 5/group). GW4869‐treatment in NOD mice ( n = 8‐9/group) reduced cEVs, improved glucose and β‐cell homeostasis in vivo, and diminished β‐cell cytotoxicity of serum ex vivo, supporting cEV contribution to T1D pathology. Of clinical relevance, serum and cEVs ( n = 7–8/group) from Aab+ subjects also induced human β‐cell cytotoxicity, implying that humoral β‐cell cytotoxicity precedes clinical disease‐onset. cEVs from post‐islet transplant T1D patients under immunosuppressive‐regimen showed reduced β‐cell cytotoxicity, suggesting that immune cells may be one source of cytotoxic cEVs in T1D. Supporting this, PBMC‐derived EVs from T1D but not HD subjects ( n = 4/group), like cEVs, induced human β‐cell cytotoxicity, which was reduced with in vitro immunosuppression.

Summary/Conclusion : Our findings demonstrate a critical role of T1D‐cEVs in disease pathology through their, selective cytotoxicity to human β‐cells; distinctive proinflammatory protein cargo mediating the cytotoxicity; in vivo role in T1D pathogenesis; cytotoxic phenotype acquired prior to disease‐onset; cytotoxicity modulated by immunosuppression; and potential source being PBMC.

Funding : This study was supported by NIH/NIDDK, Grant Number: R01DK125856.

The VAR2CSA malaria protein selectively detects tumour‐derived extracellular vesicles in blood of pancreatic cancer patients

Agustin Enciso‐Martinez 1,2 , Caroline Løppke 3 , Joyce J. Koene †1 , Jade Ebbelaar †2 , Meike van der Geest 1 , Mandy Los 2 , Robert Dagil 3 , Tobias Gustavsson 3,4 , Nick van Es 2,6 , Ton van Leeuwen 2 , Roman I. Koning 5 , Ali Salanti 3 , Edwin van der Pol 2 , Rienk Nieuwland 2 , Mette Ø. Agerbæk 3,7 , Peter ten Dijke 1

1 Oncode Institute and Leiden University Medical Center, Leiden, The Netherlands; 2 Amsterdam University Medical Center, Amsterdam, The Netherlands; 3 University of Copenhagen, Copenhagen, Denmark; 4 VAR2 Pharmaceuticals ApS, Copenhagen, Denmark; 5 Leiden University Medical Center, Leiden, The Netherlands, 6 Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands; 7 VarCT Diagnostics, Copenhagen, Denmark

Edwin van der Pol, Rienk Nieuwland, Mette Ø. Agerbæk, and Peter ten Dijke are co‐senior authors

Introduction : Despite the promise of tumour‐derived extracellular vesicles (tdEVs) as cancer biomarkers, straightforward identification of tdEVs is difficult due to the lack of tumour‐specific markers. We demonstrate that tdEVs present on their surface proteoglycans displaying oncofoetal Chrondroitin Sulphate (ofCS), a malignancy‐associated glycosaminoglycan modification, which enables robust and specific identification of tdEVs. Using the VAR2CSA protein, which selectively binds to ofCS, we successfully identified tdEVs in blood plasma samples of pancreatic adenocarcinoma (PDAC) patients.

Methods : The recombinant VAR2CSA protein (rVAR2) was fluorescently labelled, and used to detect tdEVs from cancer cell lines, tdEVs spiked into healthy plasma, and tdEVs in plasma from advanced‐stage PDAC patients. For validation, we used orthogonal single EV detection techniques, including calibrated flow cytometry and super‐resolution fluorescence microscopy. The staining specificity was validated using a non‐ofCS‐binding rVAR2 mutant, and enzymatic digestion of ofCS chains by chondroitinase treatment prior to staining.

Results : We identified ofCS‐positive tdEVs in conditioned media from multiple cancer cell lines. Furthermore, we detected tdEVs spiked into healthy donor plasma, as well as ofCS‐positive tdEVs in plasma from PDAC patients. In contrast, only minimal rVAR2 levels were detectable in plasma samples from healthy controls. These findings were consistent across the different methods, and the ofCS specificity of the rVAR2 staining was validated using the rVAR2‐mutant controls and chondroitinase treatment, both of which led to a significant decrease in particle counts and fluorescence signal.

Summary/Conclusion : Our study demonstrates that tdEVs display ofCS on their surface, allowing for selective targeting by the malarial VAR2CSA protein. This approach enables the detection of tdEVs in blood plasma, offering a promising strategy for liquid biopsy applications.

Funding : A.E.M. acknowledges funding from Oncode Institute and the Dutch Cancer Society (KWF) grant 15079. A.E.M. and E.v.d.P. acknowledge funding from the Dutch Research Council (NWO), VIDI grant 19724. C.L. and M.Ø.A. were funded by the Innovation Fund Denmark (grant number 9090‐00024B).

Urinary extracellular vesicle metabolomics analysis based on an optimized isolation strategy reveals diagnostic biomarkers for glioma

Presenter: Haitao Sun

Southern Medical University, Guangzhou, People's Republic of China

Introduction : Glioma, the most aggressive malignant tumour of the central nervous system, is currently diagnosed primarily through invasive biopsy procedures, and effective early screening methods remain elusive. Brain‐derived extracellular vesicles (EVs) can traverse the blood–brain barrier into the systemic circulation and are subsequently excreted into urine via renal filtration. As these EVs carry tumour‐derived metabolites that can be efficiently enriched and detected from noninvasively collected urine, they represent promising carriers for constructing liquid biopsy biomarkers for glioma.

Methods : In this study, we systematically evaluated four EV isolation techniques—kit‐based methods, the EXODUS method, ultracentrifugation, and size‐exclusion chromatography—in order to identify the optimal method for isolating urinary EVs. Subsequently, we conducted a case‐control study including 30 age‐, sex‐, and BMI‐matched healthy controls, 30 meningioma patients, and 30 patients each with WHO grade I–II and grade III–IV gliomas. Following EV isolation from urine samples, we performed untargeted metabolomic analysis using ultra‐high‐performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS) to identify differential metabolites and construct a diagnostic model.

Results : The findings indicate that size‐exclusion chromatography offers significant advantages in purifying urinary EVs. Transmission electron microscopy (TEM) revealed that the isolated EVs possess a typical bilayer membrane structure with diameters ranging from 30 to 150 nm. Nanoparticle tracking analysis (NTA) demonstrated a particle concentration of 1.9 × 10 1 ⁰ particles/mL, reflecting a significantly higher enrichment compared to other methods ( p < 0.05). Furthermore, purity analysis showed that EVs isolated via size‐exclusion chromatography were over ten times purer than those obtained by alternative techniques ( p < 0.0001). A total of 120 samples have been collected and processed using this standardized urinary EV isolation method. Based on an OPLS‐DA model, glioma‐specific metabolites were identified (VIP > 2.0, p < 0.005), and a multi‐marker diagnostic model constructed using a random forest algorithm achieved an area under the curve (AUC) exceeding 0.80, with both sensitivity and specificity above 80%.

Summary/Conclusion : This study represents the first systematic comparison of urinary EV isolation methods, revealing that size‐exclusion chromatography yields superior purity and enrichment. Our optimized approach enabled the discovery of robust glioma‐specific metabolic biomarkers, underscoring the potential of urinary EV‐based liquid biopsies for noninvasive early diagnosis and targeted treatment of glioma.

Development of bladder cancer liquid biopsy technology using mutant proteins in urinary extracellular vesicles

Presenter: Kazuma Sugimoto

Japanese Foundation for Cancer Research, Tokyo, Japan

Introduction : The widely used urinary biomarker for bladder cancer diagnosis is urine cytology, but its sensitivity and specificity are not sufficient. In this study, we focused on mutant proteins in extracellular vesicles (EVs) as novel urinary biomarkers.

Methods : DNA was extracted from bladder cancer tissues and blood of 15 cases, and whole exon sequencing was performed to identify somatic mutation sequences in each case. Tumour tissue, tumour culture supernatant‐derived EVs (Te‐EVs), and urinary EVs were analysed by LC/MS, and the mutant proteins were identified by adding the mutant sequence information to a reference database during database search. The mutant proteins detected were evaluated in time‐collected urine samples using absolute quantitative mass spectrometry and compared with the cystoscopy results to assess their diagnostic potential as a liquid biopsy.

Results : The number of identified proteins was 11,207 in tumour tissue, 9809 in Te‐EVs, or 5828 in urinary EVs, in which, importantly, 38, 33, or 4 mutant proteins were identified, respectively. Absolute quantitative measurement of LCP1‐D321H, TKT‐K120N, and PLCD1‐R639H in urinary EVs showed significant reduction in 3 and 6 months after surgery, which was well concordant with the cystoscopy results.

Summary/Conclusion : We developed a novel liquid biopsy technique to detect bladder cancer mutant proteins from urinary EVs and demonstrated the possibility of quantitative monitoring of treatment prognosis by urinalysis.

Tissue factor‐positive EVs at admission indicate traumatic brain injury and accompany acute Haemostatic‐immune shifts in severe trauma

Pia Lenz 1 , Johannes Oesterreicher 1 , Johannes Grillari 1,3 , Franz‐Josef Nierscher 2 , Alexandra Pomberger 2 , Hubert Hetz 2 , Heinz Steltzer 2 , Roman Ullrich 2 , Carolina Zeller 1 , Wolfgang Holnthoner 1 , Stephan Müller‐Botz 1,4 , Marcin F. Osuchowski 1 , Gerhard Fritsch 1,4 , Johannes Zipperle 1

1 Ludwig Boltzmann Institute for Traumatology, the Research Center in cooperation with AUVA, Vienna, Austria; 2 Department of Anesthesiology and Intensive Care Medicine, AUVA Trauma Center Vienna, Vienna, Austria; 3 Institute for Molecular Biotechnology, BOKU University, Vienna, Austria; 4 Department of Anesthesiology and Intensive Care Medicine, AUVA Trauma Center Salzburg, Academic Teaching Hospital of the Paracelsus Medical University, Salzburg, Austria

Introduction : Traumatic brain injury (TBI) exposes tissue factor (TF, CD142)‐rich neuronal tissue, triggering robust coagulation and immune responses. We examined whether TF‐bearing EVs in plasma could serve as a clinically relevant indicator for confirming TBI and whether temporal fluctuations in platelet‐, innate‐, and adaptive‐immune markers on EVs mirror the evolving host response to a major traumatic incident.

Methods : We analysed 42 severely injured adults (Injury Severity Score > 15), enrolled in the bicentric PRIME study ( NCT06314841 ), classifying them as TBI ( n = 21) or non‐TBI ( n = 21) based on clinical and imaging assessments. Blood samples were obtained at admission (0 h), 24, 48, and 96 h. EV characterization in plasma used the MACSPlex EV Kit, measuring markers of haemostasis (CD41b, CD42a, CD62P, CD142), innate immunity (CD14, CD56), adaptive immunity (CD3, CD4, CD8, etc.), and cellular regulation (CD9, CD63, and CD81). Semi‐quantitative epitope‐specific signals were recorded as median fluorescence intensity (MFI). Kruskal–Wallis tests with Dunn's post hoc correction determined significance ( p < 0.05).

Results : At 0 h, TBI patients showed higher CD142+ EV levels than non‐TBI (MFI, median [IQR]:1814 [1531–3354] versus 3450 [2047–5278], p = 0.024), and receiver operating characteristic (ROC) analysis confirmed moderate diagnostic value (AUC = 0.703) for TBI. No differences were observed in other antigens or at 24, 48, and 96 h. In all patients, platelet‐associated EV markers were elevated at admission, indicating an acute procoagulant state. Over 24–96 h, markers of innate immunity (e.g., myeloid, NK cell–associated signals) and later adaptive and regulatory pathways (T‐cell and antigen presentation) increased, reflecting a shift from immediate haemostatic activation toward inflammatory and reparative processes.

Summary/Conclusion : A transient surge in TF‐bearing EVs at admission prompts an early procoagulant activation secondary to TBI. Subsequent changes in platelet, innate, and adaptive EV markers in the entire cohort suggest complex immune and regenerative dynamics following major trauma. EV‐based phenotyping may complement existing neuronal markers for rapid TBI detection and better characterize evolving host responses.

Funding : This study was supported by the Austrian Workers’ Compensation Board (AUVA), institutional funds and Particle Metrix GmbH.

Subtopic EV clinical trials 2

sEV‐PD‐L1: a promising new circulating biomarker to monitor duodeno‐pancreatic neuroendocrine tumours (DPNET) in PRODIGE 31‐REMINET cohort

Presenter: Isen Naiken

CHU Dijon, Dijon, France

Introduction : There is a critical need for reliable biomarkers enable to monitor neuroendocrine tumours and refine therapeutic strategies. Chromogranin A (CgA) and other biomarkers used in current clinical practice lack specificity, making computed tomography (CT) and magnetic resonance imaging (MRI) the primary methods for disease monitoring. However, those methods can miss small pancreatic or gastrointestinal lesions and can be costly. New markers must be explored, among which immunosuppressive molecules the like of PD‐L1 and HSP70. Our study concentrates on markers found in liquid biopsies obtained through simple blood collection.

Methods : In that prospect, we conducted a retrospective multicentric clinical study to determine whether the levels of PD‐L1 and HSP70 in sEV (sEV‐PD‐L1 and sEV‐HSP70) isolated from patients’ plasma could predict disease progression. Patients from the REMINET clinical studies were treated by placebo or lanreotide. Plasma samples were drawn at various stages of the disease, allowing results to be linked to disease progression. Plasma‐EVs where isolated using a precipitation kit. EV markers were tested by western blotting to control the isolation. Size and concertation were measured by nanoparticle tracking and transmission electron microscopy. ELISA assays were performed on isolated EVs and patient's plasma to quantify immunosuppressive molecules.

Results : HSP70 and PD‐L1 were identified in DPNET‐patient's peripheral blood, as well as sEVs isolated from said blood samples. We show that molecules circulating in the blood or isolated from EVs are of different origins. The eventual linked between the variation of those molecules and disease progression was tested, revealing a significant correlation between levels of sEV‐PD‐L1 and disease progression in patients treated with lanreotide.

Summary/Conclusion : Circulating sEVs where analysed and proved to be pertinent in the monitoring of disease progression in DPNET patients. A first promising circulating molecular marker was identified, strongly correlated to cancer progression. Further studies are needed to confirmed sensitivity and specificity of our marker and its potential use as a predictive marker of disease progression in DPNET. Those findings could also contribute to the advancement of rapid and non‐invasive diagnostic tests in DPNET.

Subtopic EV therapeutics development 2

Highly efficient and easy‐to‐operate HDR gene knock‐in editing is achieved by ‘All‐in‐One’ Delivery Vehicles (AiDVs)

Presenter: Yawen Fu

Karolinska Institutet, Stockholm, Sweden

Introduction : CRISPR‐Cas9 genome editing technology has revolutionized the landscape of gene therapy, immunotherapy, and regenerative medicine. Clinical trials using CRISPR‐Cas9 for gene therapy are currently in progress or approved, such as in the treatment of patients with hemoglobinopathies. However, repairing a large defect or inserting a whole gene is not possible using CRISPR‐Cas9, base editing, or prime editing. Hence, there is growing interest in homology‐directed repair (HDR)‐mediated precise gene knock‐in to correct loss‐of‐function mutations and ultimately cure genetic diseases. However, the HDR efficiency is currently lower than indels (insertions and deletions), which has become a bottleneck for the application.

Methods : Here, we leverage advanced extracellular vesicle (EV) engineering methods to develop a novel modality for delivering Cas9‐ribonucleoproteins (RNPs) as well as templates of a gene to integrate the correcting‐cDNA at a specific locus by HDR simultaneously, all of which have been achieved by an ‘All‐in‐One’ Delivery Vehicle (AiDV).

Results : Our highly efficient nanoparticles can deliver the complete HDR machinery to multiple clinically relevant cell types, including T cells and hematopoietic stem cells (HSCs). With a simple addition to cell cultures, we achieve robust HDR editing rates (e.g., 40% precise gene knock‐in in Jurkat cells). Notably, in combination with the small molecule compound M3814, which inhibits non‐homologous end‐joining (NHEJ) repair, HDR is enhanced to over 90% at one endogenous locus.

Summary/Conclusion : This approach significantly improves HDR‐mediated gene knock‐in efficiency, making it applicable for clinical treatment. The straightforward operation and the possibility of in vivo application would make this treatment more economical than other options, paving the way for its development and improvement as an off‐the‐shelf treatment.

Funding : This study was supported by the Svenska Sällskapet för Medi­cinsk Forskning (SSMF) Postdoc Grant 2024.

Laminin‐binding extracellular vesicles from adipose‐derived Stem cells enhance peripheral nerve regeneration in a rat model

Presenter: Mai Quyen Nguyen

Paracelsus Medical University, Salzburg, Austria

Introduction : Peripheral nerve injuries (PNIs) lead to sensory and motor impairments, with current treatments often causing suboptimal recovery. Schwann cells (SCs) support axonal regeneration via their laminin‐rich basement membrane. Extracellular vesicles (EVs) facilitate intercellular communication and show promise for regenerative medicine. However, precise EV targeting remains a challenge. This study investigates genetically modified laminin‐binding EVs for targeted nerve repair in a rat PNI model with direct and delayed reconstruction.

Methods : Stable ASC lines expressing CD81‐LEL fusion proteins were generated using a lentiviral system. EVs were isolated, characterized (size, fluorescence, binding affinity), and tested for SC uptake and pro‐regenerative gene expression. In vivo, male Lewis rats underwent median nerve transection, followed by immediate or delayed (8 weeks) reconstruction using an autologous graft (ANT) or muscle‐vein conduit (MVC). Six groups ( n = 8 each) received either modified EVs, wild‐type EVs, or Vehicle Control (VC). Functional recovery was assessed weekly for 12 weeks using CatWalk gait analysis and Grip Strength tests. Electrophysiology was conducted before euthanasia, and muscle, median nerve, DRGs, and spinal cord were collected for molecular and histological analysis.

Results : EV characterization confirmed a 100–150 nm size range and 75% GFP positivity. Fluorescence imaging showed enhanced laminin binding, with EV uptake by SCs. qRT‐PCR revealed increased p75, BDNF, and c‐Fos expression, suggesting a pro‐regenerative effect of modified EVs. Functional tests demonstrated better motor recovery in the ANT + modEVs group compared to controls. While wild‐type EVs also promoted good recovery, vehicle controls exhibited the poorest outcomes. Electrophysiology confirmed enhanced reinnervation in the modified EV group, correlating with improved nerve conduction velocity and muscle response. Post‐mortem analysis revealed that DRG and spinal cord molecular changes mirrored functional test results, with higher neuroregenerative marker expression in the ANT + modEVs group. Additionally, autologous nerve repair significantly outperformed MVC‐based repair, suggesting that the EV therapy was most effective in a structurally optimal regeneration environment.

Summary/Conclusion : Laminin‐binding EVs enhance nerve regeneration by promoting SC repair, functional recovery, and reinnervation. Future research will focus on assessing EV homing after intravenous administration in a PNI model to optimize targeted delivery and therapeutic efficacy.

Dual‐ligand engineered exosome regulates WNT signalling activation to promote tissue regeneration

Presenter: Lingyan Yang

Guangzhou National Laboratory, Guangzhou, People's Republic of China

Introduction : WNT signalling is an essential pathway regulating tissue morphogenesis and regeneration across multiple organs through a large family of ligands. However, harnessing the WNT signalling pathway for regenerative medicine has been challenging due to the lack of approaches to identify specific WNT ligands and deliver them for tissue‐specific regeneration.

Methods : Herein, we developed a panel of WNT signalling‐inducing exosomes as a protein delivery system and uncovered their therapeutic potential.

Results : We showed that WNT signalling‐activating ligands, including the WNT and R‐spondin (RSPO) proteins, could be transported on engineered exosomes and synergistically achieve efficient pathway activation. Through systematic screening of human liver progenitor cell cultures, we identified a specific combination (WNT3A and RSPO1) that could effectively regulate hepatic cell fate and uncovered potential functional crosstalk with the PPARα signalling pathway. We also observed that dual ligands loaded on a single exosome (exoWNT3A/RSPO1) hyperactivate the WNT pathway and promote efficient adult liver organoid growth compared to the small molecule inhibitor CHIR99021. After exoWNT3A/RSPO1 administration, we confirmed the activation of WNT signalling in the liver using a transgenic Axin2‐mGFP mice model. Importantly, exoWNT3A/RSPO1 could accelerate liver repair and regeneration under various conditions, including acute and chronic injuries and aged‐associated phenotypes.

Summary/Conclusion : Collectively, our work revealed the broad therapeutic effects of WNT signalling activation in the liver through the dual‐ligand‐carrying exosomes, which activate PPAR signalling for hepatic lineage maintenance and proliferation.

Artificial intelligence framework for predicting exosomal miRNA therapeutic effects: from discovery to validation

Presenter: Wen‐Yu Lien

Bionet Therapeutics, Taipei, Taiwan (Republic of China)

Introduction : Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degradation, joint inflammation, and chronic pain, leading to significant functional limitations. With an ageing population and increasing healthcare burdens, new treatment approaches are urgently needed. Exosomes, extracellular vesicles carrying bioactive molecules such as miRNAs, have emerged as potential therapeutic tools for OA due to their ability to modulate gene expression and mediate intercellular communication. ExoVia, an exosome product derived from umbilical cord mesenchymal stem cells (UCMSCs) with a defined miRNA profile, offers a promising therapeutic approach for OA. This study investigates ExoVia's miRNAs to regulate key OA‐related genes through AI‐driven deep learning models.

Methods : The miRNA profile of ExoVia exosomes has been compiled and cross‐referenced with miRBase for functional annotation. OA‐related genes and pathways were identified by mining public databases such as KEGG, Reactome, and GeneCards. AI‐driven deep learning models, integrating tools like TargetScan, miRDB, and DIANA‐microT, were used to predict miRNA‐mRNA interactions. We applied CNN, RNN, and Transformer architectures, optimizing the models through feature selection and hyperparameter tuning. Predicted interactions were prioritized, and biological validation was conducted in OA chondrocyte cultures to assess changes in gene expression.

Results : Our analysis identified a comprehensive miRNA profile from ExoVia, revealing several miRNAs with strong potential to regulate key genes involved in OA, such as those related to cartilage degradation and inflammation. AI‐driven models successfully predicted high‐confidence miRNA‐mRNA interactions, highlighting several miRNAs with significant regulatory potential for OA‐related genes. Additionally, in our in vitro OA model using chondrocyte cultures, treatment with ExoVia exosomes led to a significant downregulation of OA‐associated inflammatory markers. These findings confirm the therapeutic potential of ExoVia's miRNAs in modulating gene expression and reducing inflammation, further supporting its promise as a novel OA therapy.

Summary/Conclusion : This study demonstrates the therapeutic potential of the ExoVia exosome product in OA, driven by miRNA regulation. AI models successfully identified key miRNA‐mRNA interactions, and in vitro validation confirmed ExoVia's ability to reduce inflammation in OA. These findings support ExoVia as a promising therapeutic approach for OA and highlight the value of combining exosome‐based therapies with AI to accelerate the development of treatments for degenerative diseases.

Cx43 enhances response to BRAF/MEK inhibitors by reducing DNA repair capacity

Presenter: María D. Mayán Santos

Universidade de Vigo, Vigo, Spain

Introduction : BRAF and MEK inhibitors (BRAF/MEKi) have radically changed the treatment landscape for advanced BRAF mutation‐positive tumours. However, limited efficacy and the emergence of drug resistance remain major challenges for successful treatment.

Methods : Here, using relevant preclinical models, we found that Connexin43 (Cx43), a protein involved in cell‐to‐cell communication, enhances the effectiveness of BRAF/MEKi by recruiting DNA repair complexes to lamin‐associated domains, thereby promoting persistent DNA damage and cellular senescence.

Results : The nuclear compartmentalization induced by Cx43 contributes to genome instability and synthetic lethality due to excessive DNA damage, suggesting a novel therapeutic approach to overcoming drug resistance in these tumours. Based on these findings, we designed an innovative drug combination using small extracellular vesicles (sEVs) to deliver Cx43 mRNA in combination with BRAF/MEKi.

Summary/Conclusion : This study demonstrates that this strategy enhances Cx43's antitumoural activity, increases the synergy of BRAF/MEKi, and resensitizes double‐resistant cells to cell death. These findings highlight the therapeutic potential of this approach in overcoming the limitations of current therapies and improving treatment outcomes for patients with advanced BRAF mutant tumours.

Engineered small extracellular vesicles from umbilical cord MSCs: A microRNA‐driven strategy to combat inflamm‐ageing

Yasiel Liste‐Dozo 1 , Enrique García‐Guillemette 1 , Pablo Fernández‐Pernas 2 , Javier De Toro 1 , María Fraga 3 , María C. Arufe 1 , Juan A. Fafián‐Laboura 1

1 Therapy Cellular & Medicine Regenerative Group, University of A Coruña (UDC), Interdisciplinary Center for Chemistry and Biology (CICA), INIBIC—University Hospital Complex A Coruña (CHUAC), A Coruña, Spain; 2 Centre of Biological Sciences, University of Veterinary Medicine of Vienna, Vienna, Austria; 3 Department of Anatomical Pathology, University Hospital Complex A Coruña, A Coruña, Spain

Introduction : Small extracellular vesicles (sEV) from umbilical cord mesenchymal stem cells (MSCs) are promising nanomedicine tools for delivering biomolecules like microRNAs (miRNAs). These vesicles facilitate intercellular communication and modulate cellular functions. miR‐30a‐5p and miR‐4436b‐5p regulate inflammation, particularly through the NF‐κB pathway, with IL‐1α being a key mediator of inflamm‐ageing and a component of the senescence‐associated secretory phenotype (SASP). Developing sEV loaded with these miRNAs offers potential therapeutic benefits for inflammation‐related ageing conditions.

Methods : This study engineered sEV loaded with miR‐30a‐5p‐ and miR‐4436b‐5p using sonication, electroporation, and Exo‐Fect. The efficiency of miRNA loading was evaluated using molecular analyses. The biological effects of the modified sEV were tested in in vitro models of inflamm‐ageing, including MSCs and chondrocytes, assessing their ability to modulate NF‐κB and IL‐1α pathways. Additionally, their therapeutic potential was explored in a progeria in vitro model.

Results : All loading methods achieved miRNA incorporation, but sonication and electroporation were the most effective. The engineered sEV demonstrated significant anti‐inflammatory effects in both preventive and therapeutic applications, alleviating cellular senescence and reducing inflammatory responses. In the progeria model, these sEV further mitigated inflammation and ageing‐related cellular dysfunctions, highlighting their potential for treating accelerated ageing conditions.

Summary/Conclusion : This study successfully developed miR‐30a‐5p‐ and miR‐4436b‐5p‐loaded sEV as a novel anti‐inflammatory and senotherapeutic strategy. The results support their role in modulating inflamm‐ageing and related conditions. Sonication and electroporation proved to be the most efficient miRNA‐loading methods, reinforcing the feasibility of this nanoplatform for therapeutic applications. Future research should explore in vivo efficacy and clinical translation.

Funding : This study was supported by Xunta de Galicia, grant number ED481D‐2021‐020, Ministerio de Ciencia e Innovación (RYC2021‐032567‐I), funded by MCIN/AEI/10.13039/ 501100011033 and from the European Union «NextGenerationEU»/PRTR», InTalent program from UDC‐Inditex for the research grant and Project PI23/01347, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. PI20/00497, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. Xunta de Galicia (ED431F 2023/30). Transference Project financed FINIBIC (RESISFERRO and ENDOPROT).

Exploring the anti‐inflammatory potential of 15d‐PGJ2‐loaded extracellular vesicles in the scope of multiple sclerosis

Presenter: Marie Auquière

UCLouvain, Louvain‐la‐Neuve, Belgium

Introduction : Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system. Various resident and peripheral immune cells are involved in MS, including microglia. Microglia can adopt different phenotypes, thus playing a dual role: ‘neuroprotective microglia’ (M2) slow MS progression, while ‘neurotoxic microglia’ (M1) favour MS progression. Extracellular vesicles (EV) have received considerable attention due to their intrinsic therapeutic properties and their use as drug delivery systems. We aim to develop a new nanomedicine‐based therapy to decrease neuroinflammation in MS by combining a bioactive lipid and the intrinsic properties of EV. Our approach is to select EV, either from resting or polarized microglia, based on their impact on microglial cell activation, and to associate them with the prostanoid 15‐deoxy‐∆‐12,14‐prostaglandin J2 (15d‐PGJ2) to dampen microglial activation.

Methods : EV were isolated from resting (M0) and polarized (M1 and M2) microglia cells (BV2) and were characterized by NTA (size and concentration), NanoFCM (markers) and TEM (morphology). We compared the effect of these EV (10,000 EV/cell) on mRNA cytokine expression in microglia cells (BV2) and on primary rat mixed glial cells (MGC). Then, the bioactive lipid 15d‐PGJ2, selected for its anti‐inflammatory effect, was loaded in EV (by incubation at 37°C). The impact of 15d‐PGJ2‐loaded EV on pro‐ and anti‐inflammatory cytokines was evaluated in BV2 cells and in MGC by measuring the mRNA expression of inflammatory markers.

Results : EV‐M0, EV‐M1, or EV‐M2 have no significant effect on the expression of inflammatory markers in activated BV2‐cells. EV‐M1 increased the mRNA expression of pro‐inflammatory markers in activated MGC. The 15d‐PGJ2‐loaded EV (using EV‐M0) significantly decreased mRNA expression of pro‐inflammatory markers in activated microglia.

Summary/Conclusion : We successfully isolated and characterized EV from resting and polarized microglia. However, none of the tested EV significantly impacted the expression of pro‐ or anti‐inflammatory markers in microglial cells. The bioactive lipid 15d‐PGJ2 encapsulated in EV shows promise for its anti‐inflammatory effects on microglial cells and warrants further investigation.

Targeting senescent cells using engineered extracellular vesicles

Presenter: Somi Park

Korea University, Seoul, Republic of Korea

Introduction : Senescent cells are those that have permanently stopped dividing but remain metabolically active. Over time, the accumulation of senescent cells secretes pro‐inflammatory factors known as senescence‐associated secretory phenotype (SASP). These factors can induce senescent phenotype in nearby healthy cells, eventually contributing to age‐related diseases. The most commonly used strategy to eliminate senescent cells involves inducing apoptosis; however, this approach faces challenges with specificity. To address the limitations, we employed senescent cell‐specific receptors on extracellular vesicles (EVs) as carriers for efficient drug delivery. Here, we present a novel EV‐based system to selectively target senescent cells.

Methods : Senescent cells were confirmed via SA‐β gal and rt‐pcr. EVs were isolated using serial centrifugation, tangential flow filtration and ultracentrifugation following the MISEV2023 guidelines. The function of the engineered EVs targeting senescent cells was assessed using FACS and IVIS.

Results : We induced senescent cells with H 2 O 2 , UV, replicative culture, and observed a high expression of pro‐inflammatory cytokines compared to young cells before use. We also observed that nearly 50% of EVs were ligand‐positive through nano FACS. We then incubated the engineered EVs with senescent cells and confirmed that the uptake of engineered EVs was significantly higher in senescent cells than in young cells by measuring mean fluorescence intensity change. Furthermore, the function of the engineered EV was also performed in vivo using young and old mice and showed its effectiveness in targeting senescent cells through IVIS.

Summary/Conclusion : We propose that the engineered EV could effectively target senescent cells and consequently can be used therapeutically to alleviate senescent cells.

Priming mesenchymal stem cells with osteogenesis imperfecta patients sera induces the secretion of EVs enriched in collagen‐targeting miRNAs

Arantza Infante 1 , Leire Cabodevilla 1 , Natividad Alcorta‐Sevillano 1,2 , Iratxe Macías 1 , Blanca Gener 1,3 , Javier Aldazabal 4 , Esperanza González 5 , Ana Talamillo 5 , Daniela Gerovska 6 , Marcos Araúzo‐Bravo 2,6,7 , Ibai Diez 1,7,8 , Juan M. Falcón 5,7 , Clara I. Rodríguez 1

1 Biobizkaia Health Research Institute, Barakaldo, Spain; 2 University of Basque Country (UPV/EHU), Leioa, Spain; 3 Cruces University Hospital, Barakaldo, Spain; 4 University of Navarra, Pamplona, Spain; 5 Center for Cooperative Research in Biosciences (CIC bioGUNE), Derio, Spain; 6 Biogipuzkoa Health Research Institute, Donostia, Spain; 7 IKERBASQUE, Bilbao, Spain; 8 Harvard Medical School, Boston, Massachusetts, USA

Introduction : Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range of clinical manifestations. The majority of cases are due to mutations in the COL1A1 or COL1A2 genes, which encode type I collagen, the main protein of bones. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cells, have been tested as an innovative therapy for OI with promising but transient outcomes. Concerning the mechanism of action of cell therapy in OI, the paracrine hypothesis has superseded the initially expected host tissue replacement by donor MSCs. In this regard, an emerging concept is gaining attention, the ability of MSCs to change their paracrine responses depending on environmental clues.

Methods : In this work we have tested this hypothesis by priming MSCs with serum samples coming from either severely affected OI adults or control, age‐matched donors. Then, we have isolated, characterized and functionally tested the EVs secreted by MSCs under these conditions (OI‐priming‐EVs, CTRL‐priming‐EVs). In addition, the miRNoma of their cargo has also been analysed. For the isolation of EVs, conditioned medium from MSCs was subjected to tangential flow filtration, followed by size exclusion chromatography and ultracentrifugation. Then, isolated EVs were characterized by standard methods and EVs cargo was evaluated by miRNAseq.

Results : We found a significant enrichment of miRNAs which target type I collagen in OI‐priming‐EVs, an interesting finding since the downregulation of the mutant type I collagen is being considered a possible new strategic approach for OI. Moreover, when administered in vivo in an OI mouse model, mice treated with OI‐priming‐EVs showed enhanced improvements in bone quality parameters when compared to those treated with CTRL‐priming EVs.

Summary/Conclusion : These results provide evidence that the priming of MSCs with disease‐relevant signals can induce the secretion of EVs with enhanced therapeutic potential for OI.

Funding : This work was supported by the Spanish Ministry of Health, Instituto de Salud Carlos III through the project “PI21/0077” (co‐funded by European Regional Development Fund; “A way to make Europe”), the Basque Government Health Department 2023333014; Gangoiti Foundation, AHUCE Foundation and the Spanish Federation of Rare Diseases (FEDER).

Engineered extracellular vesicles for delivery of therapeutic small RNA

Julia Rädler 1 , Giulia Corso 2 , Omnia M. Elsharkasy 1 , Antje Zickler 1 , Noriyasu Kamei 3 , Wenyi Zheng 1 , Dhanu Gupta 4 , Samir E. L. Andaloussi 1

1 Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden; 2 Evercyte GmbH, Vienna, Austria; 3 Faculty of Pharmaceutical Sciences, Laboratory of Drug Delivery Systems, Kobe Gakuin University, Kobe, Japan; 4 Institute of Developmental and Regenerative Medicine, Department of Paediatrics, University of Oxford, Oxford, UK

Introduction : RNA interference (RNAi)‐based drugs hold significant therapeutic potential due to their high specificity for gene silencing. However, the delivery of naked RNAi agents is limited by their hydrophilicity and low bioavailability. Here, we use extracellular vesicles (EVs) as a delivery vehicle for short‐hairpin RNA (shRNA), by employing a novel approach using Argonaute‐2 protein to actively load the cargo into EVs for efficient delivery to recipient cells.

Methods : In order to load shRNA into EVs, EV‐producing cells were engineered to express AGO2 as an RNA binding protein fused to an EV sorting domain, a fusogenic viral glycoprotein, in addition to the shRNA of interest. EVs were isolated from the conditioned media of producer cells and concentrated by spin filtration before further purification by size exclusion chromatography. The absolute copy number of shRNA in EVs was quantified by RT‐qPCR. Additionally, isolated EVs were added to various recipient cells, and knockdown efficiency was assessed based on mRNA levels using RT‐qPCR.

Results : EVs were engineered to load shRNA targeting Gapdh (shGapdh). RT‐qPCR analysis revealed a more than 290‐fold enrichment of shGapdh molecules in EVs compared to the controls. Furthermore, quantification of the absolute copy number of shGapdh showed an average of one copy of shGapdh per two EVs. Isolated EVs exhibited an average IC50 of 1.79 pM and a 95% reduction in Gapdh mRNA expression. Additionally, we observed significant knockdown of the target gene in eight different cell lines, highlighting the efficiency and broad applicability of this loading strategy.

Summary/Conclusion : Here, we describe a novel platform for loading shRNA into EVs. Using this approach, shRNA was successfully delivered, resulting in efficient gene knockdown. Additionally, this strategy can be adapted for the functional delivery of other RNA therapeutics, such as siRNA. Moreover, these data indicate that engineered EVs offer a promising platform for RNAi agent delivery.

Polyelectrolytes are effective cryoprotectants for extracellular vesicles

Elżbieta Karnas 1 , Mateusz Zając 1 , Katarzyna Kmiotek‐Caller 1 , Kamil Kamiński 1 , Shin‐Ichi Yusa 2 , Sylwia Kędracka‐Krok 1 , Patrycja Dudek 1 , Krzysztof Szczubiałka 1 , Maria Nowakowska 1 , Ewa K. Zuba‐Surma 1

1 Jagiellonian University, Kraków, Poland; 2 University of Hyogo, Himeji, Japan Maria Nowakowska and Ewa K. Zuba‐Surma are co‐senior authors

Introduction : Extracellular vesicles (EVs) are currently recognized as potential new‐generation cell‐free therapeutic factors in the field of tissue regeneration. However, clinical application of EVs requires the development of standardized procedures of their long‐term storage without the loss of structural integrity and biological activity following cryopreservation. Thus, the aim of this study was to develop a method of EV cryoprotection based on their coating with bilayer of biocompatible polyelectrolytes.

Methods : EVs secreted by human umbilical cord‐derived mesenchymal stem/stromal cells (AT‐MSCs) were isolated using ultracentrifugation method and examined according to their size distribution and presence of selected surface markers. Subsequently, EVs were coated with ultrathin bilayer of designed polyelectrolytes, including (1) cationic poly(ethylene glycol‐block‐(3‐(methacryloylamino)propyl)trimethylammonium chloride) block copolymer (PEG46‐b‐PMAPTAC52; P1) and (2) anionic of poly(2‐acrylamido‐2‐methylpropanesulfonic acid) (PAMPS18, P2), using layer‐by‐layer technique. Based on the nanoparticle tracking analysis (NTA), high‐resolution flow cytometry, as well as mass spectrometry, we analysed vesicle integrity following single or multiple freezing‐thawing cycles, as well as long‐term storage. Additionally, we evaluated the effect of cryopreservation on the functional activity of EVs in vitro, using human osteoblasts as exemplary target cells.

Results : Obtained data indicate, that coating with P1 and P2 polymers stabilizes structural integrity of EVs following deep freezing and thawing, preserving their biological activity in vitro. Additionally, proteomic analysis has confirmed an effect of particle stabilization, as well as an enrichment in EV‐ and MSCs‐related proteins in samples cryopreserved in the presence of tested polymers, indicating protective effect on EV protein cargo.

Summary/Conclusion : Taking together, our study indicates that the use of P1/P2 polyelectrolyte bilayer may be a novel, effective way of long‐term storage of EV preparations for the purpose of basic research and their clinical applications.

Funding : This study was funded by NCBR grant STRATEGMED III (STRATEGMED3/303570/7/NCBR/2017) and NCN grant MAESTRO 11 (2019/34/A/NZ3/00134) to E.Z.S.

Development of conductive tetra‐aniline‐grafted chitosan hydrogels containing extracellular vesicles derived from Wharton's jelly mesenchymal stromal cells for the treatment of chronic wound

Yung‐Hsin Cheng, Hao‐Zhong Lu, Tzu‐Hsiang Chen, Anh Nhat Thuy Ta

National Taiwan University of Science and Technology, Taipei, Taiwan

Introduction : In chronic wounds, excessive levels of inflammatory cytokines and reactive oxygen species affect the wound healing process. Recent studies showed the positive therapeutic effects of using extracellular vesicles (EVs) in the wound healing. Loading EVs into carriers can improve the bioavailability and prolong the half‐life, maintaining the effective therapeutic concentration at the target site. Mesenchymal stromal cells derived from umbilical cord Wharton's jelly (WJ‐MSCs) are easily accessible, with no invasive isolation procedures or ethical concerns. In this study, the conductive tetra‐aniline (TA)‐grafted chitosan (TAC) hydrogels containing EVs were developed and utilized as a wound dressing. The conductive TAC hydrogels may promote the efficiency of electrotherapy and sustain the release of EVs to accelerate wound healing.

Methods : In the study, EVs were derived from WJ‐MSCs (WJ‐MSC‐EVs) and isolated by size exclusion chromatography column. The isolated EVs were characterized with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot. Conductive TAC hydrogels were evaluated by 1H‐nuclear magnetic resonance spectroscopy (NMR), Fourier‐transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), conductivity, swelling and in vitro biocompatibility test. The possible therapeutic effects of the optimized TAC hydrogels containing WJ‐MSC‐EVs in L929 fibroblasts under H2O2‐induced oxidative stress were investigated.

Results : The isolated WJ‐MSC‐EVs, with the mean diameter ∼131 nm, exhibited bilayer membrane structure and were positively expressed for CD81 and Flotillin‐1. The characteristic peaks of synthesized materials were confirmed by 1H‐NMR and FTIR. The TAC hydrogels showed interconnected porous structure, allowing the encapsulated EVs to diffuse freely through the hydrogels matrix. The conductivity of developed hydrogels was increased with the rising TA content. The swelling capability of TAC hydrogels containing 15% and 20% of TA was dramatically reduced, which may affect the diffusion of EVs. The results of cell viability showed no cytotoxicity of TAC hydrogels containing 5% and 10% of TA. In the oxidative damage model of fibroblasts, WJ‐MSC‐EVs‐loaded TAC hydrogels were able to rescue the damaged cells.

Summary/Conclusion : The results suggested that WJ‐MSC‐EVs‐loaded TAC hydrogels with conductivity and sustain release properties may have potential applications in the future for treatment of chronic wounds.

Funding : This study was supported by the National Science and Technology Council (NSTC 113‐2221‐E‐011‐024), Taiwan.

Subtopic Biologic activity and therapeutic potency of EVs 2

Extracellular vesicles from Lactobacillus paracasei prime tonsil‐derived mesenchymal stem cells to accelerate osteogenic differentiation via mitochondrial biogenesis

Presenter: Da Hyeon Choi

Korea Institute of Toxicology, Daejeon, Republic of Korea

Introduction : The interaction between stem cells and microbial communities plays a crucial role in stem cell functionality in the tissue environment. Extracellular vesicles (EVs) are secreted by prokaryotic and eukaryotic cells, and the biomedical potential of the interaction between the stem cell microenvironment and microbial EVs (mEVs) has recently been highlighted.

Methods : Investigation of the tonsillar microbiome in normal and obese children with tonsillar hypertrophy demonstrated differences with regard to the presence of Lactobacillus paracasei. Isolated L. paracasei mEVs (LpEVs) were shown to induce mitochondrial biogenesis in tonsil‐derived mesenchymal stem cells (TMSCs), leading to increased ATP production and oxygen consumption rates, with enhanced mitochondrial biogenesis shortening the osteogenic differentiation period and improving differentiation efficiency.

Results : A biocompatible and effective regenerative therapeutic system that incorporated LpEVs within collagen sheets, mimicking the extracellular matrix structure (LpEVs+TMSCs/Collosheet), was designed, which demonstrated significant efficacy in regenerating damaged bone while shortening the osteogenic differentiation period.

Summary/Conclusion : Derived from a biocompatible microenvironment, LpEVs support cell‐free therapeutic agents particularly when used in conjunction with collagen sheets.

Funding : This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (RS‐2023–00210100).

Umbilical cord blood cells‐derived extracellular vesicles as an effective immunomodulatory drug for tissue regeneration applications

Patrícia Freire, Sílvia C. Rodrigues, Renato Cardoso, Cláudia Oliveira, Tânia Lourenço, Andreia M. Silva, Joana Simões Correia

Exogenus Therapeutics, Cantanhede, Portugal

Introduction : Umbilical cord blood cells (UCBC) have been successfully used in the clinic for over 20 years. Their foetal origin and naïve status render these cells as interesting sources of extracellular vesicles (EVs) with enhanced pro‐regenerative properties. Herein, we describe how Exogenus Therapeutics developed an innovative product based on UCBC‐derived EVs (Exo‐101) and demonstrate its immunomodulatory and regenerative potential for different therapeutic uses.

Methods : UCBCs were obtained from certified public/private banks and cultured under hypoxia (0.5% oxygen) for 18 h. Secreted EVs were isolated by ultrafiltration and chromatography, following a workflow developed for scalability and transfer to GMP‐compliant facilities. Exo‐101 critical quality attributes were defined upon characterization of particles, proteins, lipids and RNA content. Exo‐101's safety was evaluated in rats by repeated intravenous injection of different doses up to 12 weeks. Exo‐101's efficacy was determined using in vivo models of skin and lung injury. Exo‐101's mechanism of action (MOA) was tested in in vitro models of differentiation of macrophages cell line and peripheral blood‐derived human primary lymphocytes.

Results : No major alterations in blood biochemical parameters, neither in morphology of clearance organs was detected upon repeated injection of high doses of Exo‐101 in rats, indicating systemic tolerability. Functionally, Exo‐101 significantly reduced epidermal thickening in an in vivo model of imiquimod‐induced psoriasis, and accelerated closure and re‐epithelialization of skin excision wounds in diabetic and healthy animal models. In a bleomycin‐induced lung fibrosis mice model, Exo‐101 delayed disease progression, improved animal well‐being and decreased collagen levels in the lung. In vitro studies support in vivo findings indicating that Exo‐101 has immunomodulatory functions, inducing the polarization of macrophages into the pro‐regenerative M2 phenotype, while attenuating M1 macrophages pro‐inflammatory phenotype. Exo‐101 also promoted the differentiation of Treg cells, diminishing the percentage of effector T cells in culture.

Summary/Conclusion : By leverageing UCBCs as a powerful EVs source, we developed Exo‐101 as an innovative biological drug for advanced regenerative therapeutics. Tolerability and bioactivity in vivo, combined with a multifactorial MOA, suggest that it may become an effective therapy in different disease contexts.

Funding : This work was co‐funded by Centro 2020 ‐ Regional Operational Program, Portugal 2020 and the European Union through FEDER.

Extracellular vesicle bioactivity and preclinical efficacy is determined by mesenchymal stromal cell subtype: development of a scalable EV therapeutic

Presenter: Savvas Ioannou

University of York, York, UK

Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) demonstrate potential as a therapeutic for numerous disease conditions. However, the use of primary MSCs can lead to variation in the therapeutic effect due to differences in the donor source and inherent MSC heterogeneity. Here we used immortalised clonal MSC lines, termed Y201 and Y202, to examine how MSC phenotype influences EV character and function.

Methods : EVs were isolated by ultracentrifugation and characterised according to recommended MISEV guidelines, including nanoparticle tracking analysis, protein and miRNA analysis and transmission electron microscopy. In vitro analysis of the functional properties of these EVs included chondrocyte proliferation assays, activated T‐cell suppression assays, and cell signalling analyses. The in vivo efficacy of Y201‐EVs was examined using two independent models of arthritis, an antigen‐induced arthritis (AIA) model and a collagen‐induced arthritis (CIA) model.

Results : Both EV subtypes were morphologically similar; however, Y201‐EVs were more abundant in EV markers (CD63 & CD81) with an enhanced miRNA and proteomic content, predicted to contribute to an elabourate EV corona, particularly abundant in RGD‐containing proteins fibronectin and MFG‐E8. EVs from both cell subtypes could significantly suppress proliferation of activated T‐cells, however, only Y201‐EVs could significantly stimulate chondrogenesis and proliferation of primary human articular chondrocytes, mechanistically through an RGD (integrin)‐FAK‐ERK1/2 signalling axis. Considering the apparent anti‐inflammatory and pro‐regenerative properties of Y201‐EVs, they were tested in two mechanistically different in vivo models of arthritis (AIA and CIA), performed 2 years apart, to determine therapeutic scalability using the same EV‐producer cell. Y201‐EVs significantly reduced all measures of disease activity compared to vehicle controls, including inflammation, synovial infiltration, joint exudate, pannus formation, cartilage lesions and bone resorption. The in vivo immunomodulatory effect of Y201‐EVs was at least in part mediated by promoting T‐cell polarisation towards a pro‐resolving Treg phenotype.

Summary/Conclusion : Comparing EVs derived from two different MSC clonal lines demonstrates that EV therapeutic potential is significantly affected by cell subtype. EVs derived from immortalised Y201 MSCs have immunomodulatory and pro‐regenerative properties, with in vivo efficacy in preclinical models of inflammatory arthritis over an extended timeframe, demonstrating suitability as a scalable EV therapeutic.

Funding : This study was supported by BBSRC, Versus Arthritis, Northern Accelerator, and Mesenbio.

Subtopic Disease/therapy relevant mechanism(s) of action 2

Delivery of multiple spleen‐tropic extracellular vesicles reveals the dynamic biodistribution of large and small EVs

Anthony Yan‐Tang Wu, Wendy Wan‐Ting Wong, Phyllis Shao‐Fang Pan, Shannon Yu‐Hsuan Yeh, Felicia Djurijanto, Jou‐Min Wang, Christian Hanson, Charles Pin‐Kuang Lai

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China)

Introduction : Extracellular vesicles (EVs) can be categorized by size into large EVs (lEVs; > 200 nm) and small EVs (sEVs; < 200 nm), both of which transport bioactive cargo, including nucleic acids and proteins, to facilitate intercellular communication under (patho‐) physiological processes. While lumen cargo contributes to functional effects, our studies showed that EV membrane proteins affect EV organotropism in vivo. Although cells are reported to release both sEV and lEVs, simultaneous tracking and biodistribution analysis of multiple EV subpopulations remain largely unexplored.

Methods : We developed PalmSORBET, a spectrally multiplexed EV reporter system composed of two bioluminescence resonance energy transfer (BRET)‐based reporters, PalmSORET and PalmGRET, to concurrently track organotropism and biodistribution of labelled PalmGRET‐lEVs and PalmSORET‐sEVs. Each BRET reporter comprises a paired bioluminescent and fluorescent protein: PalmSORET (Rluc8.6‐535SG and iRFP713), PalmGRET (nanoluciferase and GFP). By labelling EV inner membranes via a palmitoylation motif, PalmSORBET enables multimodal and multi‐resolution EV imaging through bioluminescence, BRET‐induced fluorescence, and fluorescence. To investigate the dynamics between lEVs and sEVs, we co‐administered both populations overexpressing CD13, a protein we identified to reduce the lung organotropism of liver cancer‐derived EVs, followed by the spleen organotropism change.

Results : PalmSORBET successfully labelled lEV and sEV inner membranes without altering EV biophysical properties and biodistribution. Intravenous co‐administration of lEVs and sEVs showed that CD13 overexpression (CD13‐OE) confers EV spleen tropism at 0.5 and 72.5 h post‐injection. A single CD13‐OE in lEV or sEV shared a similar organotropism pattern to that of CD13‐OE in both lEV and sEV. Notably, co‐administration of lEV (wildtype) and sEV (CD13‐OE) leads to lEV distribution to the kidney and plasma when compared to co‐injected lEV (CD13‐OE) and sEV (CD13‐OE). On the other hand, spleen‐tropic lEV (CD13‐OE) did not significantly affect the biodistribution of the sEV (wildtype or CD13‐OE).

Summary/Conclusion : We identified CD13 as a key EV membrane protein for spleen tropism in both lEVs and sEVs. Detailed biodistribution analysis via the PalmSORBET system first discovered the interdependent behaviour of lEV and sEV populations under altered spleen tropism, suggesting potential long‐term effects on their biodistribution and functional roles.

Funding : This study was supported by the NSTC 113‐2628‐B‐001‐003, i‐MATE Program AS‐iMATE‐107‐33, IAMS 30‐08, Academia Sinica Career Development Award 109‐M04.

Subtopic Immunotherapy 2

Direct delivery of immune modulators to tumour‐infiltrating lymphocytes using engineered EVs

Presenter: Rikinari Hanayama

Kanazawa University, Kanazawa, Japan

Introduction : EVs are important mediators of cell–cell communication, including immune regulation. Despite the recent development of several EV‐based cancer immunotherapies, their clinical efficacy remains limited.

Methods : We created antigen‐presenting EVs (AP‐EVs) to express peptide‐major histocompatibility complex class I, costimulatory molecule, and IL‐2. This enabled the selective delivery of multiple immune modulators to antigen‐specific CD8+ T cells, promoting their expansion in vivo without severe adverse effects.

Results : AP‐EVs accumulated in the tumour microenvironment, increasing IFN‐γ+ CD8+ T cell and decreasing exhausted CD8+ T cell numbers, suggesting that AP‐EVs transformed the ‘cold’ tumour microenvironment into a ‘hot’ one. Combination therapy with AP‐EVs and anti‐PD‐1 demonstrated enhanced anticancer immunity against established tumours.

Summary/Conclusion : Engineering EVs to co‐express multiple immunomodulators represents a promising method for cancer immunotherapy.

Funding : This study was supported by the Japan Agency for Medical Research and Development

Subtopic EV therapeutics development 2

Time for GMP? Progressing manufacturing of bacterial‐derived extracellular vesicles to clinical stage

Ola Tuvesson, Agnes Zimmer, Isa Lindgren

NorthX Biologics, Matfors, Sweden

Introduction : Extracellular vesicles (EVs) have lately raised as an attractive new therapeutic modality for both immunotherapy and as vectors for various cargos such as nucleic acids and proteins. This new class of therapies is emerging as a transformative area of biopharmaceutical innovation, with potential applications in therapeutics, drug delivery, and vaccines. As for all new modalities the translation from research to clinical and commercial use is not without challenges, particularly in developing effective manufacturing processes and qualified analytical methods meeting the CMC requirements of Good Manufacturing Practices (GMP). This talk aims to put the light on some of the challenges that need to be addressed for progressing the scientific novelty of bacterial EVs and the practicalities of modern GMP‐compliant manufacturing.

Methods : Drawing on the extensive GMP expertise of a NorthX Biologics, a leading CDMO with several EV projects in development, this presentation will outline some of the principles of GMP as applied to the production of bacterial EVs, emphasizing the distinct challenges and considerations unique to this field. Topics will include the development of robust and reproducible upstream processes for vesicle production, scalable purification strategies tailored to the specific properties of EVs, and strategies to characterize the complex products to verify consistency, safety, and efficacy.

Results : Special focus will be given to: Describing the road map for the translation from bench to bedside. Critical quality attributes (CQAs) for EVs and their alignment with regulatory expectations. Analytical challenges in characterizing EVs for purity, potency, and sterility. • Risk management and contamination control strategies tailored to bacterial systems.

Summary/Conclusion : By addressing these key topics, this presentation seeks to give inventors working in the EV field some useful practical insights into the GMP landscape for bacterial EVs. As the field advances, leverageing GMP expertise and collabourative partnerships early in the product development journey will be essential to translating the promise of bacterial EVs into real‐world therapeutic breakthroughs.

Acknowledgements

ISEV gratefully acknowledges the support for the 2025 Annual Conference from all our sponsors.