Late Breaking Orals.

Thursday 26 May 2022

OT05: EVs in Immunity and Inflammation

Chair: Steffi Bosch, PhD – IECM, Oniris, INRAE, USC1383, Nantes, France

Chair: Duaa Dakhlallah

Extracellular vesicles released from Leishmania infected cells (LiEVs) induce macrophage polarization to the M2 type in infected tissues

Peter E. Kima ; Lisa E. Emerson; Austin Sheppe; Mariola J. Edelmann

University of Florida, Gainesville, USA

Introduction : Visceral leishmaniasis is a neglected tropical disease that causes significant morbidity and mortality. Iconic images of the distended abdomen of infected individuals due to an enlarged liver and spleen underscores the tissue remodeling in these infections. It is not known how Leishmania‐derived molecules contribute to this disease presentation. To address this knowledge gap, we hypothesized that extracellular vesicles (EVs) released from Leishmania‐infected cells (LiEVs) that contain parasite derived molecules, induce cellular responses that promote tissue remodeling.

Methods : Infection in animals was tracked by changes in liver weight and by histochemical analyses of infected livers. To evaluate the responses of macrophages in vitro, peritoneal exudate cell (PEC) responses to LiEVs was obtained by RT‐PCR amplification of M1 targets (iNOS, IFNγ and TNFα) and M2 targets (Arginase 1 (Arg‐1), IL‐10 and IL‐4R). For in vivo analysis, F4/80+ cells were monitored by immunohistochemical staining for markers of M1 or M2 polarization subsets.

Results : After overnight incubation of PECs, EVs prepared from Salmonella‐infected RAW264.7 macrophages or LPS treatment induced robust expression of the M1 markers iNOS, IFNγ or TNFα. In contrast, LiEVs induced the expression of Arg‐1, IL‐10, and IL‐4. The Salmonella derived EVs did not induce expression of M2 targets in PECs. Next, in vivo studies revealed a 3‐ and 4‐fold increase of F4/80+ cells in L. donovani‐infected livers at 20‐ and 42‐days Post Infection (PI), respectively, as compared to uninfected livers. Immunohistochemical analysis showed that IL‐4R expression by F4/80+ cells in infected livers was widespread as compared to iNOS that was expressed more discretely by cells within granulomas.

Summary/Conclusion : The in vitro studies suggested that LiEVs released from Leishmania‐infected cells have the capacity to induce macrophage polarization to the M2 type. The in vivo studies that showed more widespread IL4R expression of macrophages in infected tissue, provided strong evidence of M2 polarization in L. donovani infected tissues, which would ensure parasite persistence.

Funding : This work was supported by U. S. Public Health Grants R03 AI‐135610 (MJE) and R56 AI143293 (PEK).

Keywords : Infection; tissue distribution; macrophage polarization, Leishmania

Inflammatory extracellular vesicle subpopulations contribute to blood–brain barrier dysfunction

Baharak Hosseinkhani 1 ; Gayel Duran 1 ; Cindy Hoeks 1 ; Isabel Pintelon 2 ; Jean‐Pierre Timmermans 2 ; Doryssa Hermans 1 ; Kübra Bekar 1 ; Melissa Schepers 1 ; Paulien Baeten 1 ; Luc Michiels 3 ; Niels Hellings 1 ; Bieke Broux 1

1 Neuro‐Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium, Hasselt, Belgium; 2 Laboratory of Cell Biology & Histology Antwerp Centre for Advanced Microscopy (ACAM) University Antwerp Universiteitsplein 1, Antwerp 2610, Belgium, Antwerpen, Belgium; 3 Biomedical Research Institute, UHasselt, Diepenbeek, Belgium, Hasselt, Belgium

Introduction : Introduction: In multiple sclerosis (MS), migration of autoreactive T lymphocytes across the leaky and inflamed blood‐brain barrier (BBB) into the CNS is a key initiation event of demyelination and neurodegeneration. However, it remains unclear whether and how certain extracellular vesicle (EV)‐subpopulations contribute to blood‐brain barrier dysfunction in the early stages of MS. To this end, we aimed to unravel the content of BBB endothelial cell (EC)‐ derived EV subpopulations and their function in the breaching of the BBB.

Methods : Methods: Following MISEV2018, TEM, NTA, and western blot were used to characterize the purified inflammation‐associated BBB‐EV subpopulations from TNF‐α/ IFN‐γ treated BBB‐EC (hCMEC/D3 cells) based on their sedimentation speeds (2K, 10K, and 100K). Protein arrays were used to discover the immunomodulatory content of subpopulations and their target cells. In addition, the functional impact of EV‐size‐based subpopulations on the integrity of BBB and the loss of barrier properties were assessed using TEER, qPCR, and western blotting.

Results : Results: We demonstrated that BBB‐ECs, upon inflammation, release two distinct populations of heterogeneous EV, with a tendency towards higher secretion of small EV. Moreover, the expression pattern of cytokines and adhesion markers were significantly different in BBB‐EV subsets, which are biologically packed with key chemoattractant cytokines mediating T cell migration. In addition, BBB‐EV subpopulations (particularly large EV) can cause opening of the BBB and loss of barrier properties through affecting the expression of the junctional molecules Claudin‐5 and ZO‐1.

Summary/Conclusion : Conclusion: Further research into the underlying mechanisms of BBB‐EV in changing the paracellular permeability of BBB‐ ECs might unravel novel therapeutic targets in MS.

Funding : This work was co‐financed by the Belgian Charcot Foundation, Fonds voor Wetenschappelijk Onderzoek (FWO), Stichting MS Research, Multiple Sclerosis International Federation (MSIF), MoveS

Keywords : multiple sclerosis, blood‐brain barrier dysfunction, Inflammation, extracellular vesicle subpopulations

OT09: Various Approaches for Therapeutic Applications of EVs

Chair: Yanis Mouloud, n/a – Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany

Chair: Soazig Le Lay, n/a – Inserm U1087 Institut du Thorax Nantes & SFR ICAT Univ Angers

Fibroblast Growth Factor‐2 bound to specific dermal fibroblasts‐derived extracellular vesicles is protected from degradation: relevance to skin wound healing

Isabelle Petit 1 ; Ayelet Levy 2 ; Soline Estrach 3 ; Chloé Féral 4 ; Andrea Gonçalves Trentin 5 ; Florent Dingli 6 ; Damarys Loew 6 ; Jieqiong Qu 7 ; Huiqing Zhou 7 ; Clotilde Théry 8 ; Céline Prunier 1 ; Daniel Aberdam 9 ; Olivier Ferrigno 1

1 Sorbonne university, INSERM U938, CRSA, Paris, France; 2 INSERM U976, Paris, France; 3 INSERM, Nice, France; 4 Université Côte d'Azur, INSERM, CNRS, IRCAN, Faculté de Médecine, Nice, France; 5 Federal University of Santa Catarina, Rio de Janeiro, Brazil; 6 Institut Curie, PSL Research University, LSMP ‐ Curie CoreTech Mass Spectrometry Proteomics, Paris, France; 7 Radboud University, Nijmegen, Netherlands; 8 Institut Curie, PSL Research University, INSERM U932, Paris, France; 9 INSERM U1138, Centre des Cordeliers, Paris, France

Introduction : Fibroblast growth factor‐2 (FGF2) plays key roles in cutaneous tissue repair but its natural low stability prevents the development of its use in therapeutic for impaired skin repair (diabetic limb ulcers, burned epidermis). In light of the emerging roles of extracellular vesicles (EVs) in the secretion and transport of cytokines, and the importance of FGF2 in skin repair, we investigated the interplay between EVs and FGF2.

Methods : EVs were isolated by classical differential centrifugation from human dermal fibroblasts (DF) cultured in absence (CTL‐EVs) or presence of FGF2 (FGF2‐EVs) to mimic fibroblast activation during skin wound repair. Size‐exclusion columns were used to further purify EVs from soluble proteins. EVs were analyzed by western blot, ELISA and cytometry and tested in proliferation and migration assays. Immunoprecipitation with CD63 or CD81‐coated beads was performed to isolate EV populations. RNA‐seq analysis on EV‐treated fibroblasts and proteomic profile of EVs were also performed.

Results : We found that EVs isolated from DF cultured in presence of FGF2 harbor FGF2 at their outer surface. Moreover, FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2‐EVs activated AKT and ERK signaling pathways and increased in vitro cell proliferation and migration. Local injection of FGF2‐EVs improved cutaneous wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function.

Summary/Conclusion : These results reveal a novel aspect of EV function suggesting that a subpopulation of dermal fibroblast‐derived EVs can protect FGF2 from degradation thus increasing its stability. They also suggest EVs as potential tool for delivering FGF2 in skin healing therapies.

Funding : This work was supported by grants from the French Agency for Research "Oh Risque" (iTraNS), FRM, “Région Ile‐de‐France” and SILAB industry.

Keywords : skin, FGF2, wound healing, extracellular vesicles

Targeted Therapy by Antibody Displaying Extracellular Vesicles

Oscar P.B Wiklander 1 ; Doste Mamand 2 ; Rim Jawad Wiklander 1 ; Angus Lennaard 2 ; Heena Sharma 3 ; Dhanu Gupta 2 ; Xiuming Liang 4 ; Jeremy Bost 2 ; Antje Zickler 1 ; Wenyi Zheng 4 ; Joel Nordin 2 ; André Görgens 1 ; Samir El Andaloussi 4

1 Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, Stockholm, Sweden; 2 Department of laboratory medicine, Karolinska Institutet, Stockholm, Sweden; 3 Evox Therapeutics Limited, Oxford Science Park, Oxford, UK., Oxford, United Kingdom; 4 Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, Huddinge, Sweden

Introduction : Extracellular vesicles (EVs) have emerged as important mediators of biological signals between cells that regulate several important biological processes. EVs can be engineered to display targeting and therapeutic moieties and can shuttle cargo over biological barriers. EVs can thus act as natural vectors and offers a unique platform for a new class of therapeutics. Here, we introduce the use of antibody (mAb) decorated therapeutic EVs (Fc‐EVs).

Methods : Following optimization by a systematic comparison of different Fc‐binding moieties and EV sorting domains, the broad potential of this platform is shown by mAb guided targeting in vitro and in vivo with focus on using the Fc‐EVs platform as a targeted cancer therapy.

Results : We show that this approach renders highly efficient targeting of EVs to HER2‐positive breast cancer cells and to malignant melanoma cells by the display of HER2‐ and PDL1‐mAb, respectively. As proof of concept, Fc‐EVs were loaded with the chemotherapeutic drug doxorubicin and decorated with the immune checkpoint inhibitor PD‐L1‐Ab to treat malignant melanoma in vivo. The targeting approach with tumor‐specific mAb allows for a dual therapeutic role as it 1) directs the doxorubicin loaded EVs to the tumor and 2) the PD‐L1‐Ab itself function as a therapeutic intervention by blocking the immunosuppressive PD1/PD‐L1 axis. In fact, Fc‐EVs displaying PDL1‐Ab significantly accumulated in tumor tissue and, when loaded with doxorubicin, resulted in a significant lower tumor burden, and a pronounced increased survival of tumor bearing mice.

Summary/Conclusion : Here, we take advantage of the ability of EVs as a delivery vector and decorate them with mAb by equipping the EVs with an mAb binding moiety. We here show efficient tumor targeting and treatment in vitro and in vivo. Considering that these Fc‐EVs can be decorated with different types of mAb, they have the potential to targeted virtually any tissue of interest.

Funding : Center for Medical Innovation (CIMED, FoUI‐963452), KI Research Foundation Grant.

Keywords : Exosomes, Extracellular vesicles, EVs, HER2, PD‐L1, PD1, melanoma, Targeting, cancer, immunotherapy

Friday 27 May 2022

OF13: Neuronal and Neurodegenerative Diseases

Chair: Andrew F. Hill, PhD, DVC Research and Impact, Victoria University

Chair: Graça Raposo, Institut Curie UMR144, PSL Research University

Helicobactor pylori‐derived outer membrane vesicles contribute to Alzheimer's disease pathogenesis via C3‐C3aR signaling

Junhua Xie 1 ; Freddy Haesebrouck 2 ; Lien Van Hoecke 1 ; Roosmarijn E. Vandenbroucke 1

1 Ghent University and VIB, Zwijnaarde, Belgium; 2 Ghent University, Zwijnaarde, Belgium

Introduction : The gut microbiota represents a diverse and dynamic population of microorganisms that can influence the health of the host. Increasing evidence supports the role of the gut microbiota as a key player in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Unfortunately, the mechanisms behind the interplay between gut pathogens and AD is still elusive. It is known that bacteria‐derived outer membrane vesicles (OMVs) act as natural carriers of virulence factors that are central players in the pathogenesis of the bacteria. Helicobacter pylori (H. pylori) is a common gastric pathogen and H. pylori infection has been shown to increase the risk of AD.

Methods : Using Cre loading, TdT reporter mice and confocal imaging, we studied whether orally gavaged, Cre recombinase loaded, H. pylori‐derived OMVs reach the brain and specific brain cells. AppNL‐G‐F mice Alzheimer's disease mice were used to study the impact of the OMVs on disease progression, including amyloid beta (Aβ) analysis, via staining and ELISA, and cognitive functioning, using the novel object recognition test. Microglial activation and complement component 3 (C3)‐C3a receptor (C3aR) signaling was studied via imaging and inhibitor studies. Synaptic function was assessed via immunostainings and long‐term potentiation (LTP) measurements on brain slices.

Results : We show that H. pylori OMVs reach the brain, induce activation of glial cells and neuronal dysfunction, ultimately leading to exacerbated amyloid beta (Aβ) pathology and cognitive decline. Mechanistically, we identified a critical role for the C3/C3aR signaling in mediating the interactions between astrocytes, microglia and neurons upon the presence of gut H. pylori OMVs.

Summary/Conclusion : Taken together, our study reveals that H. pylori‐derived OMVs have a detrimental effect on brain functionality and accelerate AD development via C3‐C3aR signaling.

Keywords : Helicobacter pylori, OMVs, Alzheimer's disease, complement

OF14: Novel Approaches in Studying EV Nucleic Acids

Chair: Metka Lenassi, PhD, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

Chair: Dr. Esther N.M Nolte‐'t Hoen, Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University

RNA editing‐based assay for precise evaluation of EV‐mediated mRNA transfer

Masaharu Somiya 1 ; Shun'ichi Kuroda 2

1 Osaka University, Seattle, USA; 2 SANKEN, Osaka University, Ibaraki, Japan

Introduction : Extracellular vesicles (EVs) are expected to deliver various species of RNA including mRNA. EV‐mediated mRNA delivery has been usually evaluated using reporter assays which use luminescence or fluorescence proteins as a reporter. In the reporter assay, if EVs deliver mRNA into recipient cells the translation and expression of the reporter gene can be detected. However, conventional mRNA reporter assay can easily overestimate the efficiency of mRNA transfer because of the “pseudo‐transfer” of reporter proteins from donor cells to recipient cells. In this study, we developed the mRNA delivery assay which uses RNA editing to eliminate the contamination of reporter proteins in the EV samples from donor cells.

Methods : Human embryonic kidney HEK293T cells were transfected to express reporter gene (NanoLuc) and EVs were isolated from the culture supernatant by ultracentrifugation. Raw supernatant or isolated EVs were applied to the recipient HEK293T cells and the NanoLuc activity in the recipient cells was measured after 24 hours. For the RNA editing‐based assay, recipient cells were transfected to express an RNA editing enzyme (dPspCas13b‐hADDR2), single guide RNA, and siRNA before the treatment with supernatant or EVs.

Results : We confirmed that the supernatant and isolated EVs from donor cells expressing NanoLuc contained substantial amounts of NanoLuc proteins, and the contaminated proteins can be transferred to the recipient cells regardless of the functional mRNA delivery. To avoid the contamination of NanoLuc protein in EV samples, the 12th Trp codon (UGG) of NanoLuc was replaced to stop codon (UAG), so that donor cells only express reporter non‐functional mRNA but proteins. When the NanoLuc (W12stop) mRNA is delivered to the recipient cells that express RNA editing enzyme, the stop codon was converted to the original UGG codon by A‐to‐I(G) editing and functional NanoLuc expression can be expressed. This system dramatically decreased the background signal derived from contaminating NanoLuc proteins over 100‐fold to a negligible level. By using this assay, even in the presence of a known delivery enhancer of EVs, a virus‐derived glycoprotein VSV‐G, we did not observe evidence of EV‐mediated functional mRNA delivery into recipient cells.

Summary/Conclusion : RNA editing‐based assay developed in this study would be a convenient way to verify the EV‐mediated mRNA delivery in virtually any cell line.

Funding : This study was supported by JSPS KAKENHI (18K18386 and 20K15790) and “Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials” (MEXT).

Keywords : mRNA delivery; RNA editing; cargo delivery

Studying small extracellular vesicles DNA function and packaging mechanisms

Venkatesh Kumar Chetty 1 ; Jamal Ghanam 2 ; Dirk Reinhardt 2 ; Basant Kumar K. Thakur 2

1 University Hospital Essen, Essen, Germany; 2 Universitätsklinikum Essen, Essen, Germany

Introduction : Although DNA has been associated with small EVs (EV‐DNA), little is known about the mechanisms of EV‐DNA packaging and their biological function. These studies are hampered due to the lack of an efficient EV isolation method allowing separation of small EVs from free‐circulating DNA and apoptotic bodies, and a reliable tool for accurate EV‐DNA tracking and quantitation. We aimed to develop a monitoring and analysis tool to understand the mechanism(s) of EV‐DNA packaging and its downstream functions.

Methods : To isolate sEVs devoid of free‐circulated components, we combined Tangential flow filtration, Size exclusion chromatography, and Ultrafiltration (TSU). TSU‐sEVs were characterized according to MISEV2018 guidelines. EdU was utilized to label EV‐DNA cargo released from host cells; thereby, EV‐DNA function can be evaluated in the recipient cells. Furthermore, we implemented 3D imaging tools for accurate EV‐DNA quantitation by avoiding artifacts generated from 2D microscopy. Next, HEK293T‐TSU‐sEVs were enriched after knocking down 30 genes related to tumor suppression, DNA repair, and hypoxia to study the possible mechanisms of EV‐DNA packaging.

Results : Our results showed that the TSU method provides sEVs free from apoptotic bodies and cell‐free DNA with high yield and purity. Next, we revealed that DNA‐associated with sEVs is double‐stranded, predominantly linked with histones, and localized inside the vesicles. Using 3D analysis tool, we found the distribution of EV‐DNA localized in various recipient cell compartments overcoming the cell membrane barrier. This analysis tool can be implemented to understand the exact EV‐DNA function in cancer.

Analysis of enriched HEK293T‐TSU‐sEVs after the knockdown showed that EV‐DNA yield is increased significantly after deactivating hypoxia‐related genes like VHL, VEGFA, and VEGFB. To further examine the role of hypoxia on EV‐DNA release and packaging, we isolated sEVs under hypoxic conditions and determined that the EV‐DNA yield of hypoxic sEVs was significantly higher than normoxic sEVs revealing the hypoxia involvement in EV‐DNA packaging.

Summary/Conclusion : Altogether, we demonstrated an efficient sEV isolation method with new tools to understand EV‐DNA function. Furthermore, we determined the role of hypoxia in regulating EV‐DNA packaging.

Funding : This research was funded by the Stiftung Universitätsmedizin Essen; Deutsche Forschungsge‐meinschaft, DFG TH2012/1‐1 and Deutsche Kinderkrebsstiftung, DKS 2018.17.

OF15: Metabolism Obesity Nutrition

Chair: Soazig Le Lay, n/a – Inserm U1087 Institut du Thorax Nantes & SFR ICAT Univ Angers

Chair: Olivier P. BLANC‐BRUDE, PhD – Paris Center for Cardiovascular Research, Institut National de la Santé et de la Recherche Médicale U970, Université de Paris Cité, France

Reprogramming of endothelial cell metabolism by cancer cell derived extracellular vesicles

Joël E.J Beaumont 1 ; Lydie Barbeau 2 ; Kim Savelkouls 2 ; Kim Kampen 2 ; Marijke I. Zonneveld 2 ; Tom G.H Keulers 3 ; Kasper M.A. Rouschop 2

1 Department of Radiotherapy, GROW‐School for Oncology and Reproduction, Maastricht University, Stein, Netherlands; 2 Department of Radiotherapy, GROW‐School for Oncology and Reproduction, Maastricht University, Maastricht, Netherlands; 3 Department of Radiotherapy, GROW‐School for Oncology and Reproduction, Maastricht University, maastricht, Netherlands

Introduction : Tumor cells manipulate their surroundings via the release of EV to provide a growth supportive tumor microenvironment (TME), e.g. by stimulating angiogenesis to increase delivery of nutrients/oxygen. Activation of angiogenesis is associated with a metabolic switch in endothelial cells (EC), yet mechanisms driving this switch are relatively unknown. Hypoxia in solid tumors is associated with an aggressive disease. It induces angiogenesis and alters the molecular EV cargo. Here, we investigate the effects of (hypoxic) EV on the metabolic reprogramming of the TME.

Methods : Colorectal cancer‐ (HT29), glioblastoma‐ (U87), and breast cancer‐ (MDA‐MB‐231) cells were cultured under normoxia, moderate hypoxia (0.2% O2) or severe hypoxia (< 0.02% O2) for 24h with 5% EV depleted serum. EV were isolated by size exclusion chromatography. EV uptake was analysed by FACS and confocal imaging using CFSE labelling. Glucose uptake and lactate secretion were measured after 24h EV stimulation. For metabolomics, EC were stimulated for 48h, supplemented with 13C‐labeled glucose. De novo protein synthesis was assessed by click‐it chemistry.

Results : HT29, U87 and MDA derived EV increase glucose uptake in EC, cancer cells, monocytes and fibroblasts without increasing lactate production. The magnitude of this effect is cell type dependent. EV uptake analysis showed that all EV types are taken up by EC. Interestingly, HT29/U87 EV and MDA EV show clear differences in staining pattern, suggesting differences in uptake mechanism. Metabolic analysis of EC shows increased synthesis and uptake of amino acids (AA) after stimulation with HT29 and MDA, derived EV. Despite this increase, intracellular AA levels remain unaltered suggesting increased usage. In line, EV stimulated EC demonstrate increased de novo protein synthesis.

Summary/Conclusion : Depending on the specific needs of a tumor and its environmental conditions (oxygenation), cancer cells manipulate the metabolism of TME cells to support tumor growth.

Funding : Zeldzame Ziekten Fonds

KWF Kankerbestrijding

Saturday 28 May 2022

OS28: Late‐Breaking Session: EV Methodology Advances

Chair: Tanina Arab, PhD – Johns Hopkins University School of Medicine

Chair: David Juncker, MDPhD – Department of Biomedical Engineering, Faculty of Medicine and Health Sciences, McGill University

Assessing lipophilic dye promiscuity in staining extracellular vesicles and lipoprotein particles

Olesia Gololobova 1 ; Wyatt Vreeland 2 ; Kenneth W. Witwer 1

1 Johns Hopkins University School of Medicine, Department of Molecular and Comparative Pathobiology, Baltimore, USA; 2 NIST, Gaithersburg, USA

Introduction : Extracellular vesicle (EV) uptake and biodistribution studies have commonly used fluorescent lipid dyes for EV labeling and tracking. However, confounders to the interpretation of lipid dye studies include the presence of free fluorescent particles, dissociation of fluorescence tag from the extracellular vesicles, and possible exchange of dye between extracellular vesicles and co‐isolated particles such as lipoproteins and protein aggregates. In this study, we evaluated EV and lipoprotein labeling by several lipid dyes. We also tested signal release and mixing.

Methods : ACD‐anticoagulated human plasma was processed per ISTH/ISEV recommendations to remove cells, then platelets. HDL and LDL were prepared from 1 mL of human plasma using LDL/VLDL and HDL Purification Kits (Cell BioLabs). EVs were also prepared from Expi293F cell culture (Thermo Fisher) by differential ultracentrifugation, size exclusion chromatography with qEV10 70nm SEC columns (Izon), and Amicon 15 Ultra 100 kD filters. LDL and HDL were characterized by asymmetric flow field‐flow fractionation (AF4) and transmission electron microscopy. Lipoproteins and EVs were characterized by Western blot for the presence of ApoB100 (LDL), ApoAI (HDL), and CD9/CD81 (EVs). All particles were labeled with PKH67, MemGlow 488 or 560, DiO, and Exoria. We attempted to remove unincorporated dye using Sephadex G‐25 columns. Signals were read by a fluorescence plate reader and single‐particle tetraspanins assays (SP‐IRIS: ExoView).

Results : 40‐45% of particles were labeled by PKH67, DiO, and Exoria, and up to 80% by MemGlow and the majority of LDL and HDL were also labeled. In up to 72 hours of storage at 4C, the signal release was not observed from lipoprotein particles or EVs. After mixing two populations of EVs stained with dyes with different excitation wavelengths, SP‐IRIS detected a small population of events with dual signals.

Summary/Conclusion : Our results suggest that strict purification of EVs after labeling with lipophilic dyes is required before mixing with other particles, especially in biodistribution studies, where unincorporated dye may label lipoprotein particles and possibly other entities. Dye transfer between particles is also possible, although our current results cannot distinguish between association, fusion, and transfer. Additional investigations of incubation temperature, time, and orthogonal labeling are warranted.

Keywords : EV labeling, Lipophilic dyes, Biodistribution, Lipoproteins

Capture and detection of extracellular microvesicles based on coordination complexes/phosphatidylserine interactions

Phoulinh chantavong 1 ; Jérôme Dejeu 1 ; Aurore Thibon‐Pourret 1 ; Hugues Bonnet 1 ; Catherine Belle 2 ; Angéline VAN DER HEYDEN 3

1 University Grenoble alpes, Grenoble, France; 2 Centre national de la recherche scientifique, Grenoble, France; 3 Université Grenoble Alpes, GRENOBLE Cedex 9, France

Introduction : Extracellular Vesicles (EVs) are currently the subject of intense investigations because of their high potential for diagnostic and therapeutic applications. Among these EVs, microvesicles (MVs) are of particular interest in the early diagnosis of cardiovascular diseases. While targeting of externalized phosphatidylserine (PS) by Annexin V is a common approach to analyse MVs, this approach suffers of several drawbacks, especially Ca2+ dependence. In this context, we propose the use of coordination complexes as new chemosensors for MVs capture.

Methods : A set of bimetallic complexes based on a ligand with dipicolylamine arms linked by a phenoxo spacer has been synthesized. The recognition between complexes and PS was studied using phospholipids under different types of organizations: monomers using short‐chain phospholipids, model vesicles formed by extrusion and MVs. MVs were isolated from human plasma by differential centrifugation and characterized by DLS, TRPS and prothrombinase assay. Interaction in solution with monomers were studied by 31P NMR. After immobilization of the complexes on solid surfaces, interactions were studied by Surface Plasmon Resonance (SPR).

Results : Specific recognition of PS monomers by the coordination complexes was demonstrated by 31P NMR and SPR. After optimisations of the sensing surface, SPR was used to evaluate the capture of model vesicles of defined size and PS compositions. Distinction of the interactions between model vesicles containing or not PS was demonstrated. Finally, MVs capture by the bimetallic complexes grafted on the surface was also evidenced.

Summary/Conclusion : The coordination complexes have demonstrated their capacity to recognise PS presented as monomers or entrapped on lipid bilayers. These recognition properties were successfully used for MVs capture and detection.

Funding : This work was supported by the French research agency (ANR‐16‐CE29‐0009‐01) including PhD fellowship for P. Chantavong.

Keywords : coordination complexes phosphatidylserine recognition microvesicles

Differences in tetraspanin distribution on EVs from paired human milk and serum samples identified by multiplexed flow cytometry

Alberta Giovanazzi 1 ; Marca H.M Wauben 2 ; Martijn van Herwijnen 3

1 Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands; 2 Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, Utrecht, Netherlands; 3 Utrecht University, Utrecht, Netherlands

Introduction : EV surface protein signatures are related to their cell of origin and biogenesis route. In recent years, it became clear that tetraspanins (e.g. CD9, CD63 and CD81) heterogeneously distribute on EVs from diverse cellular sources and intracellular origin. In this study, we assessed the distribution of tetraspanins and specific surface proteins on EVs isolated from milk and serum obtained from the same donor.

Methods : EVs were isolated from nine paired human milk and serum samples by differential centrifugation, density gradient and size exclusion chromatography. After NTA quantification, 5*10⁸ EVs were subjected to MACSPlex Exosome multiplex platform analysis and simultaneous capture of EVs on 37 antibody‐coated bead populations was detected using either a cocktail of CD9, CD63, and CD81 antibodies or the individual tetraspanin antibodies. Protein signals with a mean fluorescence intensity (MFI) > MFI of isotype controls were considered positive.

Results : The proteins identified on milk EVs only (i.e. EPCAM, MCAM, CD14, CD3) are mainly co‐expressed with CD81, while proteins unique for serum EVs (i.e. CD31, CD40, CD41b, CD42a, CD62P) are exclusively co‐expressed with CD9 and CD63. In general, MFI signals using the CD81 detection antibody, but not the CD9 or CD63 antibody, was significantly higher on EVs from milk compared to serum.

Irrespective of the biofluid analysed, CD44 is co‐expressed exclusively with CD81, while CD63 is absent on HLA‐I expressing EVs. CD105 MFI was comparable on milk and serum EVs when detected with the tetraspanin antibody cocktail, however in serum EVs CD105 is co‐expressed with CD63 and CD81, while in milk EVs CD105 is co‐expressed with CD9 and CD81. Conversely, for HLA‐II expressing EVs, which overall signal was higher in milk EVs compared to serum EVs, a similar distribution pattern on single tetraspanins was observed between the two biofluids.

Summary/Conclusion : In conclusion, the tetraspanin distribution and specific tetraspanin‐protein co‐expression varies on EVs purified from paired milk and serum samples. This information can be used to define specific EV surface protein profiles for biomarker discovery.

Funding : This research is conducted under TRAIN‐EV project, funded by EU Horizon2020 Marie Sklodowska Curie Innovative Training Network (MSCA‐ITN) programme (grant agreement No 722148).

Keywords : Tetraspanins, multiplexed analysis, milk EVs, serum EVs, biomarkers, surface protein profiling

Make do and mend ‐ Free Flow Electrophoresis (FFE) as a novel approach for isolating extracellular vesicles

Christian Preußer 1 ; Tobias Tertel 2 ; Gerhard Weber 3 ; Johannes Graumann 4 ; Bernd Giebel 2 ; Elke Pogge von Strandmann 5

1 Institute for Tumor Immunology, EV Core Facility, Center for Tumor Biology and Immunology (ZTI), Philipps University of Marburg, Marburg, Germany, Marburg, Germany; 2 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany, Essen, Germany; 3 FFE Service GmbH, Feldkirchen, Germany; 4 Institute of Translational Proteomics, Biochemical/Pharmacological Center, Philipps University Marburg, Marburg, Germany; 5 Institute for Tumor Immunology, Center for Tumor Biology and Immunology (ZTI), Philipps University of Marburg, Marburg, Germany, Marburg, Germany

Introduction : Extracellular vesicles (EVs) are ubiquitous particles released by all cells. Although EVs are well‐known, the isolation of EVs, especially from primary biofluids remains challenging. Most isolation methods rely on the biophysical properties of EVs, such as size or density. However, a balance must be found between quality and quantity, and the functional integrity of EVs is not always preserved by all methods. In addition, the specific isolation of various subpopulations is a major task.

Methods : To isolate EVs in high purity from the peritoneal fluid (ascites), of ovarian cancer patients, the ascites was pre‐cleared by a centrifugation step followed by free‐flow electrophoresis (FFE). This method represents an established, matrix‐free approach for separating different analytes with inherent differences in charge density and/or pI value. Here we applied a protocol using a series of buffer media with different pH values which allowed us to collect 96 fractions. To characterize the composition of the individual samples in more detail, we have used different state‐of‐the‐art methods such as nano‐flow and imaging flow cytometry in addition to classical approaches.

Results : We have successfully applied the FFE approach for the isolation of EVs from ascites. We could show that the tetraspanin‐positive events were mainly found to be enriched in a few distinct fractions. This observation is further supported by the analysis of the respective fractions using nano‐flow cytometry, western blot analysis, and electron microscopy. A detailed analysis of the EV‐associated proteins by mass spectrometry will be discussed.

Summary/Conclusion : Here we described a novel approach for isolating EVs from ascites. This methodical approach applies to other biofluids such as plasma, thereby representing a new efficient and rapid way of isolating native and high‐purity EVs from intricate primary samples.

Funding : This work was supported by the Deutsche Forschungsgemeinschaft (GRK 2573/1)

Keywords : free‐flow electrophoresis, native EV isolation

Accurate differentiation of extracellular vesicles and lipoproteins by interferometric nanoparticle tracking analysis

Anna Kashkanova 1 ; Jan Van Deun 2 ; Martin Blessing 1 ; Marie Reischke 1 ; Vahid Sandoghdar 1 ; Andreas Baur 2

1 Max Planck Institute for the Science of Light, Erlangen, Germany; 2 Universitätsklinikum Erlangen, Erlangen, Germany

Introduction : Pathological conditions often lead to a dysregulation of the circulating extracellular vesicle (EV) population, including in number and size. While nanoparticle tracking analysis (NTA) is widely used for EV measurement, it is biased towards larger sizes and cannot distinguish between similarly‐sized particles of different origin. This is especially problematic for plasma‐derived EVs, as lipoproteins outnumber EVs by several orders of magnitude and often contaminate EV preps. We developed a novel technique called interferometric NTA (iNTA) and show that it can accurately distinguish EVs from lipoproteins across a wide size range.

Methods : In iNTA, the light back‐scattered by a particle is interfered with light reflected by the coverglass, leading to enhanced sensitivity for small particle sizes. Recording trajectories at high frame rates (5 kHz) and measuring the amount of back‐scattered light allows for accurate estimation of both size and refractive index (RI) for each individual particle. iNTA was compared to classical NTA by using artificial mixtures of density gradient‐purified cell culture EVs (RI < 1.4) and VLDLs (RI>1.4) as well as plasma samples, purified by both size‐exclusion (SEC) and dual mode chromatography (DMC). ApoB100 and CD63 ELISA were used to quantify levels of VLDL and EV, respectively.

Results : Using mixtures of purified EVs and VLDLs, we found that iNTA can accurately measure their relative concentrations, as opposed to NTA. We show that iNTA can clearly measure an increase in lipoproteins in chromatography‐purified plasma samples of healthy volunteers after a lipid‐reach meal. EVs purified by DMC, which is designed to remove VLDLs, resulted in significantly lower lipoprotein numbers than the same samples processed by SEC. Purified EVs spiked into these samples were accurately measured by iNTA only. In addition, we demonstrated that iNTA is applicable to the whole plasma.

Summary/Conclusion : iNTA can accurately quantify bona fide EVs in blood plasma‐derived samples of varying complexity. The ease of implementation and measurement could make it a tool in any EV lab.

Funding : AK acknowledges support from Christiane Nüsslein‐Volhard‐Stiftung.

Keywords : NTA, Size, Refractive index

Sunday 29 May 2022

OS31: Late‐Breaking Session: Inflammation and immune cells

Chair: Hernando A del Portillo – ISGlobal Institute for Global Health, Hospital Clínic ‐ Universitat de Barcelona, Barcelona, and Institut d'Investigació Germans Trias i Pujol (IGTP), Badalona/Barcelona, Spain, and Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelo

Chair: Kenneth W. Witwer, n/a – Johns Hopkins University School of Medicine

A Protein Corona on Human Platelet‐Derived Extracellular Vesicles can Promote Regenerative Functions

Fausto Gueths Gomes 1 ; André Cronemberger Andrade 2 ; Martin Wolf 2 ; Sarah Hochmann 2 ; Linda Krisch 2 ; Christof Regl 3 ; Nicole Maeding 2 ; Rodolphe W. Poupardin 2 ; Patricia Ebner‐Peking 2 ; Christian G Huber 4 ; Nicole Meisner‐Kober 3 ; Katharina Schllmoser 5 ; Dirk Strunk 2

1 Department of Transfusion Medicine, Paracelsus Medical University, Salzburg, Austria, Salzburg, Austria; 2 Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI ‐ TReCS), Paracelsus Medical University (PMU), Salzburg, Austria, Salzburg, Austria; 3 Department for Biosciences, Paris Lodron University Salzburg, Austria, Salzburg, Austria; 4 Dept. of Biosciences, Paris Lodron University Salzburg, Salzburg, Austria; 5 Department of Transfusion Medicine and SCI‐TReCS, PMU, Salzburg, Austria, Salzburg, Austria

Introduction : Human‐platelet lysate (HPL) is gaining attention as a raw material for regenerative medicine. HPL is rich in growth factors and known to promote cell growth more efficiently than fetal bovine serum in vitro. We separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin organoid formation and immunomodulation as model systems for tissue regeneration.

Methods : EVs were isolated from HPL supernatant concentrated by tangential‐flow filtration (TFF‐EVs) and further purified by size‐exclusion chromatography (TSEC‐EVs) separating EVs from (lipo) protein‐enriched soluble fractions (TSEC‐sol.F.). Samples were characterized by tunable resistive pulse sensing, western blot, tandem mass‐tag proteomics, and super‐resolution microscopy, and functionally tested during organoid formation and immunomodulation.

Results : We identified three major protein clusters by proteomics separating TSEC‐EVs from HPL clustering with TFF‐sol.F. and TFF‐EVs clustering with TSEC sol.F. Functional evaluation of HPL‐derived TFF‐EVs showed significantly improved skin‐organoid formation and inhibition of T‐cell proliferation compared to TSEC‐EVs lacking the corona or to TSEC‐sol.F. Reconstituting the corona on TSEC‐EVs with TSEC soluble fractions re‐established a functionality comparable to TFF‐EV. Zeta potential and super‐resolution imaging confirmed corona formation. Thus, corona depletion on SEC‐EVs could be artificially reconstituted by TSEC late fractions in add‐back experiments showing a similar effect compared to TFF‐EVs.

Summary/Conclusion : HPL‐derived TFF‐EVs are highly efficient in promoting skin organoid formation and inhibiting T‐cell proliferation. TFF is permissive technology enabling scalable enrichment and separation functional EVs and soluble factors. Mechanistically, depletion of the TFF‐EV protein corona by SEC or ultracentrifugation abrogated functionality indicating a novel mode of action. This also opens the possibility to engineer EVs with selected proteins for specific therapeutic applications.

Funding : The authors were supported by funding from the European Union's Horizon 2020, EV‐TT‐Bpro, Cancer Cluster II, EV‐Quant, STEBS, and EV‐TT.

Keywords : human platelet lysate (HPL); extracellular vesicles (EVs); EV corona; skin regeneration; immune modulation; angiogenesis

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

S. Rubina Baglio 1 ; Michel Tsang‐A‐Sjoe 2 ; M.A.J. van Eijndhoven 3 ; Leontien Bosch 3 ; Niala Masoumi 3 ; Joris J. Roelofs 4 ; Ekaterina S. Jordanova 5 ; Kirstin M. Heutinck 6 ; Kenny de Wildt 3 ; Nils J. Groenewegen 3 ; R.J.M. ten Berge 6 ; Raymond M. Schiffelers 7 ; J.R.T. van Weering 8 ; Sandra A. Verkuijlen 3 ; I.E.M. Bultink 2 ; Yongsoo Kim 3 ; Jaap M. Middeldorp 3 ; Alexandre E. Voskuyl 2 ; Dirk M. Pegtel 3

1 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, amsterdam, Netherlands; 2 Department of Rheumatology and Clinical Immunology, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, Amsterdam, Netherlands; 3 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, Amsterdam, Netherlands; 4 Department of Pathology, Amsterdam UMC location AMC, Amsterdam, The Netherlands, Amsterdam, Netherlands; 5 Center for Gynaecologic Oncology Amsterdam, Department of Gynaecology, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, Amsterdam, Netherlands; 6 Department of Experimental Immunology, Amsterdam UMC location AMC, Amsterdam, The Netherlands, Amsterdam, Netherlands; 7 University Medical Center Utrecht, Utrecht, The Netherlands, Utrecht, Netherlands; 8 Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, Netherlands, Amsterdam, Netherlands

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, these inflammatory EVs disproportionately reach the tubular epithelium inducing an IFN response that worsens the disease.

Methods : Gene set enrichment analysis was applied to identify key pathways and cell signatures in proliferative LN. RNA‐seq was used to define the pol‐III trascriptome 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 unravel the mechanism 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, including (EBV‐infected) B lymphocyte, transcripts 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 from Lupus but not IgA nephritis biopsies. Functional assays showed uptake of B cell EVs by primary TECs in a phosphatidylserine (PtdSer) and KIM‐1‐dependent manner. Crucially, B cell EVs deliver EBERs into endosomes of primary TECs driving IFNb, TNFa and IL6 production through TLR3 activation.

Summary/Conclusion : In SLE patients, blood cell‐secreted, PtDSer+, inflammatory EVs target TEC cells via KIM‐1 inducing an IFN‐mediated inflammatory response.

Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation

Hyun Young Kim 1 ; Min‐Kyoung Song 2 ; Younggap Lim 3 ; Ji Sun Jang 4 ; Hong‐Hee Kim 4 ; Bong‐Kyu Choi 3

1 Department of Oral Microbiology and Immunology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2 Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea, Seoul, Republic of Korea; 3 Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul, Republic of Korea., Seoul, Republic of Korea; 4 Department of Cell and Developmental Biology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea., Seoul, Republic of Korea

Introduction : Dysbiosis of oral microbiota is play an important role in progression of periodontitis which is characterized by chronic inflammation, alveolar bone loss and systemic diseases. Bacterial extracellular vesicles (EVs) contain various bioactive molecules and showed diverse effects on host environments depending on the bacterial species. Recently, we reported that EVs derived from Gram‐positive periodontal pathogen, Filifactor alocis have osteoclastogenic activity. In the present study, we determined the osteoclastogenic potency and immunostimulatory activity of EVs derived from Gram‐negative periodontal pathogens (Porphyromonas gingivalis and Tannerella forsythia), an oral commensal (Streptococcus oralis) and a gut probiotic strain (Lactobacillus reuteri).

Methods : Bacterial EVs were purified by density gradient ultracentrifugation using OptiPrep reagentTM. Morphology and size of bacterial EVs were analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), respectively. Osteoclast differentiation by bacterial EVs was determined by tartrate‐resistant acid phosphatase (TRAP) staining. Immunostimulatory activity of bacterial EVs was determined by cytokine array and enzyme‐linked immunosorbent assay (ELISA). Toll‐like receptor (TLR)‐activating ability of bacterial EVs was measured using CHO/CD14 reporter cells. Inactivation of bacterial lipoprotein and lipopolysaccharide (LPS) from EVs was performed using lipoprotein lipase and polymyxin B, respectively.

Results : EVs from P. gingivalis, T. forsythia, and S. oralis increased osteoclast differentiation and osteoclstogenic cytokine expression in osteoclast precursors, whereas EVs from L. reuteri did not. EVs from P. gingivalis, T. forsythia, and S. oralis preferentially activated TLR2 rather than TLR4 and induced osteoclsatogenesis mainly through TLR2. Osteoclastogenic effects of EVs from P. gingivalis and T. forsythia were reduced by both lipoprotein lipase and polymyxin B, while osteoclastogenic effects of EVs from S. oralis and F. alocis were reduced by lipoprotein lipase alone.

Summary/Conclusion : EVs from periodontal pathogens and oral commensal, but not probiotic strain, showed osteoclastogenic potency. TLR2 is major receptor for osteoclast differentiation by EVs from oral bacteria including periodontal pathogens.

Funding : This work was supported by grants from the National Research Foundation of Korea (NRF‐ 2021R1A2C1003952 and NRF‐2022R1C1C2007752).

Keywords : Bacterial extracellular vesicle, Periodontal pathogen, Osteoclast, Toll‐like receptor 2

Heterogeneus Induction of Interferon Pathway in the Tumor Microenvironment by Different Populations of Cancer‐Derived Extracellular Vesicles

Krizia Sagini 1 ; Diana Kitka 2 ; Blandine F. Victor 3 ; Minhyung Kim 4 ; Sungyong You 4 ; Fayyaz Sutterwala 3 ; Catherine Grasso 3 ; Caroline Jefferies 5 ; Jlenia Guarnerio 3 ; Paola de Candia 6 ; Karen Cavassani 3 ; Tatyana Vagner 4 ; Dolores Di Vizio 4

1 Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 2 cedars sinai medical center, Los Angeles, USA; 3 cedars sinai medical center, los angeles, USA; 4 Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars‐Sinai Medical Center, Los Angeles, CA, USA, Los Angeles, USA; 5 cedars‐sinai medical center, los angeles, USA; 6 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Degli Studi di Napoli, Naples, Italy

Introduction : Cancer‐derived EVs come in different sizes that can be separated by differential centrifugation followed by density gradient resulting in different fractions containing different cargo. Along with nanovesicles, cancer cells shed abnormally large EVs (> 1 m in diameter) whose role in cancer metastasis has not been fully elucidated, since most of the attention has been placed to small EVs.

Methods : UC followed by iodixanol gradient centrifugation to purify the vesicles. TRPS, RNA‐seq, PCR flow cytometry, single cell RNA‐Seq

Results : We demonstrated by RNA‐Seq that bone stromal cells exhibit an anti‐viral, type I interferon, response when exposed to EVs from cancer cells. While protein amount was similar in the two EV fractions obtained from the same number of cells, the number of L‐EVs was 3 orders of magnitude smaller than the number of S‐EVs. Therefore, we dosed them for our experiments based on the number of donor cells. Both S‐ and L‐EVs induced a profound alteration of transcripts in stromal cells but the L‐EV elicited a significantly more remarkable response. Several of the mRNAs induced by L‐EVs codify for cytokines involved in tumor progression (CXCL1, CXCL10, CCL5, IL6). Additionally, neutrophils exposed to the conditioned medium of stromal cells treated with EVs exhibited increased expression of Arg1, Lox1, and IL10, which suggests a switch to an immune‐suppressive phenotype. The result was stronger for L‐EVs vs S‐EVs. These neutrophils also gained stronger migratory abilities toward the stroma when exposed to L‐EVs. We are now comparing the role of specific nucleic acids in L‐ and S‐EVs and of key nuclei acid sensor pathways (TLRs, RIG‐I, MDA5, cGAS/STING and AIM2) in mediating the interferon response induced by L‐ and S‐EVs to understand whether different EV populations have specific pathways of activation of the anti‐viral response.

Summary/Conclusion : Our results provide novel insight into how tumor derived EVs and in particular L‐EVs alter bone stroma cells by prompting a pro‐inflammatory type‐I interferon response and recruiting neutrophils having an immune‐suppressive phenotype, which may drive bone metastasis in prostate cancer.

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

Keywords : large and small extracellular vesicles, EV heterogeneity, cancer progression

Morphine‐mediated neuroinflammaging: Role of Astrocyte‐derived extracellular vesicles

Shilpa Buch 1 ; Susmita Sil 2 ; Divya Chemparthy 3 ; Seema Singh 3 ; Muthukumar Kannan 3 ; Shannon Callen 3

1 University of Nebraska Medical Center, Omaha, USA; 2 Department of Pharmacology & Experimental Neuroscience, Omaha, USA; 3 UNMC, Omaha, USA

Introduction : Heroin abuse has been implicated in accelerated aging, specifically impacting age‐sensitive brain functional networks, in heroin addicts. Extracellular vesicles (EVs) are well‐recognized conduits for transferring cellular cargo across recipient neuronal cells, leading to neuronal synaptodendritic injury. While the direct role of morphine‐induced neuronal synaptodendritic injury has been well‐documented, whether glial‐neuronal interactions also play a role in neuronal senesenscenece remains an enigma. We thus sought to examine whether morphine‐stimulated astrocyte‐derived EVs (M‐ADEVs) could carry the SASP cargoes to recipient neurons and mediate synaptodendritic injury.

Methods : We have employed EV isolation by ultracentrifugation, neuronal injury by Neurolucida and behavior assessment by frailty assay, maze study & locomotor behavior.

Results : Morphine exposure induced senescence phenotype (upregulation of p16, p21, inflammatory cytokines, oxidative stress, β‐ gal activity and cell cyclce arrest) & inflammation in human primary astrocytes in vitro. Intriguingly, astrocytic SASP upregulation was regulated by long noncoding (lnc)RNA BACE1AS, which was proven by gene silencing approach. Further, M‐ADEVs were also demonstrated to carry the SASP cargoes , a process that was again regulated by lncRNA BACE1AS. Uptake of M‐ADEVs by neurons was shown to induce neuronal senescence & synaptodendritic injury. In vivo validation further demonstrated that morphine administered mice exhibited inflammageing phenotype and cognitive deficits in both the frontal cortex and hippocampus.

Summary/Conclusion : This study underscores the role of lncRNA BACE1AS in regulating astrocytic‐neuronal senescence and the role of M‐ADEVs in mediating synaptodegeneration leading to cognitive impairments associated with ageing.

Funding : This work was supported by NSCAR to Dr. S Buch.

Keywords : BACE1AS, Morphine, ADEVs, ageing, synaptodegeneration, cognitive study

OS32: Late‐Breaking Session: Techniques and Reporting

Chair: Caterina Nardella – Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, 38123 Trento, Italy

Chair: Bas W. van Balkom, n/a – UMC Utrecht

EV‐TRACK 2.0: an optimized framework for transparent and reproducible extracellular vesicle research

Quentin Roux 1 ; Sarah Deville 1 ; Sandor Dedeyne 1 ; Jan Van Deun 2 ; Sofie Van Dorpe 1 ; Nele De Langhe 1 ; Niké Guilbert 1 ; Amélie Vander Cruyssen 1 ; Felix De Vuyst 1 ; Cláudio A. Pinheiro 3 ; Robin Boiy 1 ; Mohammad Ghiasloo 4 ; Sam Calis 1 ; Jodi Moons 1 ; Phaedra Van de Vyver 1 ; Olivier De Wever 1 ; An Hendrix 3

1 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Cancer Research Institute Ghent, Ghent Belgium., Gent, Belgium; 2 Universitätsklinikum Erlangen, Erlangen, Germany; 3 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Cancer Research Institute Ghent, Ghent Belgium., Ghent, Belgium; 4 Department of Plastic and Reconstructive Surgery, Ghent University Hospital, Gent, Belgium

Introduction : EV‐TRACK ( https://evtrack.org/ ) is a free online community‐powered knowledgebase endorsed by ISEV. It centralizes curated experimental parameters of EV experiments reported in literature in the ‘EV‐METRIC’ as a guidance tool to support transparency and reproducibility. To keep up with the latest advances in EV research, we present EV‐TRACK 2.0 with seven upgraded enabling features to stimulate and further facilitate its use by EV researchers.

Methods : To launch EV‐TRACK 2.0, we developed a user‐friendly submission platform to facilitate study reporting. We updated the queried experimental parameters considering emerging methodologies, MISEV guidelines, reports from ISEV task forces, and other reporting initiatives. We revised the EV‐METRIC to inform researchers and provide guidance about all experimental parameters entered to the submission platform. Finally, driven by community participation the number of entries in the knowledgebase substantially enlarged.

Results : From the 115 experimental parameters surveyed for 1,226 studies originally included in the EV‐TRACK knowledgebase released in 2017, the experimental parameter checklist and the number of reported EV studies have more than doubled with now over 5000 experiments accessible. Yearly an increasing number of pre‐publications has been submitted, resulting in over 500 submissions of which more than half have been published to date. Both the number of experiments per study and the EV‐METRIC increased over time, reflecting increased adherence to the community's reporting and minimal information guidelines.

Summary/Conclusion : Developed for and endorsed by the EV community, EV‐TRACK 2.0 aims to continue supporting an interpretable and reproducible EV research where experiments are transparently reported and guidelines timely centralized.

Funding : European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No [722148].

Keywords : EV‐TRACK; Transparent Reporting; Reproducibility; Rigor and Standardization; Extracellular vesicles

Filtering out platelets from human plasma to reveal extracellular vesicle‐associated miRNA profiles

Jillian Bracht 1 ; Mandy Los 1 ; M.A.J. van Eijndhoven 2 ; Britta A. Bettin 3 ; Edwin van der Pol 3 ; Dirk M. Pegtel 2 ; Rienk Nieuwland 4

1 Department of Clinical Chemistry, Vesicle Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; 2 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, Amsterdam, Netherlands; 3 Department of Clinical Chemistry, Vesicle Observation Center, Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; 4 Department of Clinical Chemistry; Vesicle Observation Center; Amsterdam University Medical Center, University of Amsterdam, Amsterdam, Netherlands

Introduction : To prepare plasma for studies of extracellular vesicles (EVs), blood is centrifuged to remove cells and cellular debris. To which extent the removal of cells, including platelets, is successful, depends on the applied centrifugation protocol. In daily practice, plasma still contains platelets and erythrocyte ghosts, which may contain genetic material and fragment during freeze‐thaw cycles. Because cells and ghosts co‐migrate with EVs during size‐exclusion chromatography (SEC), their presence can interfere with downstream analysis of EVs. Here we studied the effect of filtration to remove platelets and ghosts from plasma and SEC fractions on downstream miRNA analyses.

Methods : EDTA blood was prepared by (1) 2x centrifugation (2500g, 15 min, no brake; plasma collected 10 mm above the buffy coat/cell pellet), and by (2) 2x centrifugation (900g, 7 min + 2500g, 10 min; max brake; plasma collected approx. 10 mm above buffy coat/cell pellet). Cell and EV concentrations were measured by flow cytometry (BD FACSCanto and Apogee A60‐Micro), and let7a‐5p, miR‐21‐5p, and miR‐223‐3p were measured by qPCR in fresh and frozen‐thawed human plasma before and after SEC2B, with and without filtration using 800 nm polycarbonate filters.

Results : Plasma prepared by protocol 1 and 2 contained ∼10ˆ6 and 10ˆ7 platelets/mL, respectively. Plasma also contained ∼10ˆ6 erythrocytes ghosts/mL. The concentration of platelets decreased after one freeze‐thaw cycle, which coincided with an increase in “platelet‐derived EVs” of 150‐1000 nm. EV‐enriched SEC2B fractions contained ∼10ˆ5 platelets‐ and erythrocyte ghosts/mL. Filtration of fresh plasma or SEC fractions lowered platelet and ghost concentrations >98% without affecting the EV concentration. Filtering resulted in ∼2 to 4‐fold (ΔCt=1‐2) reductions in let7a‐5p, miR‐21‐5p and platelet‐associated miR‐223‐3p. The miRNA quantities correlated with high (>10ˆ6/mL) platelet concentrations in plasma (R2=0.68; p< 0.001), but not with erythrocyte ghosts (R2=0.02; p=0.62) or total EV concentration (R2=0.01; p=0.92). In corresponding SEC fractions (platelet concentration < 10ˆ6/mL) the correlation was less apparent.

Summary/Conclusion : EDTA plasma contains 10ˆ6‐10ˆ7 platelets/mL and 10ˆ6 erythrocytes ghosts/mL after a 2‐step centrifugation protocol, of which a proportion ends up in SEC fractions. This could result in an overestimation of EV‐miRNA content. Therefore, we recommend to measure and report concentrations of platelets and erythrocyte ghosts, and to remove them before freezing samples.

Funding : This study was funded by Health Holland (AQrate project).

Keywords : EVs; Plasma preparation; Platelets; Filtration

New Methods to Study the Composition and Structure of T cell trans‐Synaptic Particles

Pablo F. Céspedes‐Donoso 1 ; Ashwin K. Jainarayanan 1 ; Michael L. Dustin 1 ; Lola Fernandez‐Messina 2 ; Francisco Sanchez‐Madrid 2 ; Ben Peacock 3 ; Roman Fischer 4

1 University of Oxford, Oxford, United Kingdom; 2 Universidad Autonoma de Madrid, Madrid, Spain; 3 NanoFCM Co., Ltd., Nottingham, United Kingdom; 4 Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, Oxford, United Kingdom

Introduction : Here we present bead‐supported lipid bilayers as synthetic antigen‐presenting cells (sAPCs) to study the output of T cell synapses, which contains different extracellular particles, including trans‐synaptic vesicles (tSV) and supramolecular attack particles (SMAPs).

Methods : We developed bead supported lipid bilayers containing physiological densities of immune receptors and adhesion molecules to instigate the formation of T cell immunological synapses and capture the trans‐synaptic particles released by T cells. Using this methodology we quantitatively study the composition and biogenesis of trans‐synaptic vesicles (tSV) using TIRF and Airyscan microscopy, Multicolor, Nano and Spectral Flow Cytometry, and proteomics and small RNA sequencing.

Results : We discuss the various applications of sAPCs to characterise trans‐synaptic vesicle composition including flow cytometry, proteomics and sncRNA sequencing. Among others, we used sAPCs to compare the compositional hallmarks of trans‐synaptic vesicles and steadily released EVs. We also discuss the use of the quantitative flow cytometry data to reconstitute sAPCs as well as artificial vesicles. As a proof of concept, we show how the application of the quantitative information extracted using sAPCs helps to generate synthetic trans‐synaptic vesicles working as growth factors to establish human lymphoid organoids.

Summary/Conclusion : APCs represent a good model to dissect elements participating in the biogenesis of T cell trans‐synaptic particles. The application of different OMICs also enables the dissection of key trans‐cellular effectors, including juxtacrine signals recruited at the cell‐cell interface and sorted into trans‐synaptic vesicles as a result of their binding to cognate ligands.

Funding : P.F.C.D was supported by EMBO Long‐Term Fellowship (ALTF 1420‐2015, in conjunction with the European Commission (LTFCOFUND2013, GA‐2013‐609409) and Marie Sklodowska‐Curie Actions) and Oxford‐Bristol Myers Squibb Fellowship.

Keywords : Helper T cells, cytotoxic T lymphocytes, regulatory T cells, TSG101, ESCRT, CD81, CD63, CD40L, ADAM10, BST2, CAR T cells, CD4, BSLBs, microRNAs, target discovery platform, CRISPR/Cas9, Human immunology.

Protocol to measure the concentration and size distribution of extracellular vesicles with microfluidic resistive pulse sensing

Mona Shahsavari 1 ; Rienk Nieuwland 2 ; Ton van Leeuwen 3 ; Edwin van der Pol 4

1 Department of Biomedical Engineering and Physics & Department of Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; 2 Department of Clinical Chemistry; Vesicle Observation Center; Amsterdam University Medical Center, University of Amsterdam, Amsterdam, Netherlands; 3 1. Amsterdam UMC location University of Amsterdam, Biomedical Engineering and Physics, Meibergdreef 9, Amsterdam, the Netherlands 2. Amsterdam Cardiovascular Sciences, Atherosclerosis and Ischemic Syndromes, Amsterdam, the Netherlands 3. Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, the Netherlands, Amsterdam, Netherlands; 4 Department of Clinical Chemistry, Vesicle Observation Center, Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands

Introduction : Microfluidic resistive pulse sensing (MRPS; nCS1, Spectradyne) is based on the Coulter Principle, the most accurate technology to size single particles in solution. However, MRPS is laborious because the pore and interior of the microfluidic chip need to be wet. To wet the chip, a wetting agent is added to reduce the surface tension of biological samples. Previously, we used bovine serum albumin (BSA), which leaves extracellular vesicles (EVs) intact but insufficiently decreases the surface tension, thereby hampering measurements. Here, a practical and reliable MRPS protocol was developed using non‐ionic surfactants, Poloxamer‐188, and Tween 20, as wetting agents added to an EV‐containing test sample.

Methods : A cell‐free outdated erythrocyte concentrate sample was used as the EV test sample. To find the percentage of labeled EVs out of all particles, EVs were labeled with CD235a or Lactadherin and measured with flow cytometry (A60‐Micro, Apogee; lower detection limit ∼145 nm). To study the effect of surfactants on EVs, the unstained sample was diluted in filtered Dulbecco's phosphate‐buffered saline (DPBS), or DPBS containing 0.1% BSA (w/v), 0.05% Poloxamer‐188 (v/v), 0.1% Triton X‐100 (v/v), or 1% Tween 20 (v/v), and measured with flow cytometry.

Results : Of all particles, 65% were CD235a+ or Lactadherin+ EVs. About 90% of these particles were susceptible to detergent lysis with Triton X‐100, confirming they are indeed EVs. The total concentration of unstained particles was 1.89E+10 in DPBS, 1.97E+10 in BSA, 1.94E+10 in Poloxamer‐188, and 2.07E+10 in Tween 20. Whereas the total concentration of EVs was unaffected by the presence of Poloxamer‐188 or Tween 20, the size distribution in Tween 20 differed from DPBS, and DPBS containing BSA or Poloxamer‐188. The concentration of EVs smaller than 235 nm and of EVs larger than 235 nm increased by 54% and decreased by 34% in Tween 20 compared to positive controls (DPBS, and DPBS containing BSA), respectively.

Summary/Conclusion : To investigate the effect of surfactants on EVs, not only the total concentration of particles should be taken into account, but also the size distribution should be considered because variations in size distribution do not necessarily affect the total particle concentration. Thus, Poloxamer‐188 can safely be used in MRPS to size particles, including EVS, in suspension.

Funding : M. Shahsavari acknowledges funding from the Dutch Research Council (NWO), grant P18‐26: Medical Photonics (MEDPHOT)

Removal and identification of protein corona members of extracellular vesicles using surface covering antimicrobial peptides

Imola Csilla Szigyártó 1 ; Priyanka Singh 2 ; Mayra Queme Pena 2 ; Anikó Gaál 3 ; Lívia Fülöp 4 ; Lilla Turiák 5 ; Zoltán Varga 3 ; Tamas Beke‐Somfai 2

1 Research Centre for Natural Science, Institute of Materials and Environmental Sciences, Budapest, Hungary; 2 Research Centre for Natural Science, Budapest, Hungary; 3 Biological Nanochemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest, Hungary; 4 Department of Medical Chemistry, University of Szeged, Szeged, Hungary; 5 Research Centre for Natural Sciences, Institute of Organic Chemistry, Budapest, Hungary, Budapest, Hungary

Introduction : Extracellular vesicles (EVs), these cell‐derived nanoparticles, in the last years have shown great interest and extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on EV function, on their targeting and thus on their therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve.

Methods : Here we have employed three well‐characterized membrane active peptides: CM15, LL‐37 and FK‐16, which were recently reported to preferentially exert their membrane‐related activity via the surface‐covering carpet model. To test their removing capacity of EVs protein corona, red blood cell‐derived vesicles (REVs) were isolated. The interaction between peptides and EVs were followed by flow‐linear dichroism spectroscopy, which provided insight on the effective applicable concentration for EVs treatment. Samples were then subjected to subsequent size exclusion chromatography, microfluidic resistive pulse sensing measurements and proteomics analysis.

Results : Based on the results, antimicrobial peptides have reduced the protein content of the treated REV fractions, where TEM images clearly demonstrate a smooth vesicle surface lacking protein aggregates. Comparing the identified proteins for the control and peptide treated EVs, fifteen proteins were assigned as corona members, which were removed from surface by at least two antimicrobial peptides in separate experiments. 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 antimicrobial peptides as a useful tool in specific surface engineering of nanoparticle from both synthetic and natural origin.

Funding : This work was funded by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021‐EGA‐31 and the 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021 funding schemes. Support from Eötvös Loránd Research Network, grant n.o. SA‐87/2021, is also acknowledged.

Keywords : red blood cell derived vesicles, protein corona, membrane active peptides, proteomic analysis

OS33: Late‐Breaking Session: EVs and Cancer I

Chair: Emma Symonds, n/a – Department of Surgery and Anaesthesia, University of Otago, Wellington, New Zealand

Chair: Carlos Salomon, MSc, DMedSc, PhD – The University of Queensland

Charge matters – identifying extracellular vesicle subpopulations in melanoma patients by free‐flow electrophoresis

Miriam Zentgraf 1 ; Zeldali Lourens 1 ; Jan Van Deun 1 ; Gerhard Weber 2 ; Andreas Baur 1

1 Universitätsklinikum Erlangen, Erlangen, Germany; 2 FFE Service GmbH, Feldkirchen, Germany

Introduction : The biomarker potential of extracellular vesicles stems from a dysregulated secretion in pathological conditions. While the identification of EV subpopulations based on size, density or surface proteins is well established, an underexposed characteristic of EVs is their surface charge. Free‐flow electrophoresis (FFE) is a gentle, matrix‐free and semi‐preparative method that isolates analytes according to their isoelectric point. Here, we report the use of FFE for the charge‐based separation of EV subpopulations in melanoma. This technology opens new perspectives for establishing EV‐based biomarkers.

Methods : EVs were isolated from cell culture by size‐exclusion chromatography (SEC) or density gradient (DG), resulting in preparations of variable purity. Plasma samples of control subjects and melanoma patients were pre‐processed by dual‐mode chromatography (DMC) to enrich EVs. The FFE instrument was primed with separation buffers spanning a pH range of 4.8‐7.9 and calibrated using pI marker dyes. Concentrated EV samples were introduced into the chamber, separated according to pI and collected into 96‐well plates. Engineered fluorescent EVs were used to optimize the separation conditions. Presence of EV‐associated proteins in different fractions was measured using a plate reader. Relevant fractions were pooled, concentrated by ultrafiltration and characterized by Western blotting and nanoparticle tracking analysis.

Results : By comparing the separation of SEC and DG EVs, we could demonstrate that a successful separation of EV populations by charge depends on their purity. SKMEL‐37 melanoma cell EVs were found to split into different populations, which had a variable relative expression of tetraspanins CD63, CD9 and CD81. Next, comparing DMC‐purified EVs extracted from plasma of control subjects and melanoma patients before and after excision of the primary tumor, we found that melanoma patients are characterized by an EV subpopulation with an increased negative charge.

Summary/Conclusion : Our results qualify DG/DMC‐FFE as a novel procedure for the charge‐based separation of EV populations. These previously unexplored subpopulations could function as promising biomarkers in melanoma.

Keywords : extracellular vesicles, surface charge, subpopulations, melanoma, dual‐mode chromatography, free‐flow electrophoresis

Definition of HDL and EV particles in prostate cancer monitoring

Liliia Paniushkina 1 ; Martin Wolf 2 ; Laura Bianciardi 3 ; Natasa Zarovni 4 ; Dirk Strunk 2 ; Irina Nazarenko 5

1 Paracelsus Medical Private University, Salzburg, Salzburg, Austria; 2 Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI ‐ TReCS), Paracelsus Medical University (PMU), Salzburg, Austria, Salzburg, Austria; 3 Exosomics, Siena, Italy; 4 HansaBioMed Life Sciences Ltd, Mäealuse 2/1, 12618 Tallinn, Estonia, Tallinn, Estonia; 5 Institute for Infection Prevention and Hospital Epidemiology, Faculty of Medicine, University Medical Center Freiburg, Freiburg, Germany, Freiburg, Germany

Introduction : Isolation of purified EV populations from plasma remains a conceptual and methodological topic of discussion in the field. While some studies exclude lipoprotein‐enriched fractions from characterization, we compared proteins and oncogenic signatures in low and high HDL fractions from different EV subtypes in plasma from prostate cancer (PC) and castration‐resistant prostate cancer (CRPC) patients. Our results demonstrate the specific formation of HDL‐EV complexes in progressing prostate cancer.

Methods : ACD plasma samples from patients with BPH, PC and CRPC were analysed regarding platelet and haemolysed RBC. Four size‐based EV subtypes were isolated from contamination‐free samples using differential centrifugation (DC) followed by iodixanol density gradient purification. Each of the fractions was characterized by Nanoparticle Tracking Analysis (NTA), Tunable Resistive Pulse Sensing (TRPS), Interferometric light microscopy (ILM) followed by ELISA, MACSPlex and Transmission electron microscopy (TEM). The CD63, CD9 and CD81 were used as EV markers, ApoB and ApoA1 ‐ for lipoproteins, CD3, CD41 and PSMA as a tissue‐specific biomarker. HDL and EV‐contained fractions were subjected to Next Generation Sequencing (NGS). Differentially expressed genes were characterized using the oncogenic signature pathways library.

Results : High‐ and low‐density fractions from PC and CRPC plasma have different EVs and lipoproteins levels. CRPC plasma‐isolated EV samples contain more CD41 and ApoA1 proteins than PC. The fraction at 1,10 g/ml density was highly enriched on oncogenic signatures (OS) in the CRPC case only. The OS present in the EV5 sample from CRPC plasma is involved in immune system suppression, activation of scavenging by Class A receptors, increase of post‐translational protein modification and activation of vesicle‐mediated transport.

Summary/Conclusion : The characterisation of high‐density particles from plasma samples with PC and CRPC diagnoses is informative. Specific formation of HDL‐EV complexes might signify prostate cancer progression.

Funding : European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska‐Curie grant agreement No 722148.

Keywords : prostate cancer, ACD plasma, oncogenic signatures, HDL

Diagnostic performance evaluation of small RNA biomarkers in urinary EVs from prostate cancer patients

Elena S. Martens‐Uzunova 1 ; Natasja Dits 1 ; Jozien Heleman 1 ; Martin E. E. van Royen 2 ; Irene Bijnsdorp 3 ; Gerald Verhaegh 4 ; Connie Jimenez 5 ; Jack Schalken 4 ; Guido W. W. Jenster 1

1 Department of Urology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands; 2 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands, Rotterdam, Netherlands; 3 Department of Urology, Amsterdam UMC, Location VUMC, Amsterdam, Netherlands; 4 Department of Urology, Radboud UMC, Nijmegen, Netherlands; 5 Department of Medical Oncology, OncoProteomics Laboratory, Cancer Center Amsterdam, Amsterdam UMC, Location VUMC, Amsterdam, Netherlands

Introduction : Early identification of clinically significant disease remains a major challenge in the treatment of prostate cancer (PCa) patients. A warranted solution is the development of a minimally‐invasive test that can detect aggressive PCa and prevent repeated biopsies. Here, we explored the potential of a group of small RNA (sRNA) biomarkers (IP evaluation pending) previously identified in PCa tissue for their presence in urinary extracellular vesicles (uEVs) and their performance as minimally‐invasive, liquid biopsy biomarkers.

Methods : Spot urines were collected at 3 Dutch medical centers after digital rectal examination and prior to diagnostic biopsy. Informed consent was obtained from all patients. Urines were precleared by 20 min centrifugation at 500 G within 4 h after collection. Creatinine and urinary PSA (uPSA) were determined by standard clinical laboratory tests. Total uEV RNA was isolated from 2x4 ml supernatant aliquots with Norgen urine exosome RNA isolation kit. uEV quantity, CD9, and CD63 were determined by (immuno)EVQuant and TR‐FIA. Duplex qPCR was done with TaqMan advanced kit and custom probes. Differences between groups were assessed by Kruskal‐Wallis test with Dunn's test for multiple comparisons. Biomarker performance was assessed by receiver operating characteristic (ROC) curve analysis.

Results : Evaluation of sRNA performance was done in 3 assessment rounds. 2 out of 11 tested sRNAs (sRNAx2) were selected based on their expression and ability to detect aggressive PCa (Gleason score >7) for final evaluation in a cohort of 107 PCa negative and 221 PCa positive patients (Gleason score: 6, n=64; 7, n=98; >7, n=59). There was no significant difference in creatinine levels among the groups. However, uPSA levels were significantly lower in patients with aggressive PCa, indicating lower proportion of prostate fluid and prostate derived EVs in the urine of patients from this group. Therefore sRNAx2 values were normalized to uPSA. Normalized sRNAx2 values were significantly higher for patients with aggressive PCa compared to indolent PCa (Gleason score 6, adjusted p = 0.002) or no PCa (adjusted p = 0.011). ROC curve analysis of the diagnostic ability of sRNAx2/uPSA ratio returned an AUC= 0,682 (p = 0.001) and an AUC = 0,655 (p = 0.0005) for the indolent PCa and for the no PCa groups, respectively.

Summary/Conclusion : Urinary EV sRNAx2/uPSA ratio can classify patients with high grade PCa and has a potential as an early liquid biopsy biomarker for aggressive PCa.

Funding : The IMMPROVE consortium (Innovative Measurements and Markers for PROstate cancer diagnosis and prognosis using extracellular VEsicles) is supported by an Alpe d'HuZes & Dutch Cancer Society grant (KWF grant number #EMCR2015‐8022).

Keywords : prostate cancer, diagnosis, urinary extracellular vesicles, small RNA

Extracellular vesicles released from leptin‐treated breast cancer cells rewire cell metabolism: a proteomic approach

Luca Gelsomino 1 ; Amanda Caruso 2 ; Francesca Giordano 2 ; Matteo Brindisi 2 ; Giovanna Morello 3 ; Felice Maria Accattatis 2 ; Ines Barone 2 ; Salvatore Panza 2 ; Daniela Bonofiglio 2 ; Sebastiano Andò 2 ; Cinzia Giordano 2 ; Stefania Catalano 2

1 University of Calabria, arcavacata di Rende, Italy; 2 University of Calabria, Arcavacata di Rende, Italy; 3 National Research Council, Catania, Italy

Introduction : The obesity hormone leptin acting in autocrine, paracrine and endocrine manner deeply influences breast cancer (BC) biology thus recognized as a crucial mediator in cell‐to‐cell communication. Recently, we demonstrated that leptin increases extracellular vesicle (EV) biogenesis and release in BC cells. Here, we focused on the identification of a specific leptin‐induced EV proteomic signature in BC cells in attempt to find molecular effectors associated with BC progression.

Methods : EVs from conditioned media of MCF‐7 BC cells have been isolated by ultracentrifugation method and, accordingly to MISEV guidelines, characterized by transmission electron microscopy, nanoparticle tracking analysis and immunoblot. EV proteomic profile was obtained by liquid chromatography‐tandem mass spectrometry analysis (LC‐MS/MS), and analyzed by MaxQuant Software. Gene set enrichment analysis (GSEA) was used to explore gene ontology (GO) categories enriched in leptin‐derived EVs. ATP assay, MitoTracker Deep Red/Orange and Seahorse XFe96 analysis were performed to assess energy production and mitochondrial metabolism.

Results : LC‐MS/MS of EV cargo allowed the identification of 38 differentially expressed proteins (FDR< 0.2) in EV from leptin treated MCF‐7 cells considering |1.5| fold as a cut‐off. GSEA revealed an enrichment in GO involved in energy production and mitochondrial metabolism in leptin‐derived EVs compared to the untreated one. Functional studies revealed that leptin‐derived EVs significantly increased ATP production, mitochondrial activity and oxidative phosphorylation in BC cells.

Summary/Conclusion : Our data indicate that leptin through the release of EV‐enriched mitochondrial proteins may rewire metabolism of BC cells. Characterization of tumor‐derived EV protein cargo in obesity‐associated milieu, as in the presence of elevated leptin levels, might allow to identify unique features and specific biological processes to be targeted in clinical management of disease.

Funding : Department of Pharmacy, Health and Nutritional Sciences of University of Calabria (Department of Excellence, Italian Law 232/2016) MIUR; AIRC IG #21414; PRIN 2017 #2017EKMFTN_001 to S. C.; PRIN 2017 #2017WNKSLR_005 to I.B; SILA PONa3_00341.

Keywords : leptin, breast cancer, cell metabolism

Heparin treatment prevents tumour‐derived EV‐induced epithelial‐to‐mesenchymal transition: A novel bioelectronic platform for screening anti‐cancer drugs

Walther Traberg‐Christensen ; Roisin Owens

University of Cambridge, Cambridge, United Kingdom

Introduction : Tumor cell‐derived EVs (TEVs) have recently been reported to play an active role in tumorigenesis and metastasis owing to their ability to transmit oncogenes. One hypothesis is that TEVs contribute to metastasis by inducing the epithelial‐to‐mesenchymal transition (EMT), characterized by the loss of barrier function in barrier tissue‐forming cells. Developing drugs that directly target TEVs and block TEV‐induced EMT is crucial for improving clinical outcomes in cancer.

Methods : Current methods for measuring EV function lack the ability to obtain quantitative data in a non‐disruptive, label‐free, and continuous manner. We have shown that organic electronic devices based on the conducting polymer PEDOT:PSS can be interfaced with biological systems of varying complexity allowing for quantitative real‐time monitoring of biological interactions with excellent sensitivity. The optical transparency of organic electrochemical transistors (OECTs) based on PEDOT:PSS allows for multiplex analysis with both optical and electrical readouts. The malignant transformation of healthy breast epithelial cells (MCF‐10A) after exposure to EVs derived from triple‐negative metastatic breast cancer cells (MDA‐MB‐231) was monitored using OECTs and orthogonal validation by immunoblotting, immunofluorescence imaging, DNA methylation, and ELISA was performed to validate the electrical measurements.

Results : We show that CD63‐positive TEVs can induce an EMT‐like process in MCF‐10A cells as demonstrated by changes to cell morphology, loss of apico‐basal polarity, reorganization of filamentus actin, and gain of mesenchymal proteins, such as vimentin and N‐cadherin. The decrease in barrier integrity of the cell monolayer was monitored electrically using OECTs, gaining of truly quantitative insight into TEV‐induced EMT with higher temporal resolution than conventional, orthogonal methods. Having established this, we demonstrated that heparin ‐ a common anticoagulant drug ‐ successfully abrogates this malignant transformation and prevented TEV‐induced EMT.

Summary/Conclusion : Importantly, our OECT platform allowed us to monitor the transformational state of the cells in real‐time, providing unprecedented temporal resolution of EMT and revealing that persistent heparin treatment is necessary to prevent TEV‐induced EMT. The non‐disruptive and continuous measurements enabled by this bioelectronic platform allow researchers to test the efficacy of anti‐cancer, and especially anti‐metastatic drugs, faster and using fewer resources than conventional methods, opening up the possibility for high‐throughput drug screening.

Keywords : Cancer metastasis, EMT, heparin, anti‐cancer drug, drug screening, screening platform

OS36: Late‐Breaking Session: EVs and Cancer II

Chair: Liliia Paniushkina, PhD – Paracelsus Medical Private University, Salzburg

Chair: Nicholas Peake – Sheffield Hallam University

Extracellular vesicle‐based delivery of therapeutic siRNAs alleviates skeletal muscle loss and prolongs survival of cancer cachectic mice

Boya Peng 1 ; Yuqi Yang 1 ; Migara Kavishka K. Jayasinghe 1 ; Rachel Tan 1 ; Marco Pirisinu 2 ; Thach Tuan Pham 1 ; Kun Liang 3 ; Shyh‐Chang Ng 3 ; Minh T.N. T.N Le 1

1 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, Singapore; 2 Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, Hong Kong, Hong Kong; 3 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China (People's Republic)

Introduction : RNA drug delivery to the muscle is a promising strategy for the treatment of muscle degenerative diseases but it is challenging because myocytes are hard to transfect. Here, we utilize red blood cell extracellular vesicles (RBCEVs) to deliver siRNAs to skeletal muscles for cancer cachexia treatment based on our previous observation that small RNAs can be loaded efficiently into RBCEVs. Cancer cachexia is the loss of skeletal muscle commonly seen in cancer patients. It not only has an adverse impact on quality of life, but is also associated with poor responses to chemotherapy and poor survival.

Methods : RBCEVs were purified from RBCs of healthy donors by ultracentrifugation and characterized by nanoparticle tracking analysis, western blotting and nanoanalyzer flow cytometry. We studied the uptake of RBCEVs by myotubes both in vitro and in vivo using immunostaining. siRNAs were designed to target Myostatin (Mstn), a negative regulator of skeletal muscle growth and differentiation, and Malonyl‐CoA decarboxylase (Mlycd), an enzyme driving the malfunctioned metabolism of cancer cachexia. We assessed the therapeutic effects of siRNAs against Mstn and Mlycd when delivered using RBCEVs via intramuscular administration in a mouse model of lung cancer with cachexia.

Results : RBCEVs were robustly taken up by C2C12 myotubes in culture and by primary myotubes in the quadriceps upon intramuscular injection. The EV uptake was mediated by endocytosis. Delivery of Mstn siRNA and Mlycd siRNA using RBCEVs notably inhibited endogenous Mstn and Mlycd, and increased skeletal muscle growth without considerable side effects in wild‐type mice. In cancer cachectic mouse model, we observed significant alleviation of skeletal muscle loss and prolonged survival rate after multiple intramuscular administrations of Mstn siRNA‐loaded and Mlycd siRNA‐loaded RBCEVs. Furthermore, blood chemistry tests of the mice demonstrated that the intramuscular delivery of siRNAs using RBCEVs was non‐toxic and non‐inflammatory.

Summary/Conclusion : In this study, we have characterized the uptake of RBCEVs by myotubes and demonstrated potential therapeutic properties of RBCEV‐delivered Mstn siRNA and Mlycd siRNA in cancer cachexia treatment. Hence, this is a safe and efficient RNA drug delivery strategy for muscle degenerative diseases.

Influence of age on fibroblast‐derived extracellular vesicles and their role in melanoma progression

Laura Hueser 1 ; Yash Chhabra 1 ; Olesia Gololobova 2 ; Mitchell Fane 1 ; Kenneth W. Witwer 2 ; Ashani Weeraratna 1

1 Johns Hopkins University, Baltimore, USA; 2 Johns Hopkins University School of Medicine, Department of Molecular and Comparative Pathobiology, Baltimore, USA

Introduction : Age is an important prognostic factor in cutaneous melanoma, commonly arising in the elderly. Melanoma progression is the concerted outcome of cellular and molecular changes occurring in tumor cells and within the milieu of the stromal cells, collectively referred to as the tumor microenvironment (TME). Also, TME‐secreted growth factors, cytokines, and extracellular matrix (ECM) are vital to tumor progression and therapy resistance. Aging causes remodeling of the TME making it conducive to melanoma progression. Extracellular vesicles (EVs) have been shown to transport lipids, mRNAs, and functional proteins across several cell types. Considering their important role in tumor progression in different cancer types, investigating how an aged tissue microenvironment can alter EV attributes and cargo remains to be evaluated.

Methods : Conditioned media of age‐stratified dermal fibroblasts (dFs), the predominant stromal cell population of the melanoma TME, was concentrated and subsequently EVs were isolated using size separation via Izon (consistent with MISEV2018). Next, size and concentration of EVs were analyzed via NTA and mass spectrometry was performed to investigate how systemic aging changes the EVs released by dFs. We also sought to evaluate the influence of these EVs on melanoma progression.

Results : Our study shows that the aged secretome significantly promotes melanoma cell invasion and that this effect can be significantly reduced by EV depletion. We found that the tetraspanin CD9 was decreased in aged dFs and their released EVs versus young dFs. Reduction in CD9 expression in dFs was sufficient to significantly reduce the proliferation of melanoma cells while significantly increasing melanoma cell invasion.

Summary/Conclusion : Thus, we can conclude that with age, EVs released by dFs experience cargo changes and promote tumor progression. EVs are thus another mechanistic avenue to age‐associated melanoma progression and therapy resistance.

Funding : Deutsche Forschung Gesellschaft (DFG) Post Doc Fellowship

Keywords : Melanoma, tumor microenvironment, age

RNA‐Seq and Mass Spec profiles of cancer‐derived EVs overlap in distinct EV fractions

Tatyana Vagner 1 ; Elizabeth Hutchins 2 ; Wenyan Zhao 3 ; Yari Ciani 4 ; Federico Vannuccini 4 ; Andrew R. Chin 1 ; Javier Mariscal 1 ; Minhyung Kim 1 ; Sungyong You 1 ; Agnes Kittel 5 ; Krizia Sagini 6 ; Marco De Simone 7 ; Edit I. Buzás 8 ; Wei Yang 1 ; Clotilde Théry 9 ; Francesca Demichelis 4 ; Paul Boutros 10 ; Kendall Jensen 2 ; Dolores Di Vizio 1

1 Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars‐Sinai Medical Center, Los Angeles, CA, USA, Los Angeles, USA; 2 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA; 3 Department of Human Genetics, Jonsson Comprehensive Cancer Center, Institute for Precision Health, University of California Los Angeles, Los Angeles, USA; 4 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, 38123 Trento, Italy, Trento, Italy; 5 Institute of Experimental Medicine, Budapest, Hungary; 6 Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 7 Department of Radiation Oncology, Cedars‐Sinai Medical Center, Los Angeles, USA; 8 Semmelweis University Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary; 9 Institut Curie, PSL Research University, INSERM U932, Paris, France; 10 Department of Human Genetics, Jonsson Comprehensive Cancer Center, Institute for Precision Health, Broad Stem Cell Research Center, Department of Urology, University of California Los Angeles, Los Angeles, USA

Introduction : To identify general and EV population‐specific proteins and transcripts, we performed quantitative proteomic and transcriptomic analyses of 3 EV fractions obtained from prostate cancer, glioma, and breast cancer cell lines.

Methods : Differential centrifugation at low, medium, and high speed (2.8K, 10K, 100K) and density gradient, TRPS, TEM, LC‐MS/MS, RNA‐Seq, Flow Cytometry, SP‐IRIS.

Results : Quantitative proteomic analysis of 3 EV fractions showed that proteins considered to be enriched in exosomes are present in all EVs and can be used as general EV markers. The most distinct protein and RNA expression patterns were identified in the 2.8K and 100K fractions, while the 10K EVs represented a mixture of the two. The correlation between the proteins and protein‐coding mRNA transcripts was higher in 2.8K vs 10K, and higher in 10K vs 100K, with the 2.8K and 10K EVs exhibiting the mRNA profiles more similar to each other than to the 100K EVs. Additionally, the protein cargo correlations between the 2.8K and 10K (0.64) and between the 10K and 100K (0.84) were lower than the mRNA correlations between these fractions (0.94 and 0.91, respectively), potentially indicating that the identity of the EV populations is defined by their protein cargo more than by their mRNA cargo. We also noticed that the mRNA transcripts in 2.8K and 10K are more similar to each other than the mRNA transcripts in 10K and 100K, while the proteins in 10K and 100K are more similar to each other than the proteins in 2.8K and 10K, indicating that 2.8K and 10K are more similar to each other at the mRNA level, whereas 10K and 100K are more similar at the protein level. Pathway analysis demonstrated that large EVs (2.8K and 10K) are enriched in pathways involved in essential metabolic activities including mitochondrial functions. The enrichment of mitochondria transcripts was also supported by single vesicles RNA sequencing of the 10K EVs. Finally, we identified several internal and surface proteins as novel markers of EV populations.

Summary/Conclusion : Comprehensive proteomic and transcriptomic analyses identify specific protein and mRNA profiles in EVs suggesting that cancer pathways are conserved at the protein and RNA level in EVs.

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

Keywords : EV heterogeneity, mass spectrometry, RNA‐seq, proteomics, transcriptomics, cancer

The role of small extracellular vesicles from head and neck cancer patients in tumor angiogenesis

Luisa Tengler 1 ; Marie‐Nicole Theodoraki 2 ; Julia Schütz 3 ; Frederic Jungbauer 3 ; Anne Lammert 3 ; Nicole Rotter 3 ; Sonja Ludwig 3

1 Medical Faculty Mannheim of Heidelberg University, Germany, Mannheim, Germany; 2 Department of Otorhinolaryngology, Head and Neck Surgery, Ulm University Medical Center, Ulm, Germany, Ulm, Germany; 3 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Mannheim, Germany, Mannheim, Germany

Introduction : Angiogenesis is a key regulatory mechanism in the carcinogenesis of head and neck cancer (HNC). While small extracellular vesicles (sEVs) from HNC cell lines (TEX) reprogram endothelial cells (ECs) and promote angiogenesis, the role of patient plasma sEVs still remains unclear.

Methods : sEVs were isolated from plasma of 24 HNC patients (HNC), 10 recovered patients (NED) and 9 healthy donors (HD) by size‐exclusion chromatography. The vesicles were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blots (WBs), respectively. The interaction of sEVs with human umbilical vein ECs (HUVECs) was visualized by confocal microscopy (CM). The angiogenesis‐related protein profiles of sEVs were examined via antibody arrays. Functional assays were performed to analyze the effect of sEVs on the proliferation, migration and tubulogenesis of HUVECs.

Results : Plasma sEVs showed typical shape in TEM, size in NTA and protein markers in WBs and were effectively internalized by HUVECs. Antibody arrays confirmed an enrichment of anti‐angiogenic proteins in all sEVs, while the pro‐angiogenic protein MMP‐9 was significantly overexpressed in sEVs from HNC compared to ND sEVs. In functional assays, sEVs from HNC patients inhibited proliferation, migration and tubulogenesis of HUVECs, however, to a significantly lesser extent than HD and NED sEVs.

Summary/Conclusion : Angiogenesis is regulated by a balance between inhibitors and stimulators. In contrast to TEX, HNC sEVs show rather inhibiting effects on ECs. Since plasma contains a mixture of sEVs from different origins, HNC plasma sEVs might shift this balance in favor of a less anti‐angiogenic side in comparison to NED and HD sEVs.

Funding : German Research Foundation (DFG) and the Foundation for Cancer and Scarlet Fever Research of the University Heidelberg

Keywords : sEV, angiogenesis, head and neck cancer, HUVEC

OS34: Late‐Breaking Session: EVs and Viruses

Chair: Kenneth W. Witwer, n/a – Johns Hopkins University School of Medicine

Chair: Peter E. Kima – University of Florida

OS34 Astrocytes & astrocyte derived extracellular vesicles in HIV associated neurological disorders: HIF‐1α‐BACE1AS axis

Susmita Sil 1 ; Divya Chemparthy 2 ; Shannon Callen 2 ; Shilpa Buch 3

1 Department of Pharmacology & Experimental Neuroscience, Omaha, USA; 2 UNMC, Omaha, USA; 3 University of Nebraska Medical Center, Omaha, USA

Introduction : Although the advent of antiretoivrals has dramatically increased the lifespan of HIV+ individuals, paradoxically, long‐term dependence on these toxic medications, is also associated with an increased risk of Alzheimer's‐like pathology – an emerging comorbidity of HIV‐Associated Neurocognitive Disorders (HAND). Based on our previous findings that astrocytes play a major role in HIV Tat‐mediated amyloidosis, and, since amyloids are released in extracellular vesicles (EVs), we sought to assess whether HIV‐1‐Tat stimulated astrocyte derived EVs (ADEVs): a) could mediate synaptodendritic injury in vitro in hippocampal neurons and, b) when injected stereotactically into the brains of naïve mice could mediate cognitive impairment. Our previous studies have demonstrated the role of HIF‐1α as an upstream regulator of HIV1‐Tat mediated astrocytuc amyloidosis. We thus hypothesized that blocking HIF‐1α could likely mitigate neuronal injury.

Methods : Tat‐ADEV uptake by the neurons was assessed by mcherry plasmid tagged EVs, and synaptic injury by confocal microscopy & analysis in Neurolucida software. Behavioral assays included Y‐maze study, locomotor, anxiety & fear conditioning.

Results : Rat hippocampal neurons exposed to Tat‐ADEVs carrying amyloids led to reduction of dendritic arborization, spines, ratio of spine sub‐types, synaptic proteins & post‐synaptic currents. Silencing of astrocytic HIF‐1α not only reduced the numbers of ADEV release and their amyloid cargos, but also ameliorated Tat‐ADEV induced neuronal synaptodendritic injury. Interestingly injection of Tat‐ADEVs carrying amyloids in the hippocampus of naïve mice brains, resulted in reduction of excitatory synapse densities, increased chromatolysis in neurons, increased amyloid deposition, and enhanced glial cell activation and neuroinflammation. Concomitantly these animals showed impairment of long‐term memory, locomotor activity as well as anxiety like behavior. This impairment(s) was abrogated in mice injected with HIF‐1α silenced ADEVs.

Summary/Conclusion : This study underscores the role of amyloid carrying ADEVs in mediating synaptodegeneration leading to cognitive impairments associated with HAND, and highlights the protective role of HIF‐1α in this process.

Funding : This work was supported by grants R21AG069541 and John Hopkins University, R25MH080661 (S. Sil) from National Institute of Health.

Keywords : HIF‐1α, HAND, ADEVs, synaptodegeneration, memory

OS34 HIV‐1 Nef changes the protein content of T cells extracellular vesicles promoting the reduction of the antiviral proteins IFITMs

Mara E. da Silva‐Januário 1 ; Cristina da Costa 2 ; Ana Karina Oliveira 3 ; Lucas tavares 2 ; Yunan Januário 2 ; Andreia de Carvalho 2 ; Michael Miller 4 ; Clarice Arns 4 ; Adriana Leme 3 ; Luis L. da Silva 2

1 Ribeirão Preto Medical School ‐ University of São Paulo, Brazil, Ribeirão Preto, Brazil; 2 Virology Research Center ‐ Ribeirão Preto School of Medicine ‐ University of Sao Paulo, Ribeirão Preto, Brazil; 3 CNPEM, Campinas, Brazil; 4 UNICAMP, Campinas, Brazil

Introduction : EVs carry a repertoire of molecules involved in intercellular communication and function as vectors in the transference of proteins involved in defense against virus infections. Conversely, viruses counteract EVs with antiviral activity by modifying their content to favor infection. Nef is a multifunctional accessory protein of HIV that contributes to higher viremia and disease progression. Nef achieves these goals by modifying the levels of several antiviral proteins in different compartments of the host cell, such as the plasma membrane, Golgi complex, and EVs.

Methods : We performed quantitative proteomics of EVs isolated from T cells to characterize the global changes in EVs protein content and identify novel targets of Nef. The EVs were separated by differential ultracentrifugation, washed, and characterized by NTA, SEM, and western blot. EVs content delivery was analyzed by western blot and EVs membrane dye associated with confocal microscopy. HIV challenge in acceptor cells was monitored by luciferase assays.

Results : Proteomic data showed an evident variation in the levels of specific proteins due to Nef expression. The antiviral protein IFITM2 presented reduced levels. Further analysis showed that Nef reduced the IFITM1, IFITM2, and IFITM3 in T cells EVs. Besides, Nef altered IFITM3 subcellular localization in T cells and Hela cells, excluding IFITMs from plasma membrane and lipid rafts domains. Previous works showed that IFITM3 transference by EVs confers protection against Dengue and Zika infection. Therefore, we investigated if IFITMs transference via EVs to acceptor cells would confer antiviral activity to HIV infection. There was an efficient delivery of IFITM3 via EVs to acceptor cells. Our preliminary data showed that HIV infectivity was reduced in acceptor cells of IFITM3‐containing EVs.

Summary/Conclusion : The described findings point Nef as a global modulator of EVs protein content. Our preliminary results suggest that IFITM3 transference by EVs may confer protection against HIV infection. We propose that Nef is an antagonist of antiviral IFITMs transference mediated by EVs.

Funding : CNPq and FAPESP.

Keywords : Extracellular vesicles, HIV, Nef, IFITMs, and antiviral activity.

OS34 Milk extracellular vesicles carry ACE2 and serve as natural decoys to inhibit pseudotyped SARS‐CoV‐2 virus

Martijn van Herwijnen 1 ; Susanne G. van der Grein 2 ; Lonneke Broeks 3 ; Marije Kleinjan 1 ; Berend‐Jan Bosch 2 ; Frank J.M. Van Kuppeveld 2 ; Marca H.M Wauben 4

1 Utrecht University, Utrecht, Netherlands; 2 Virology Section, Division Infectious Diseases & Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands; 3 Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands; 4 Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, Utrecht, Netherlands

Introduction : Human milk is a rich source of immunomodulatory and anti‐viral components that reduce the risk for enteric and respiratory virus infections in breastfed infants. For instance, extracellular vesicles (EVs) from milk have been shown to reduce rotavirus and respiratory syncytial virus infection. Yet, despite considerable research on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), and indications that breastfeeding protects infants from COVID‐19, the direct role of milk‐derived EVs on corona virus infection has not been elucidated.

Methods : In this study, we used mature milk that was donated by healthy mothers prior to the emergence of SARS‐CoV‐2 and purified EVs (and a procedural EV‐depleted control) via differential centrifugation, density gradient separation and SEC. Proteomic analysis, as well as immunoblotting was used to identify SARS‐CoV‐2 binding proteins on milk EVs. Functional activity of milk EVs was tested in vitro by incubating EVs with vesicular stomatitis virus (VSV) expressing firefly luciferase and pseudotyped with the SARS‐CoV‐2 spike protein, after which these were added to Vero E6 cells. Luciferase activity was determined after overnight incubation and served as a measure for viral entry. Additionally, milk EVs were treated with blocking antibodies against ACE2 prior to virus incubation to test the involvement of ACE2 in their function.

Results : Pre‐incubation of virus and milk EVs prior to infection reduced viral entry significantly in a dose‐dependent matter, while a procedural EV‐depleted milk control did not. Using proteomic analysis together with immunoblotting, we show that milk EVs express ACE2. We therefore investigated whether the inhibition of viral entry was caused by binding of the SARS‐CoV‐2 spike protein to EV‐associated ACE2. Indeed, blocking ACE2 on the milk EVs prior to virus incubation led to a reduction in viral entry inhibition.

Summary/Conclusion : By analysing pre‐pandemic expressed milk, we show that milk EVs are naturally endowed with the capacity to neutralize viral entry of SARS‐CoV‐2 and acts as decoys potentially via ACE2.

Funding : This work was supported by the European Union's Horizon 2020 Framework Programme under the grant FETOPEN‐801367 evFOUNDRY

Keywords : human milk SEC density gradient corona virus SARS‐CoV‐2 COVID‐19 ACE2

OS34 Proteomics characterization of circulating extracellular vesicles in COVID‐19 patients with different clinical manifestations

Maria Jesus Pinazo 1 ; Núria Cortés‐Serra 2 ; Alberto Ayllon‐Hermida 2 ; Melisa Gualdrón‐López 2 ; Patricia Resa‐Infante 3 ; Joan Josep Bech‐Serra 4 ; Sergio Montaner‐Tarbes 5 ; Iris Aparici Herraiz 2 ; Marc Nicolau‐Fernandez 6 ; Itziar Erkizia 3 ; Bernat Cucurull 4 ; Lucia Gutierrez‐Chamorro 3 ; Ester Ballana‐Guix 3 ; Silvia Marfil 3 ; Edwards Pradenas‐Saavedra 3 ; Carlos Ávila‐Nieto 3 ; Julia Bauzá‐Martinez 7 ; Franziska I Völlmy 8 ; Magdalena Muelas 1 ; Ruth Sotil 1 ; Núria Izquierdo‐Useros 3 ; Lorenzo Fraile 9 ; Maria Montoya 10 ; Julia Vergara 11 ; Joaquim Segalés 11 ; Jorge Carrillo 3 ; Julia Blanco 3 ; Carmen Fernandez Becerra 2 ; Carolina De la Torre 4 ; Javier Martinez‐Picado 12 ; Hernando A. del Portillo 13

1 ISGlobal, Barcelona, Spain; 2 ISGlobal IGTP, Barcelona, Spain; 3 IrsiCaixa, Badalona, Spain; 4 Institute Josep Carreras, Badalona, Spain; 5 Innovex Therapeutics S.L., Badalona, Spain; 6 ISGlobal IGTP, Badalona, Spain; 7 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, Utrecht, Netherlands; 8 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, utrecht, Netherlands; 9 Universitat de Lleida, Lleida, Spain; 10 CSIC‐CiB, madrid, Spain; 11 IRTA, Barcelona, Spain; 12 IrsiCaixa ICREA, Badalona, Spain; 13 ISGlobal IGTP ICREA, Barcelona, Spain

Introduction : Extracellular vesicles (EVs) released by viral‐infected cells can incorporate viral peptides. In this study, we hypothesised that the molecular characterization of EVs obtained from COVID‐19 patients will reveal novel immunogenic viral peptides and molecular signatures of disease severity.

Methods : Studies were ethically approved. Blood samples from COVID‐19 patients with severe (G1, N=19) mild/asymptomatic (G2, N=26) disease and controls (G3, N=12) were collected during active infection (T0) in early 2020 and 8 months’ (T8) post‐infection. Total and neutralizing antibody levels were determined. EVs from plasma were isolated by immunocapture (CD9), ganglioside‐capture (Siglec‐1) and size‐exclusion chromatography (SEC). In addition, we isolated EVs from SARS‐CoV2 infected hamsters. EVs were characterized by Western blot, BBA‐assay, NTA, and analysed by label‐free proteomics.

Results : Clinical data showed increased inflammation markers in G1. Neutralising antibodies in G1 non‐vaccinated individuals increased at T8, indicating sustained exposure to SARS‐CoV2 antigens. Proteomic analysis of EVs from G1 and G2 patients, failed to detect viral peptides. These results were validated by Western blot against Spike‐RBD. Moreover, we also failed to detect viral peptides in plasma‐derived EVs from hamsters with active (PCR+) SARS‐Cov2 infection. CD9‐enriched EVs showed significant upregulation of 140 proteins in G1, including proteins involved in the response to viral infection. Siglec‐1‐captured EVs showed significant enrichment of EV markers and upregulation of 26 proteins in G1, including 13 of the most upregulated in CD9+EVs in G1. SEC‐enriched EVs showed no significant differences in proteins in any group. Control individuals from G3 showed dramatic changes in the protein's abundance when comparing T0 vs T8, likely due to vaccination.

Summary/Conclusion : Proteomic profiling of EVs isolated by three different methods failed in identifying viral peptides associated with circulating EVs from COVID‐19 patients. Human EV cargo identified proteins with prognostic potential in severe disease.

Funding : SGlobal, IrsiCaixa, IJC, EPIC‐XS

Keywords : Plasma circulating EVs, Covid‐19 patients, different clinical syndromes, neutralizing antibodies, proteomics

OS34 Red blood cell‐derived extracellular vesicles display endogenous anti‐viral effects and serve as effective oligonucleotide carriers for SARS‐CoV‐2 inhibition

Migara Kavishka K. Jayasinghe 1 ; Chang Gao 1 ; Gracemary Yap 1 ; Brendon Yeo 1 ; Wen Xiu Loh 1 ; Thach Tuan Pham 1 ; Dong Van Hoang 1 ; Jang Hann Chu 2 ; Minh T.N. T.N Le 1

1 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, Singapore; 2 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, Singapore

Introduction : The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the largest pandemic in recent history. Despite the success of vaccines, there still remains no widely accessible treatment that can efficiently suppress COVID‐19 infection. Several recent studies have suggested the application of extracellular vesicles (EVs) expressing ACE2 as a potential treatment for SARS‐CoV‐2. Here, we investigated the endogenous anti‐viral effects displayed by ACE2‐negative red blood cell‐derived EVs (RBCEVs) against SARS‐CoV‐2. Moreover, the use of RBCEVs to deliver anti‐sense oligonucleotides (ASOs) for SARS‐CoV‐2 treatment is explored.

Methods : RBCEVs were isolated using established protocols using a sequential workflow consisting of differential centrifugation, filtration, density centrifugation and size exclusion chromatography. The resulting EVs were assessed on their ability to suppress pseudotyped and authentic SARS‐CoV‐2 infection using plaque reduction neutralization tests, qPCR and immunofluorescent imaging. Subsequently, EVs were loaded with ASOs targeting conserved regions of key SARS‐CoV‐2 genes and assayed for the ability to inhibit authentic SARS‐CoV‐2 infection.

Results : RBCEVs were shown to be capable of inhibiting pseudotyped and authentic SARS‐CoV‐2 infection in a dose dependent manner. The neutralization effects were shown to be consistent across major variants of concern and mediated via inhibiting viral entry into cells. RBCEV‐mediated delivery of ASOs targeting SARS‐CoV‐2 genes helicase, 3CLPro, PLpro and RdRp effectively inhibited authentic SARS‐CoV‐2 infection and replication. ASO‐loaded RBCEVs administered via intratracheal delivery to K18‐ACE2 mice accumulated in lung epithelial cells and suppressed SARS‐CoV‐2 infection.

Summary/Conclusion : We leveraged the endogenous anti‐viral properties of RBCEVs together with exogenous loading of anti‐sense oligonucleotides (ASOs), to develop a potent EV‐based anti‐viral therapeutic capable of efficiently suppressing SARS‐CoV‐2 infection and replication.

Funding : This work was funded by the Institute for Digital Medicine (WisDM) and the Singapore Ministry of Education.

Keywords : SARS‐CoV‐2, COVID‐19, extracellular vesicles, RBCEVs, oligonucleotide therapy, anti‐viral therapy

OS35: Late‐Breaking Session: The EV life cycle: from biogenesis to uptake

Chair: Tom Driedonks – Johns Hopkins University

Chair: Kenneth W. Witwer, n/a – Johns Hopkins University School of Medicine

A GWC screen to unravel new molecular mechanisms involved in EV uptake

Víctor Toribio 1 ; Albert Lu 2 ; Miguel Palma‐Cobo 1 ; Carlos E. Bastús 2 ; María Yáñez‐Mó 3

1 Universidad Autónoma de Madrid (UAM), Madrid, Spain; 2 Universidad de Barcelona, Facultad de Medicina, IDIBAPS., Barcelona, Spain; 3 Universidad Autónoma de Madrid (UAM), Madrid, Spain, Madrid, Spain

Introduction : The outstanding potential of Extracellular Vesicles (EVs) in medicine deserves a detailed study of the molecular aspects regulating their incorporation into target cells. We have performed a Genome Wide CRISPR screen by flow cytometry sorting, to identify possible molecular candidates that may regulate EV uptake.

Methods : We have used a GWC cell library in K562 cells, which includes 10 sgRNAs for each gene of the whole human genome, plus negative controls, summing up a total of 250,000 individual sgRNAs. Total EVs were isolated from SKMEL147 melanoma cell line by ultracentrifugation at 100000g. EVs were characterized by NTA, Electron Microscopy, Western blot and confocal microscopy. EVs were stained with Alexa633‐C5‐Maleimide, a fluorescent compound able to covalently bind to sulphur residues of proteins and glycans, and the unbound compound was removed by passing the sample through a ExoSpinColumns (3000MW). For each assay 3.6x1012 of stained EVs were used. 500x106 cells were used per assay in order to have a 2000x coverage. After two hours of incubation with cells, the cell culture was washed and analysed with a flow cytometer for cellular sorting. 5% high and low fluorescence populations were sorted. Total genomic DNA was obtained, and sgRNAs sequences amplified, adding Illumina adaptors as overhangs, to perform NGS.

Results : The results point out to the COMMD and the WASH complex as possible candidates among others.

Summary/Conclusion : Quantitative EV uptake assays previously developed in the laboratory, based on Renilla Luciferase, will be used to validate the hits found in the Genome Wide CRISPR screen.

Funding : This project was funded by a GEIVEX mobility fellowship.

Keywords : Genome‐Wide CRISPR, EV uptake, flow cytometry

At which size does the membrane‐to‐lumen protein partition switch?

Paolo Bergese 1 ; Giorgia Guerra 2 ; Krizia Sagini 3 ; Tatyana Vagner 2 ; Dolores Di Vizio 2

1 Università degli studi di Brescia, Brescia, Italy; 2 Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars‐Sinai Medical Center, Los Angeles, CA, USA, Los Angeles, USA; 3 Institute for Cancer Research, Oslo University Hospital, Oslo, Norway

Introduction : Nanomaterials are characterized by amazing surface‐to‐volume ratio. Extracellular Vesicles (EVs) – which have been recently recognized as the universal agent of intercellular communication, being involved in many physiological and pathological processes and in interkingdom biochemical communication – are nanoparticles, but this key aspect has never been rationally addressed. We will present and discuss the first attempt to quantify the membrane‐to‐lumen (viz. the surface‐to‐bulk) partition of proteins in EVs.

Methods : A semi‐quantitative model, based on the available cellular compositional and microstructural data, was formulated. Large EVs were isolated from culture medium of human prostate cancer cells (PC3 cell line). EV separation and characterization was compliant with MISEV2018.

Results : The model allows for estimation of the partition between membrane (surface) associated and lumen (bulk) proteins, as well of the overall protein content of an EV, as a function of the EV size. Calculations identify a switch diameter at 180 nm, below which EVs carry more membrane than luminal proteins. At larger diameters the partition is reversed, surpassing 80% of luminal proteins for EVs with diameter larger 800 nm. The model has been successfully tested to analyze and describe a real preparation composed of subpopulations of small EVs and Large Oncosomes from human prostate cancer cells. Further latest results on the model validation will be also presented.

Summary/Conclusion : Overall, the model may improve experimental design and data analysis, including: (i) new ways of dosing EVs in vitro and in vivo, (ii) interpretation of EV biological effects and (iii) drawing protocols for EV engineering.

Funding : We acknowledge the grant H2020 FETPROACT 952183 “BOW‐ Biogenic Organotropic Wetsuits” and the grant NIH/NCI R01 CA234557.

Keywords : Extracellular Vesicles, Large Oncosome, Protein, Membrane, Nanoparticles, Bio‐nano Interfaces

Extracellular vesicle internalization by prostate cancer cells is cell cycle dependent and source‐independent

Anca D. Dobrian ; Allison M. Mathiesen; Noel Miller; MIchael Brown

Eastern Virginia Medical School, Norfolk, USA

Introduction : Endothelial cells (EC) represent a large proportion of the tumor microenvironment and their EVs were shown to support malignant cell growth. Also, EC EVs represent an approach to target cancer cells for delivery of therapeutics. Therefore, understanding the uptake mechanisms and cellular processing of EVs is of prime importance. In this study we examined the internalization of EVs produced by microvascular ECs in absence or presence of inflammation by metastatic prostate cancer cells.

Methods : Primary microvascular EC were cultured with or without proinflammatory cytokines. EV were isolated from media by sequential centrifugation (500 x g, 10,000 x g and 2x at 100,000 x g). EV were characterized by EM, protein markers, and NTA. EV were labeled with lipophilic dyes and incubated with PC3ML cells for 4, 12 and 24 hours. To determine mechanisms of uptake we used dynasore, genistein, fillipin and nystatin. EV internalization, cell cycle and lysosomal co‐localization were assessed by imaging flow cytometry.

Results : EV internalization was cell cycle dependent, energy dependent and independent of EV type. The proportion of PC3ML that internalized EV was independent of time and concentration of EV. The average percentage of cells that internalized EVs was 59.4% of which 55% were in G0, followed by S phase(∼35%) and G2/M (∼10%). Internalization in G0 and S phases was ∼80% inhibited by both dynasore and genistein suggesting clathrin and caveolin‐mediate endocytosis. Internalization at G2/M was not significantly affected by any of the inhibitors. Further, only 10% of the EVs were found in the lysosomal compartment in all experimental conditions.

Summary/Conclusion : Proliferative cells have minimal EV internalization making their targeting by EV limited. Modulation of endocytosis has profound impact on EV internalization but not on lysosomal localization. Internalization mechanisms and kinetics are independent of the inflammatory environment of the EV producing cells.

Funding : American Heart Association

PDZ proteins interact with tetraspanins & syndecans and regulate the production, composition and uptake of extracellular vesicles

Monica Castro‐Cruz 1 ; Anouk Oris 2 ; George Daaboul 3 ; Sofie Meeussen 2 ; Lukas Hyka 2 ; Raphael Leblanc 4 ; Guido David 5 ; Pascale Zimmermann 6

1 KU leuven, Leuven, Belgium; 2 KU Leuven, Leuven, Belgium; 3 NanoView Biosciences, Boston, USA; 4 CRCM, Marseille, France; 5 Centre de Recherche en Cancérologie de Marseille, Marseille, France; 6 KU Leuven, MARSEILLE 09, France

Introduction : PDZ proteins co‐evolved with multi‐cellularity and control multiple aspects of cell signaling and polarity. We previously established that, together with syndecan heparan sulfate proteoglycan, the PDZ protein syntenin is a rate‐limiting factor for the exchanges of small extracellular vesicles (sEV, specifically exosomes), stimulating both exosome biogenesis and uptake. Here, we evaluate whether additional proteins of the PDZ family might function in sEV biology.

Methods : We mapped interactions by yeast‐two‐hybrid; silenced the expression of 10 PDZ proteins by siRNA treatment of MCF‐7 and HEK293T cells; purified sEV by differential centrifugation, size‐exclusion chromatography and single particle interferometric reflectance imaging; characterized protein composition by western blot and immunocapture; studied subcellular localization and sEV uptake by immunocytochemistry and fluorescence microscopy; depleted membrane heparan sulfate by heparitinase treatment.

Results : We reveal that PDZ proteins establish multiple direct interactions with CD9, CD63, CD81 and with syndecan‐1/2/3/4. We illustrate that syndecans are selective markers of sEVs. We establish that PDZ proteins differentially control the number of sEVs and the exosomal accumulations of tetraspanins and syndecans. Intracellularly, PDZ proteins impact on tetraspanin/syndecan endosomal budding, subcellular localizations and steady‐state levels. Finally, we show that PDZ proteins differentially control the uptake of CD63 positive extracellular vesicles, most probably by regulating the cell surface levels of heparan sulfate.

Summary/Conclusion : Our study establishes that the extensive networking of syndecans, tetraspanins and PDZ proteins contributes to exosome heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication.

Funding : This work was supported by CONACYT, ANR‐18‐CE13‐0017 (Project SynTEV), FWO (G0C5718N & LK fellowship) and the internal funds of the KU Leuven (C14/20/105).

Keywords : tetraspanins, syndecans, PDZ proteins, EV production, EV composition, EV uptake

Single EV analysis for investigating EV biogenesis

Chungmin Han ; Jaesung Park

POSTECH, Pohang, Republic of Korea

Introduction : The most widely accepted EV classification have suggested that EVs can be classified into subpopulations according to their biogenesis pathway, for example, exosomes, microvesicles, etc. However, due to the lack of appropriate EV analysis, the relationship between EV subpopulations and their biogenesis have not yet been clearly elucidated. Therefore, in this study, we utilized a single EV analysis techniques to investigate this relationship.

Methods : EVs were isolated from cultured HEK293 cells using differential centrifugation, and characterized according to the MISEV2018 guideline. EVs were analyzed using our TIRF‐based single EV analysis technique. Briefly, EVs were immobilized on a passivated glass surface and labeled with antibodies. EV signals from four different fluorescence channels were simultaneously acquired using Quad‐View optics system. Signals located within 3‐pixels were considered co‐localized. To compare tetraspanin expression profiles of EVs and virus‐like particles (VLPs), two different VLPs (HIV and CoV) were prepared according to the methods from highly cited literatures.

Results : Single EV tetraspanin (CD9, CD63 and CD81) expression analysis of WT HEK293 showed distinct correlations between the tetraspanins. CD9 and CD81 were mostly co‐expressed in the same vesicles, while half of CD63 were expressed alone. Following single EV analysis of HEK293 cell lines that express organelle markers showed CD9 and CD81 were more correlated with plasma membrane and early endosomes, while CD63 were more correlated with late endosomes and lysosomes. These results indicated that EVs expressing CD9 and CD81 might be originated from plasma membrane and EVs expressing CD63 might be originated from endosomal organelles. To test this hypothesis, HIV and CoV VLPs, whose biogenesis were well studied, were applied to the single vesicle analysis. The single HIV VLP analysis showed high correlation with the all three tetraspanins, while single CoV VLP did not show any correlations with the tetraspanins. Considering that the biogenesis of HIV in HEK293 is more correlated with ESCRT‐dependent and endosome related exosome biogenesis, EVs expressing all three tetraspanin at the same time might be the exosome populations.

Summary/Conclusion : We identified distinct EV subpopulations from the results of single EV tetraspanin analysis of HEK293 EVs. Following analysis using HEK293 cells expressing organelle specific fluorescence markers indicated that these subpopulations might have different subcellular origination. Single EV analysis VLPs provided an evidence that support our hypothesis: EVs expressing all three tetraspanins (CD9, CD63 and CD81) might be exosomes.

Funding : National Research Foundation of Korea (NRF) (2021R1I1A1A01060075)

Korea Medical Device Development Fund grant (202011B12).

Keywords : exosomes, microvesicles, viruses, biogenesis, single‐vesicle analysis

Late Breaking Posters

Thursday 26 May 2022

PT12: Late‐Breaking Poster Session 1: Therapeutics

Chair: Johannes Grillari – Ludwig Boltzmann Institute of Traumatology in Cooperation With AUVA, Vienna, Austria .

Chair: Kenneth W. Witwer, n/a – Johns Hopkins University School of Medicine

Extracellular vesicles from clinically expired platelet concentrates as a targeted anti‐cancer drug delivery system

Ana Meliciano 1 ; Daniela Salvador 2 ; Ana Filipa D. Louro 2 ; Pedro Mendonça 3 ; Ana Paula Sousa 3 ; Margarida Serra 2

1 iBET‐ITQB/NOVA, Oeiras, Portugal; 2 iBET‐ITQB/NOVA, Lisboa, Portugal; 3 IPST, Lisboa, Portugal

Introduction : Platelet concentrates (PC) have been widely used to treat hematological diseases, but due to their short shelf‐life, several pools of PC are periodically disposed. To value this product beyond its expiration date, alternative applications in regenerative medicine have been explored, particularly their use as a source of growth factors for (stem) ‐cell culture. It is known that large amounts of platelet‐derived extracellular vesicles (pEV) are released during PC storage but few studies have explored its applicability. In fact, an attractive interaction between tumor cells and platelets membrane integrins and receptors (e.g., CD62p, GPIIb/IIIa) has already been described, and thus, in this study, we evaluated the feasibility of using pEV as targeted drug‐delivery systems for cancer therapy.

Methods : pEV isolated by density gradient were loaded with the anticancer agent paclitaxel (PTX‐pEV) by direct incubation. pEV were characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. The bioactivity of PTX‐pEV was assessed on human umbilical vein endothelial cells (HUVEC) and metastatic breast cancer cells MDA‐MB‐231, by observing its effect on angiogenesis, migration, and invasion capability.

Results : pEV were successfully loaded with PTX and entrapment efficiencies of 10% were achieved. The biological efficacy of the engineered pEV was also validated as bioactivity assays showed a greater inhibition in wound closure ability of HUVEC (21%) treated with PTX‐pEV compared to free drug. Promising results were observed in treated MDA‐MB‐231, with a decrease in cell migration (44%) and invasion (22%) abilities.

Summary/Conclusion : These data unveiled for the first time, a novel valorization of PC with no therapeutic value, using pEV as PTX‐delivery systems. PTX‐pEV can potentially provide clinical advantages over free drug or synthetic nanocarriers, regarding both efficacy of delivery and reduction of off‐target effects.

Target‐specific delivery of small RNA cargos facilitated by BioDrone® platform technology

Sung‐Soo Park 1 ; Yoonjin Baeg 2 ; Eun Hyang Jang 2 ; Dong Woo Han 2 ; Jinhee Park 2 ; Miyeon Choi 2 ; Minchul Jung 2 ; Kyeongeun Park 2 ; Jeong Eun Park 1 ; Hye Yeong Nam 2 ; Seung Wook Oh 1

1 BioDrone® Research Institute, MDimune Inc., Seoul, Republic of Korea; 2 BioDrone Research Institute, MDimune Inc., Seoul, Republic of Korea

Introduction : At MDimune, we are developing an innovative drug delivery platform technology, BioDrone®, using cell‐derived vesicles (CDVs) produced from diverse human cell sources by using our proprietary extrusion process. We have previously demonstrated that CDVs, while sharing many physical and biochemical properties with exosomes, can serve as effective biomolecular carriers with highly efficient drug loading capabilities and excellent cellular uptake and tissue penetration properties.

Methods : Small RNAs were loaded onto CDVs as efficiently as exosomes using the cholesterol conjugation method previously published. On the other hand, mRNAs were complexed with CDVs using charge interaction mediated by PEI. Finally, the surface of CDVs was modified by either engineering source cells to express the ligands specific to tumors or chemically conjugating them on the purified CDV membrane.

Results : The CDVs decorated with tumor‐specific ligands enhance the cellular uptake of CDVs to target tumor cells. We also examine how such engineered CDVs promote the delivery of RNA cargos and eventually lead to improved cellular response. Finally, such enhancement in gene modulation mediated by CDVs is compared to exosomes and other conventional gene delivery methods.

Summary/Conclusion : Along with the manufacturing‐scale extrusion process we established recently, the demonstration of targeted delivery of various gene cargos will propel the therapeutic applications of BioDrone® platform technology.

A CD73 enzyme activity assay for stability and dose‐finding of therapeutically‐active EVs and CDVs

Daniela Auer 1 ; Tanja Schally 1 ; Claudia Arzt 2 ; Han Fang 2 ; Eva Grasmann 2 ; Carina Kals 1 ; Elisabeth Bayer 1 ; Eva Rohde 3 ; Hui‐Chong Lau 4 ; Seung Wook Oh 4 ; Mario Gimona 1

1 Paracelsus Medizinische Privatuniversität Salzburg ‐ Privatstiftung, Salzburg, Austria; 2 EVTT at Paracelsus Medizinische Privatuniversität Salzburg ‐ Privatstiftung, Salzburg, Austria; 3 Transfusion Medicine at SALK / Paracelsus Medizinische Privatuniversität Salzburg ‐ Privatstiftung, Salzburg, Austria; 4 BioDrone® Research Institute, MDimune Inc., Seoul, Republic of Korea

Introduction : The translation of nanovesicle‐based therapeutics into clinical application requires the definition and quantitative, reproducible analysis of criteria of bioactivity and stability. For naive un‐manipulated extracellular vesicles (EVs) or cell‐derived vesicles (CDVs) generated by serial extrusion of cells the definition of the active substance(s) remains difficult and the intrinsic heterogeneity of nanovesicle preparations prevents dose determination that is solely based on particle number or total protein mass. The GPI‐anchored 5’‐ectonucleotidase CD73 is an established identity marker for multipotent (mesenchymal) stromal cells (MSCs) and is abundantly present on umbilical cord MSC‐derived EVs and CDVs. The exposed position at the vesicle membrane and the delicate nature of the enzymatic activity position CD73 as a suitable surrogate molecule to establish a biochemical assay that meets the above criteria and that may assist in the control of storage stability and dose definition.

Methods : CD73 hydrolyzes AMP to adenosine and inorganic phosphate. Thus, the activity of the enzyme can be determined by measuring any of the three parameters. We measured CD73 activity in various research grade and GMP‐compliant human MSC‐EV and MSC‐CDV preparations via the consumption of AMP, essentially employing the commercial AMP‐Glo™‐Assay Kit (Promega) with some modifications. The concentration of non‐hydrolyzed AMP is proportional to the detected light generated by the luciferase reaction.

Results : Reproducibility and stability of the assay system was confirmed over a range of buffers (including phosphate‐containing systems) and the sensitivity was suitable for both naturally secreted EVs and extruded CDVs. We reproducibly obtained values of 2‐4 x 10‐8 mM Adenosine production / particle. Enzymatic activity was abolished by heat inactivation while vesicle aggregation or loss of particles was not observed by this treatment. Moreover, the detected enzymatic activity in correlation to the particle number was independent of the total protein concentration of the preparations.

Summary/Conclusion : The biological activity of a GPI‐anchored enzyme is sensitive to denaturing conditions and could indicate the deterioration of a given CD73+ nanovesicular preparation during the storage process. Excipients and co‐purifying protein components in the preparations do not seem to affect the activity and thus allows this assay to be used for a wide range of buffers and purification strategies.

Funding : Supported by the Projects P1812596 “EV‐TT” and 20102‐F1900731‐KZP “EV‐TT‐Bpro”.

Keywords : Biological Biochemical Activity Assay

Extracellular Vesicles

Cell Derived Vesicles

EV Mimetics

Stability

Dose Finding

Extracellular vesicles from adipose tissue‐derived stem cells alleviate osteoporosis through osteoprotegerin and miR‐21‐5p

Jeongmi Lee 1 ; Kyoung Soo Lee 2 ; Eunae Kim 1 ; Ji Suk Choi 2 ; Yong Woo Cho 3 ; Dong‐Gyu Jo 1

1 Sungkyunkwan University, Suwon‐si, Republic of Korea; 2 Exostemtech, Inc., Ansan‐si, Republic of Korea; 3 ExoStemTech Inc., Seongdong‐gu, Republic of Korea

Introduction : Antiresorptive and anabolic agents are predominantly used as osteoporosis medications. However, the limited efficacy and undesirable side effects of these drugs are obstacles to osteoporosis treatment. Because stem cell‐derived extracellular vesicles are growing attention as novel cell‐free therapeutics that have advantages over parental stem cells, the therapeutic effects of extracellular vesicles from adipose‐derived stem cells (ASC‐EVs) on osteoporosis pathogenesis were investigated.

Methods : ASC‐EVs were isolated by a multifiltration system based on the tangential flow filtration system and characterized using transmission electron microscopy, dynamic light scattering, zeta potential, flow cytometry, cytokine arrays, and enzyme‐linked immunosorbent assay. Eight‐week‐old female ovariectomized mice were used for the in vivo experimental model of osteoporosis. Small RNA sequencing data were analyzed to identify miRNA candidates related to anti‐osteoporosis effects.

Results : Extracellular vesicles are rich in growth factors and cytokines related to bone metabolism and mesenchymal stem cell (MSC) migration. In particular, osteoprotegerin (OPG), a natural inhibitor of receptor activator of nuclear factor‐κB ligand, was highly expressed in ASC‐EVs. The intravenous administration of ASC‐EVs attenuated bone loss in osteoporosis mice. ASC‐EVs significantly inhibited osteoclast differentiation of macrophages and promoted the migration of bone marrow‐derived MSCs. However, OPG‐depleted ASC‐EVs did not show anti‐osteoclastogenesis effects, demonstrating that OPG is critical for the therapeutic effects of ASC‐EVs. Additionally, miR‐21‐5p in ASC‐EVs inhibited osteoclast differentiation through Acvr2a down‐regulation.

Summary/Conclusion : OPG and miR‐21‐5p in ASC‐EVs inhibit osteoclast differentiation and reduce gene expression related to bone resorption, suggesting that ASC‐EVs are highly promising as cell‐free therapeutic agents for osteoporosis treatment.

Funding : This work was supported by the National Research Foundation (NRF) under Grant NRF‐2018M3A9H1023767, NRF‐2019R1A5A2027340 ); Ministry of Food and Drug Safety under Grant (18172MFDS173); and the startup commercialization by MMS (B0080831000460, ASC‐derived EV). This work was also supported by a grant from the Ministry of Oceans and Fisheries’ R&D project, Korea (1525011845); and Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2020R1A6C101A191).

Keywords : Extracellular vesicles, Human adipose‐derived stem cells, Osteoprotegerin, Osteoporosis, miR‐21‐5p

Therapeutic effects of extracellular vesicles from human adipose‐derived stem cells in systemic sclerosis

Eunae Kim; Hark Kyun Kim; Jae Hoon Sul; Dong‐Gyu Jo

Sungkyunkwan University, Suwon‐si, Republic of Korea

Introduction : Systemic sclerosis (SSc) is an autoimmune disease characterized by inflammatory reactions and fibrosis. SSc causes severe damage and dysfunction of multiple organs, but there is no complete cure method. Myofibroblasts are considered as one of the therapeutic targets to prevent and reverse the pathogenesis of fibrosis in SSc. Although the mechanisms that differentiate into myofibroblasts are diverse, transforming growth factor‐beta (TGF‐β) is known as a key mediator in the fibrosis of SSc. Extracellular vesicles (EVs) are 30‐200 nm nano‐sized vesicles that cells secrete into the extracellular space.

Methods : EVs were isolated from conditioned media of hADSCs with tangential flow filtration (TFF) and characterized with nanoparticle tracking analysis(NTA), dynamic light scattering(DLS), and western blotting. Subcutaneous injection of bleomycin (100μl from a stock of 0.5mg/ml) to 6 weeks old male DBA/2 mice was continued every other day for 6 weeks. At 5 weeks after the first injection of bleomycin, ADSC‐EVs were administered subcutaneously three times on week 5 and week 6. Primary human dermal fibroblasts (HDFs) were treated for 24 hours with TGF‐β1 or ADSC‐EVs for in vitro studies.

Results : The therapeutic effects of EVs on in vivo SSc model were evaluated with dermal thickness and the number of alpha‐smooth muscle actin (α‐SMA) expressing cells through hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). ADSC‐EVs decreased dermal thickness and α‐SMA expressing cell number and the mRNA level of fibrotic genes such as α‐SMA, CTGF, Col1A1, COMP, LOX. In TGF‐β‐induced dermal fibroblasts, ADSC‐EVs also downregulated the mRNA and protein levels of α‐SMA, CTGF. Furthermore, ADSC‐EVs can inhibit the phosphorylation of SMAD2 and the nuclear localization of phosphor‐SMAD2. These results indicate that ADSC‐EVs block the TGF‐β/SMAD signaling pathway and ameliorate the fibrotic symptoms.

Summary/Conclusion : ADSC‐EVs have therapeutic effects on a bleomycin‐induced mouse model and TGF‐β induced human dermal fibroblasts in vitro. ADSC‐EVs downregulated the expression of fibrotic genes and decreased the accumulation of extracellular matrix. These results indicate that ADSC‐EVs can be a therapeutic approach for preventing and reversing SSc.

Cell reprogramming via extracellular vesicles derived from adipose‐derived stem cells during white/beige adipogenic differentiation

Jihoon Han 1 ; Hark Kyun Kim 2 ; Youn Jae Jung 3 ; Yong Woo Cho 4 ; Dong‐Gyu Jo 2

1 Sungkyunkwan University, Suwon, Republic of Korea; 2 Sungkyunkwan University, Suwon‐si, Republic of Korea; 3 ExoStemTech Inc., Ansan, Republic of Korea; 4 ExoStemTech Inc., Seongdong‐gu, Republic of Korea

Introduction : Mesenchymal stem cells (MSCs) derived from human tissues such as bone marrow, adipose tissue, and placental tissue are widely used as therapeutic agents for tissue repair and regeneration. However, stem cells remain relatively rare after transplantation and differentiation is inefficient. On the other hand, stem cell‐derived extracellular vesicles (EVs) offer alternative approaches to stem cell‐based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation were used as cell‐free therapeutic systems to induce tissue‐specific differentiation.

Methods : EVs were isolated from human adipose‐derived stem cells (HASCs) during white and beige adipogenic differentiation (D‐EVs and BD‐EVs, respectively) via a multifiltration system based on the tangential flow filtration. Purified EVs were characterized by nanoparticle tracking analysis, dynamic light scattering, electrophoretic light scattering, and cryo‐TEM.

Results : Both D‐EVs and BD‐EVs successfully differentiated HASCs into white and beige adipocytes, respectively. D‐EVs were transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produced numerous lipid droplets in injected sites. Treatments of BD‐EVs attenuated diet‐induced obesity through browning of adipose tissue in mice. Furthermore, high‐fat diet‐induced hepatic steatosis and glucose tolerance were improved by BD‐EVs treatment. The miRNA expression profile of EVs indicates that miRNAs contributing to brown fat cell differentiation are enriched in BD‐EVs.

Summary/Conclusion : These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can induce cellular differentiation, suggesting a potential for cell‐free therapeutic systems by inducing tissue‐specific differentiation in defective tissues.

Funding : This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (1420390). This study was also supported by the National Research Foundation (NRF) funded by the Korean Government (NRF‐2019R1A2C3011422, NRF‐2019R1A5A2027340, NRF‐2018M3A9H1023767, and NRF‐2017R1A5A1070259), and the Industrial Core Technology Development Program (10078392) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea).

Antiinflammatory properties of ROS‐responsive tolerogenic dendritic cell‐derived exosomes in rheumatoid arthritis

Leon Felipe Palomera Sandoval 1 ; Jae Hoon Sul 2 ; Eun sook Lee 3 ; Jae Hyung Park 3 ; Dong‐Gyu Jo 2

1 SKKU, suwon, Republic of Korea; 2 Sungkyunkwan University, Suwon‐si, Republic of Korea; 3 skku, suwon, Republic of Korea

Introduction : Despite the emergence of tolerogenic dendritic cell‐derived exosomes (TolDex) as a novel treatment in models of rheumatoid arthritis (RA), their use is limited by the poor bioavailability after administration. Our group achieved lesion‐specific immunoregulation through reactive oxygen species‐responsive TolDex in RA by hydrophobic insertion of a polyethylene glycol (PEG)‐embedded thioketal (TK) linker on the surface of the TolDex. ROS overproduction causes enhanced cellular uptake of ROS‐responsive TolDEX (TKDex) through PEG detachment. This study shows the potential therapeutic application of TKDex in RA.

Methods : After Synthesis of thioketal (TK) linker TolDex were isolated from DC 2.4 cells cultured in conditioned media supplemented with exosome‐free FBS. Media were collected, centrifuged and then filtered using a 0.22‐μm filter, followed by a TFF system at 30 rpm with a TFF capsule of 300 kDa. Evs were mixed with the TK linker to form TKDEX and characterized using NMR, NTA, TEM and western blot. In vivo studies including biodistribution, ex‐vivo fluorescence and immune regulatory effects were carried out in collagen‐induced arthritis (CIA) 8‐week‐old male DBA1/J mice.

Results : CD40 downregulation as well as reduced secretion of pro‐inflammatory cytokines such as tumor necrosis factor α (TNF‐α) and interleukin 6 (IL‐6), were found in mature dendritic cells (mDCs) after TolDex and TKDex treatment. Following intravenous administration, PEG increased bioavailability of TKDex in the collagen induced arthritis (CIA) model, and enhanced joint accumulation. Regulatory T cells (Treg) expressing CD4/CD25/FOXP3 were induced as well as transforming growth factor ß elevation and IL‐6 reduction by TKDex.

Summary/Conclusion : An enhanced efficiency ROS‐sensitive TolDex was developed after investigation of the putative mechanism of action of TolDex. Interestingly, our results showed that not only a reduction in the pro‐inflammatory signals were achieved, but an anti‐inflammatory switch in the cell population occurred after CD40 downregulation. The RA microenviroment in the lesions was altered by citokyne regulation and Treg induction. This study might set a foundation for future works on stimuli‐ responsive exosomes for the treatment of disease.

Funding : Basic Science Research Programs (Grants number: 2019R1A5A2027340, 2020R1I1A1A01069178 and 2018R1A2B3006080) of the National Research Foundation (NRF) and the Korea Basic Science Institute (National Research Facilities and Equipment Center, 2020R1A6C101A191) grant from Ministry of Education, the Republic of Korea.

Keywords : ExosomeROS‐responsiveSurface modificationImmunoregulationRheumatoid arthritis

Plant extracellular vesicles as a drug delivery system for humans

Joshua Farley 1 ; Laura Pellegrini 2 ; Madeline Lancaster 2 ; Stephen Muench 3 ; Vincent Postis 4 ; Carine De Marcos Lousa 1

1 School of Health, Leeds Beckett University, Leeds, United Kingdom; 2 MRC Laboratory of Molecular Medicine, University of Cambridge, Cambridge, United Kingdom; 3 Faculty of Biological Science, University of Leeds, Leeds, United Kingdom; 4 School of Life Sciences, University of Dundee, Dundee, United Kingdom

Introduction : Ever‐growing interest has focused on human EVs (hEVs) as a drug delivery system. Plant‐derived EVs (pEVs) are an appealing alternative to hEVs due to a low cost associated with their isolation, low immunogenicity, and high engineering potential with biopharming. Here, we investigate the potential of pEVs as a drug delivery system in human cells.

Methods : EVs were isolated from apoplastic fluids from Nicotiana Benthamiana (Nb) leaves using vacuum infiltration followed by differential ultracentrifugation. EVs were characterised by Nanoparticle tracking analysis (NS300‐ Malvern) and negative stain electron microscopy (NS‐EM). The nature of EVs was confirmed via western blot with the pEV‐specific markers PEN1 and TET8. Uptake of fluorescently stained pEVs by human cells (SH‐SY5Y) was analysed through flow cytometry with free stain as controls. Toxicity of pEVs on human cells was analysed through Ready Probes™ live/dead staining.

Results : Using NTA and NS‐EM, we confirmed that nanoparticles with expected vesicular size and morphology can be isolated from Nb leaf apoplastic fluids (plant extracellular medium). We then confirmed the presence of 2 distinct populations of EVs through western blotting: a SYP121/PEN1 population from the P40 fraction and a TET8 (CD63 orthologue) positive population from the P100‐40 fraction. We demonstrated that fluorescent total pEVs (P100) are taken up by human neuroblastoma cells and human brain organoids. Additionally, we have confirmed that there is no cytotoxic effect of pEV uptake in SH‐SY5Y cell culture.

Summary/Conclusion : We can purify pEVs from plant extracellular fluids successfully and show pEVs are taken up by human cells without cytotoxicity, shedding a positive light on the use of pEVs as a drug delivery system. Further characterisation forthcoming includes density gradients, proteomics, and lipidomics. Delivery of therapeutic cargo from siRNA loaded pEVs to human cells is ongoing.

Therapeutic effect of Engineered small extracellular vesicles displaying ACE2 variants on the surface against SARS‐CoV‐2 infection

JUNSIK KIM 1 ; Hark Kyun Kim 2 ; Dong‐Gyu Jo 2

1 SKKU, Suwon, Republic of Korea; 2 Sungkyunkwan University, Suwon‐si, Republic of Korea

Introduction : Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin‐converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID‐19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. With excellent biocompatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS‐CoV‐2. Moreover, to enhance the sACE2‐S binding interaction, we employ sACE2 variants. sACE2‐loaded sEVs exhibit typical sEV characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry of wild‐type (WT), the globally dominant D614G variant, Beta (K417N‐E484K‐N501Y) variant, and Delta (L452R‐T478K‐D614G) variant SARS‐CoV‐2 pseudovirus, and protect against authentic SARS‐CoV‐2 and Delta variant infection. Of note, sACE2 variants harboring sEV show superior antiviral efficacy than WT sACE2 loaded sEV. The current findings provide opportunities for the development of new EV‐based antiviral therapeutics.

Methods : Plasmid construction Isolation and characterization of sEVs Cell culture and generation of stably transfected cell lines sEVs neutralization assay and authentic wildtype SARS‐CoV‐2 infection sEVs neutralization assay and authentic Delta variant SARS‐CoV‐2 infection ACE2/SARS‐CoV‐2 Spike S1 inhibitor screening assay Luciferase assay

Results : Characterization of engineered sACE2 sEVs. Binding properties of engineered sEVs Protection against WT and mutant S‐containing pseudovirus. Protection against authentic Delta variant SARS‐CoV‐2 infection.

Summary/Conclusion : EV engineering allows for displaying native proteins on the EV surface, with various potential therapeutic applications. Herein, we showed that CD9ΔTM4 scaffolds successfully presented sACE2 and its variants on the sEVs surface. These sEVs inhibited binding between S and ACE2 in a dose‐dependent manner by occupying available S protein as decoys. The infectivity of WT, D614G, Beta variant, and Delta variant mutant S‐containing pseudovirus was compromised by sACE2.v1 and sACE2.v2 sEVs. Furthermore, both sEVs potently blocked authentic SARS‐CoV‐2 (BetaCoV/Korea/KCDC03/2020 virus) and Delta variant infection in Vero E6 cells. Our observation that sACE2‐expressing sEVs markedly protected against S‐containing pseudovirus and authentic SARC‐CoV‐2 infection further support the potential of engineered sEVs as an efficient platform for the delivery of therapeutics.

Using Nanobody displaying EVs to target the Transferrin Receptor

Philip C. Hewlett 1 ; Arianna Coluzzi 1 ; Heena Sharma 2

1 Evox Therapuetics, Oxford, United Kingdom; 2 Evox Therapeutics Limited, Oxford Science Park, Oxford, UK., Oxford, United Kingdom

Introduction : Extracellular vesicles (EV) are naturally occurring particles that have inherent tropism for specific cells or organs. Observations from bio‐distribution experiments show (CAP)‐derived EVs are taken up predominantly by the liver and spleen. Antibodies against the transferrin receptor (TFR) have been widely shown to carry drugs to the CNS across the BBB. The aim of this study was to assess whether TFR targeting moieties can be used to redirect CAP‐derived EVs to cells of the CNS.

Methods : To this end, TFR‐targeting nanobodies were fused at the N‐terminal of the EV membrane protein Lamp2b, whereas the C‐terminal was fused to the reporter protein eGFP. Following transient transfection of CAP cells, NanoFCM and eGFP quantification were utilised to determine a ligand density of 500 molecules per EV in our engineered population of vesicles; with functional display targeting moieties confirmed with ELISA. Next, the ability of EV‐expressed nanobodies against TFR to facilitate uptake in TFR expressing cell lines was assessed through high content imaging.

Results : Both cell and cell‐free methods showed a dose‐dependent increase in receptor engagement with targeted over control EVs.

Summary/Conclusion : In the next steps, we will explore different ligand densities, scaffolds, and cell sources to see if binding and cellular uptake can be further improved.

Keywords : EVs, Nanobodies, TFR, High Content Microscopy, NanoFCM, ELISA

Hybrid extracellular vesicles for RNA delivery

Xiaoqin Wang 1 ; Kristina Friis 2 ; Andreia M. Silva 3 ; Niek Dekker 3

1 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Gothenburg, Sweden; 2 Advanced Drug Delivery, Pharmaceutical Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Gothenburg, Sweden; 3 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D AstraZeneca, Gothenburg, Sweden, Molndal, Sweden

Introduction : Extracellular vesicles (EVs) are naturally secreted nanovesicles carrying bioactive molecules and show to mediate cell‐to‐cell communication. EVs possess intrinsic features, such as low immunogenicity, innate tropism and capacity to cross physiological barriers, which emerge EV as an attractive drug delivery vehicle. Different approaches have been investigated to engineer EV for loading of diverse cargoes. Nevertheless, challenges such as low loading efficiency still remain. Here, we develop a novel approach to load and deliver RNA via hybrid EVs.

Methods : EVs purified from Expi293F cells were fused with lipid nanoparticles (LNP) carrying Cre mRNA. The resulting mixed particles are referred to as hybrid EV (HEV). Various parameters were evaluated to optimize the formulation of HEVs. The size profile, protein composition and Cre mRNA encapsulation efficiency were analyzed by nanoparticle tracking analysis, Western blot, immuno‐electronic microscopy and Qubit RNA HS assay. The functional mRNA delivery by HEV and by LNPs were tested in a Cre reporter assay. A cell viability assay was used to determine the cell tolerability.

Results : Total particle concentration and EV‐LNP ratio are key parameters influencing the encapsulation efficiency, yield and properties of HEVs. HEVs displayed increased size and carried components of EV and LNP. Cre mRNA was successfully loaded in HEVs and functionally delivered to Cre reporter cells. Moreover, HEVs showed excellent cell tolerability and outperformed delivery efficiency of LNPs at high dose.

Summary/Conclusion : We developed a novel approach for effectively loading of RNA via HEVs. HEV is a promising strategy for functional delivery of RNA with the advantages of balancing safety and efficacy.

3D bioreactor culture in chemically‐defined medium for MSC‐EV production optimization for clinical translation

Yvan Courageux 1 ; Alba López‐Fernández 2 ; Cristina Prat‐Vidal 2 ; Luciano Rodríguez 2 ; Silvia Torrents 3 ; Carolina Gálvez‐Montón 1 ; Antoni Bayes‐Genis 4 ; Joaquim Vives 2 ; Sergi Querol 2 ; Santiago Roura 4 ; Marta Monguió‐Tortajada 4

1 ICREC Research Program, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain, Barcelona, Spain; 2 Servei de Teràpia Cel·lular, Banc de Sang i Teixits, Barcelona, Spain, Barcelona, Spain; 3 Laboratori Cel·lular, Banc de Sang i Teixits, Barcelona, Spain, Barcelona, Spain; 4 ICREC Research Program, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain, Badalona, Spain

Introduction : Mesenchymal Stromal Cell‐derived Extracellular Vesicles (MSC‐EV) have increasing clinical interest as new versatile therapy against broad human conditions related to immune modulation and tissue regeneration. Nevertheless, the optimum MSC culture scale up fostering potent EV secretion is still a major concern. Also, the implemented strategy has to be designed for reproducibility and meet standards of clinical manufacturing.

Methods : We comparatively analyzed the production efficiency of EV derived from clinical‐grade Wharton jelly‐derived MSC (MSC,WJ) cultured in 2D scale up cell culture flasks or 3D microcarriers‐based bioreactor using either serum‐supplemented medium (SER) or chemically‐defined medium (CHEM). Cell growth, viability and phenotype were monitored for each condition and MSC,WJ‐EV were purified by size exclusion chromatography after concentration by ultrafiltration or tangential flow filtration. Next, MSC,WJ‐EV were characterized in terms of identity, purity, yield and functional activity to better design MSC,WJ‐EV scale‐up production, and to assess the suitability of the delineated analytical methods.

Results : MSC,WJ expressed a stable phenotype across the different culture settings. Cell recovery was lower in both CHEM conditions as well as in both 3D cultures, although aggregate formation difficulted cell harvest in the latter. Purified MSC,WJ‐EV were recovered from each condition, and bead‐based flow cytometry confirmed expression of both EV and MSC markers. CHEM‐based cultures yielded higher EV protein and RNA quantities relative to cell numbers and culture volumes. In terms of immunomodulatory activity, EV derived from 3D cultures showed greater inhibition of T cell proliferation, and 3D‐CHEM condition further correlated to superior CD73 enzyme activity.

Summary/Conclusion : While 2D and SER culture conditions yielded higher cell production, 3D‐CHEM culture conditions enabled MSC,WJ‐EV harvests with superior immunomodulatory potential.

Funding : MICINN (Madrid, Spain) ‐ PID2019‐110137RB‐I00 / PRE2020‐095357 ; Instituto de Salud Carlos III (Madrid, Spain) ‐ ICI20/00135 / RD21/0017/0022

Keywords : Bioreactor, Immunomodulation, Production optimization, Chemically defined

This abstract is about the programmed delivery and stimulate neural differentiation of Ginseng‐derived EVs

Lihua Peng 1 ; Minhong Tan 2 ; Maoze Wang 1

1 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China (People's Republic); 2 School of Materials Science and Engineering, Zhejiang University, Hangzhou, China (People's Republic)

Introduction : Inducing the neural differentiation of endogenous mesenchymal stem cells (MSCs) is a momentous strategy for promoting neural regeneration. The combined‐delivery of biological‐electrical cues has exhibited great efficiency in neurogenesis and repair. However, biological factors have always been scarce due to low delivery efficiency or immunological risk, and recently plant‐derived Exos have been explored that may have great potential in promoting neural differentiation of MSCs. Moreover, the inconvenience and immune risk of traditional electrode have always been great obstacles. Herein, a novel nanoplatform for delivering biological signals to efficiently induce rapid nerve regeneration based on ginseng‐derived EVs (G‐Exos) was constructed, assisted by self‐powered electrical signals to further accelerate sensation restoration.

Methods : The directed neural differentiation efficiency and cells genetic alterations of MSCs by the isolated G‐Exos were investigated in vitro. The effects and mechanism of nerve regeneration and sensation restoration by delivering biological‐electrical signals were evaluated in vivo.

Results : G‐Exos has been confirmed to accelerate the neural differentiation of MSCs by activating the Pi3k‐Akt signaling pathway, increasing the expression of neurotrophic factors, and promoting the activation of Ras signaling pathway. On this basis, electrical signals further promote nerve regeneration and sensory conduction recovery through calcium signaling pathway.

Summary/Conclusion : The results demonstrated that G‐Exos promote the neural restoration and increase the nerve regeneration by delivering biological signals. In this sense, plant‐Exos possess great potential to be a promising nanoplatform for disease therapy, along with the higher production yield, simple fabrication, and less immunological risk. Furthermore, the programmed delivery of multiple biological‐electrical cues also grants a universal strategy for regenerating tissues and organs.

Keywords : Ginseng‐derived EVs, bioloigcal‐electrical cues, programmed delivery, neural restoration

Exosomes mediated delivery of Recombinant Human Interleukin 24 for breast cancer

Muhammad I. Amirzada 1 ; Hamid Bashir 2

1 Department of Pharmacy, COMSATS University Islamabad, Abbottabad, Pakistan; 2 Centre for Applied Molecular Biology, University of The Punjab, Lahore, Pakistan, Lahore, Pakistan

Introduction : Interleukin 24 (IL24) has drawn attention as an antiproliferative and antitumor activity. IL24 has ability to induce the specific death of cancer cells. Application of viral vectors for delivery of recombinant interleukins reported toxicity and immunogenicity. Exosomes are cell secreted nanoparticles play an important role in intercellular communication. Exosomes has potential to targeted drug/gene delivery due to innate stability, low immunogenicity, and excellent tissue/cell penetration. The aim of the current study was to develop exosomes mediated Recombinant Human Interleukin 24 (Exo‐IL24) formulation for breast cancer.

Methods : Exosomes were isolated from plasma by modifying the isolation method of Wu X et al 2019. The isolation procedure involves ultracentrifugation and dialysis. Plasma was ultracentrifuged to remove unwanted contaminating components. Furthermore, ultrafiltration was carried out on differentially centrifuged plasma by dialysis. Exo‐IL24 nanoparticles were formulated by the modified calcium chloride transfection method of Jin Y et al 2017. Nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM) and SDS‐PAGE were used to characterize free exosomes and Exo‐IL24. The cytotoxicity of Exo‐IL24 was determined by MTT assay on MCF‐7 cells.

Results : SDS‐PAGE of free exosomes revealed the expression of CD1, CD63, CD81 and LAMP1. Nanoparticle tracking analysis (NTA) confirms that the particle size of Exo‐IL24 was 203 nm larger than free exosomes. The typical structure of the exosome can be seen in SEM. After loading with IL24, their size is greater than that of free exosomes. The cytotoxicity test showed a decrease in cell viability with increasing concentration of Exo‐IL24.

Summary/Conclusion : Exosome's incorporation in the delivery of Interleukin 24 to breast cancer cells resulted to modify the release profile and improve IC50 of IL24. Exosomes mediated Recombinant Human Interleukin 24 formulation could be a promising strategy to combat the breast cancer via targeted delivery to cancerous cells.

Funding : Self

Keywords : Exosomes, Interleukin 24, Breast cancer

Development of an exosome‐based therapy with skeletal muscle targeting capabilities for Pompe disease

Althea R. Lobo 1 ; Claudia Hill 1 ; Lydia Kgosana 1 ; Sally Allison 1 ; Lorenzo Errichelli 1 ; David Lowe 1 ; Justin Hean 2 ; Mario Buono 1 ; Virginia Castilla‐Llorente 1

1 Evox Therapeutics, Oxford, United Kingdom; 2 Evox Therapeutics Limited, Oxford, UK., Oxford, United Kingdom

Introduction : Pompe disease is a rare inherited lysosomal storage disease in which lysosomal glycogen catabolism is reduced or depleted due to deficiency in acid‐alpha‐glucosidase (GAA). The current approved enzyme replacement therapy (ERT) has clear therapeutic benefit; however, it poorly targets to skeletal muscle. Consequently, higher doses are administered leading to increased immunogenicity. To overcome these challenges, we aimed to develop a potential therapeutic candidate based on skeletal muscle targeted exosomes loaded with GAA.

Methods : Different exosome engineering strategies were utilised in order to load GAA either in the lumen or on the surface of exosomes (Exo‐GAA EVs), as well as to target the exosomes to skeletal muscle. The plasmids encoding for the engineered proteins were transfected into human cells and the exosomes were purified following Evox proprietary/standard methods. The purified material was subjected to a thorough characterization following ISEV guidelines and assessed for functional delivery in vitro.

Results : When compared with an equivalent dose of the current ERT, Exo‐GAA EVs showed a similar significant recovery of C2C12 GAA‐/‐ myotubes. Strikingly, the inclusion of an M12 muscle targeting peptide on the surface of the Exo‐GAA EVs increased post‐uptake GAA activity and led to an increase in glycogen removal in comparison with the current ERT.

Summary/Conclusion : The results highlight the potential of engineered exosomes as a modality for delivery of therapeutic proteins with the added capability to increase targeting to specific tissues often hard to reach, such as skeletal muscle.

Funding : Funded by Takeda.

Keywords : Protein loading, Targeting, Skeletal Muscle, Pompe disease, Drug delivery.

Exosomal delivery of celastrol for lung cancer management

Farrukh Aqil 1 ; Raghuram Kandimalla 2 ; Disha Moholkar 2 ; Mohd Saeed 3 ; Ahmad Almatroudi 4 ; Ramesh C. Gupta 5

1 University of Louisville, Louisville, USA; 2 University of Louisville, Louisville, KY 40202, Louisville, USA; 3 College of Sciences, University of Hail, PO BOX. 2240, Hail, Saudi Arabia, Hail, Saudi Arabia; 4 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Qassim 51431, Saudi Arabia., Qassim, Saudi Arabia; 5 3P biotechnologies and Department of Pharmecology & Toxicology and Brown Cancer Center, University of Louisville, Louisville, KY 40202, Louisville, USA

Introduction : Cancer is a growing concern and a leading cause of deaths worldwide. Celastrol (CEL), a triterpenoid, has attracted significant attention for its strong anticancer properties. However, due to low bioavailability and poor pk parameters, it has been restricted from clinical applications. Exosomes are natural nanoparticles with inherent properties such as stability, biocompatibility, low immunogenicity and ability to overcome biological barriers. Owing to these superior advantages, exosomes have been utilized as potential drug carriers to overcome the challenges of oral delivery of CEL and other drugs. In this report, we demonstrate the antiproliferative and colony forming inhibition activity of CEL and its exosomal formulations against drug‐sensitive and drug‐resistant lung cancer cells and inhibition of lung tumors by oral administration of ExoCEL.

Methods : Exosomes were isolated from bovine milk by differential centrifugation and characterized for size by Zetasizer and AFM and protein markers by Western blot. Cells were treated with CEL and ExoCEL and cell survival and inhibition of colony formation was measured by MTT assay and trypan blue staining, respectively. For antitumor activity, s.c. lung tumor‐bearing mice were treated orally with CEL, ExoCEL or vehicle; all regimen were given 3‐times a week orally and tumor growth was monitored. The mechanism of CEL activity was analyzed by RNAseq analysis followed by confirmation with western blot.

Results : CEL when tested in A549 and H1299 lung cancer cells showed time‐ and dose‐dependent antiproliferative activity with IC50 values of 0.63 and 1.8 μM against A549 and H1299 cells. The activity was enhanced with ExoCEL. The ExoCEL also showed enhanced efficacy in inhibition of A549 colony formation, would healing and cell migration assay. The ExoCEL enhanced therapeutic response compared to the CEL in tumor xenograft model. The ExoCEL showed 77% reduction of s.c. tumor growth while CEL provided 52% inhibition compared to vehicle treatment. Mechanistically, ExoCEL and CEL suppressed TGF‐β1 and TNF‐α induced NF‐κB activity and showed effect on multiple markers related to cell cycle, EMT signaling, PI3K‐Akt signaling, p53 signaling and pathways related to lung cancer and its metastasis.

Summary/Conclusion : Orally delivered ExoCEL formulation exhibit significantly higher inhibition of lung tumors compared with free CEL. Thus, oral delivery of CEL could potentially be developed against lung and other cancers to achieve a user‐friendly, cost‐effective alternative of the traditional bolus dose i.v. therapy which carries severe side effects. Furthermore, exosomal formulation of poorly bioavailable compounds such as CEL can achieve significant biological effects presumably by enhancing its bioavailability and sustained release.

Funding : Supported From RDO grant # RDO‐2003.

Keywords : Milk exosomal, oral delivery, celastrol, lung cancer.

Engineered Cas9 extracellular vesicles as a novel gene editing tool

Xabier Osteikoetxea 1 ; Andreia M. Silva 2 ; Elisa Lázaro‐Ibáñez 3 ; Nikki Salmond 4 ; Olga Shatnyeva 3 ; Josia Stein 4 ; Jan Schick 4 ; Stephen Wren 5 ; Julia Lindgren 6 ; Mike Firth 7 ; Alexandra Madsen 8 ; Lorenz M. Mayr 9 ; Ross Overman 10 ; Rick Davies 4 ; Niek Dekker 2

1 Semmelweis University ‐ Hungarian Centre of Excellence for Molecular Medicine, Budapest, Hungary; 2 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D AstraZeneca, Gothenburg, Sweden, Molndal, Sweden; 3 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Gothenburg, Sweden; 4 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Alderley Park, United Kingdom, Alderley Park, United Kingdom; 5 Global Product Development, Pharmaceutical Technology & Development, AstraZeneca, Macclesfield, United Kingdom, Alderley Park, United Kingdom; 6 Translational Genomics, Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Gothenburg, Sweden; 7 Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom; 8 Genome Engineering, Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Gothenburg, Sweden; 9 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom, Cambridge, United Kingdom; 10 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Alderley Park, United Kingdom, Cambridge, United Kingdom

Introduction : Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9‐fusions with EV sorting partners.

Methods : Cas9‐loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analyzed for CRISPR/Cas9‐mediated functional gene editing in a Cre‐reporter cellular assay.

Results : As we have shown earlier, light‐induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation‐Palmitoylation‐Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 cells. New data shows that this approach could also reach 25% editing if the Cre reporter cassette in HepG2 cells as well as targeting knock‐down of the therapeutically relevant PCSK9 gene with 6% indel efficiency. Importantly, Cas9 transfer was detergent‐sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV‐mediated transfer.

Summary/Conclusion : Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Funding : This work was funded by AstraZeneca with a part of support from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 739593.

Keywords : extracellular vesicles, exosomes, CRISPR/Cas9 delivery, gene editing, optogenetics

Exosome‐derived lipid nanoparticles for mRNA delivery

Minsub Chung

Hongik University, Bryn Mawr, USA

Introduction : Extracellular vesicles (EVs), primarily exosomes and microvesicles, hold great promise for development of new disease diagnostic techniques, drug delivery systems, and therapeutics. Exosomes can be biocompatible natural carriers of mRNA, which successfully cross the cellular membrane and bypass drug delivery obstacles. However, studies on the roles and functions of these exosome components have often encountered difficulties due to the complexity of the EVs, and the need for an artificial lipid membrane model with high reliability and utility is emerging.

Methods : HEK293T cell line, known to their high transfection efficiency, was used as source of EVs, which were prepared by sequential ultracentrifugation using the cell culture supernatant. We isolated 3 different fractions of vesicles by collecting the pellets upon 2.000xg, 12.200xg and 120.000xg spins. Each fraction of EVs were treated with 20% ethanol and ionizable lipids, followed by FLuc mRNA. After buffer exchange, integrity of different EV fractions was studied by electron microscopy, dynamic light scattering (DLS), and zeta‐potential. The mRNA encapsulation was measured by Ribogreen assay. The mRNA transfection and translation were visualized and examined by luciferase assay.

Results : EVs from different spin fraction with different average size became similar size in range of 100 ∼ 300 nm after mRNA treatment and buffer exchange. Their wide size distribution was confirmed by TEM and showed the expected size distribution by DLS. Over 80% of mRNA was encapsulated. Preliminary data showed that the mRNA‐modified EVs were successfully delivered, and their luciferase activity was proportional to the amount of mRNA.

Summary/Conclusion : A new engineering exosomes methods developed to utilize them as RNA drug delivery carriers. The physical properties, encapsulation and transfection profile of the engineered exosomes also are provided.

Keywords : Exosome, drug delivery, lipid nanoparticles, mRNA

PT13: Late‐Breaking Poster Session 2: Technologies and Methods I

Chair: Veronika Kralj Igič, n/a – Biomedical Research Group, Faculty of Health Sciences, University of Ljubljana, SI‐1000 Ljubljana, Slovenia

Chair: Olesia Gololobova, n/a – Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill

Differential detection of cancer‐derived extracellular vesicles using combined antibody functionalized magnetic beads and infrared spectroscopy

Gregory E. Rice 1 ; Thomas Stewart 2 ; Tamkin Ahmadzada 3 ; Emily Stein 4 ; Peter French 5 ; Michelle Wood 6 ; Joonsup Lee 3 ; Elham Hosseini‐Beheshti 7 ; Georges Grau 7 ; Peter Lay 8 ; Sophia Amerena‐Cowie 2

1 INOVIQ Limited, Notting Hill, Australia; 2 University of Sydney, Sydeny, Australia; 3 Sydney University, Sydeny, Australia; 4 Inoviq Limited, Minneapolis, USA; 5 Sydney University, Ballina, Australia; 6 Sydney University, Sydney, Australia; 7 School of Medical Sciences, The University of Sydney, Camperdown, NSW, Australia, Sydney, Australia; 8 Sydney Univerity, sydeny, Australia

Introduction : Extracellular vesicles (EV) play a pivotal role in cellular functions including signalling pathways involved in the maintenance of normal physiological processes. EVs surface antigens and molecular cargo are altered in response to challenges to cellular homoeostasis and disease onset and progression. Rapid and simple methods for isolating EVs and characterizing their cell type‐specific biomarkers and molecular cargo are requisite to realizing their diagnostic potential. In this study, cancer cell‐derived EVs were isolated using antibody functionalized magnetic beads and infrared spectroscopy was used to identify cell and phenotypic biomarkers. In particular, changes in the concentrations of biomolecular classes were identified that separated EVs from different cancer cell lines into different regions of the principal component analysis (PCA) scores plots.

Methods : EVs were isolated from lung cancer cell line A549 (unstimulated and IFN‐gamma stimulated) and the mesothelial cell line MeT‐5A cell‐conditioned media using EXO‐NET® (according to manufacturer's instructions). EV‐loaded beads (5 μl) was placed on a CaF2 window then air‐dried. Vibrational spectra were recorded (Bruker Lumos, Tensor or Alpha FTIR spectrometers). The Alpha and Tensor instruments measured spectra from the entire dried drop, whereas point maps across individual dried drops were recorded with the Lumos instrument. The latter enabled an examination of variability across the dried sample. Spectra were vector‐normalized to the 1800‐1050 cm‐1 region and subjected to the Savitsky‐Golay algorithm with 13 smoothing points before PCA.

Results : The spectra from both cell lines and unstimulated vs stimulated A549 cells were clearly differentiated from one another, as well as the spectrum from the beads alone. The mapping experiments showed that random points across the dried drop of EXO‐NET bead samples (loaded and unloaded) had less variation than those between different cell lines and treatment groups when appropriate spectral regions were chosen for the analysis. Spectra of EVs isolated using sucrose gradients were confounded by FTIR sucrose peaks and not‐fit‐for‐purpose.

Summary/Conclusion : On‐bead analysis using vibrational spectroscopy provides a rapid and simple method for determining the relative concentrations of EV biomolecular classes. Distinct clustering of the spectra by sample type occurred, indicating that the different sub‐populations of cell‐derived exosomes could be differentiated using the vibrational spectroscopic method. The data obtained evidence the utility of EXO‐NET combined with FTIR to differentiate cancer type from the position in PC1 and the phenotype from the position along PC2, both of which are important diagnostically and in triage to treatment.

Funding : na

Keywords : EV isolation infrared spectroscopy

A novel tool for extracellular vesicle characterization: interferometric nanoparticle tracking analysis (iNTA)

Martin Blessing; Anna Kashkanova; André Gemeinhardt; Vahid Sandoghdar

Max Planck Institute for the Science of Light, Erlangen, Germany

Introduction : Nanoparticle tracking analysis (NTA) is one of the most widely used techniques for EV characterization; the label‐free method works with small volumes, is very user‐friendly, and provides realistic size distributions. However, it falls short of expectations regarding resolution, the limit of detection, and the ability to identify EVs in complex media. Here, we present a novel tool for precise particle sizing, matching up with electron microscopy (EM) resolution. Moreover, based on their refractive indices, we distinguish urinary EVs from co‐isolates and provide additional information about their inner composition.

Methods : In iNTA, the single‐particle tracking was performed with an interferometric scattering microscope (Taylor & Sandoghdar 2019), resulting in an enhanced particle signal even at high frame rates (5kHz). We inferred size distributions and refractive indices from trajectory analysis and signal strength. Measurements of iNTA and classic NTA were compared using (i) inorganic particles, (ii) liposomes, and (iii) human urine after centrifugation. We identified urinary EVs by EM and western blot. The urine sample was further treated with detergent buffer and proteinase K.

Results : In contrast to classic NTA, our interferometric approach resolved polydispersions of gold nanoparticles < 30nm and provided size distributions comparable with EM. In human urine, iNTA revealed two subpopulations based on their refractive indices. Upon treatment, the sub‐populations differed in their response, suggesting one to be urinary EVs. Based on a spherical model, we determined their inner refractive index to be 1.373, corresponding to a protein content of about 13%. This finding was validated by measuring liposomes prepared in water.

Summary/Conclusion : iNTA can reliably identify EVs from other co‐isolates in complex media, demonstrating the potential to become the next‐generation particle sizer for extracellular vesicle research.

Funding : AK acknowledges funding from Alexander von Humboldt‐Stiftung

Keywords : NTA, iNTA, urinary EV, size, refractive index, liposome

Isolation of extracellular vesicles with high purity using mixed mode resin (MMR) method

Wendy Trieu 1 ; Dmitry Ter‐Ovanesyan 2 ; Bogdan Budnik 3 ; George Church 3 ; David Walt 3

1 Stanford, Stanford, USA; 2 Harvard University, Cambridge, USA; 3 Wyss Institute for Biologically Inspired Engineering, Boston, USA

Introduction : It is difficult to separate extracellular vesicles (EVs) from free proteins in biofluids such as plasma. We set out to develop a method for high purity EV isolation for applications such as mass spectrometry.

Methods : Therefore we developed a novel, high purity EV isolation method which uses mixed mode resin (MMR) traditionally used in chromatography columns in a slurry format. We optimized our MMR Slurry method to work by measuring the tetraspanins CD9, CD63, CD81, and albumin, as well as total protein to find the optimal parameters for isolation.

Results : We optimized our MMR Slurry isolation method to isolate EVs from human CSF as a one‐step protocol and plasma as a two‐step protocol after size exclusion chromatography (SEC) with high purity. We found that we retain high levels of EVs while eliminating quantifiable albumin. Furthermore, we can tune the purity by changing the ratio of resin to free protein.

Summary/Conclusion : We have developed a novel method, MMR Slurry, for isolation of EVs from biofluids with high yield and purity. This method is well suited for mass spectrometry on clinically relevant volumes of biofluids and can be scaled for large numbers of patient samples.

Funding : Chan Zuckerberg Initiative; Open Philanthropy Project; Wyss Institute

Development of microsphere‐based solid support for the separation of extracellular vesicles and suppression of non‐specific binding

Dario Brambilla; Marcella Chiari

National Research Council of Italy ‐ Institute of Chemical Sciences and Technology (CNR ‐ SCITEC), Milan, Italy

Introduction : To evaluate the biological roles of specific extracellular vesicles (EVs) subpopulations, the choice of a proper separation protocol is mandatory. Especially when dealing with low represented EVs subpopulations, the need for enrichment strategies that display high specificity and selectivity is crucial. Contamination might result in misleading and data.

The immuno‐affinity separation of EVs offers unmatched selectivity. The use of antibodies allows the recognition of surface antigens that potentially discriminate different populations of EVs. Unfortunately, antibodies represent only one of the factors that impact on the presence of contaminants. Indeed, the characteristics of the solid surface are equally important. The ideal surface should provide an aqueous‐like environment, an optimal orientation, and accessibility of probes, and possess antifouling properties to prevent non‐specific adhesion of biomolecules.

Within this work, the polymer MCP‐6 has been applied in the coating of microspheres. The solid support, used to immobilize biomolecules, provides the efficient separation of EVs with significant reduction of contaminants.

Methods : Microspheres were coated using MCP‐6, a polymer functionalized with azide groups. The beads were functionalized with biomolecules exploiting the strain‐promoted azide alkyne cicloaddition (SPAAC).

Beads were used to immobilize DNA antibody conjugates (DACs), consisting of a DNA able to direct the protein on the surface of beads, and an EV‐directed antibody.

Finally, DACs‐functionalized beads were used to perform the separation of EVs.

Results : Incubation of microspheres with a solution of MCP‐6 provided a stable coating. Polymeric coating confers antifouling properties to microspheres towards proteins with different isoelectric points.

MCP‐6 coated beads can immobilize biomolecules modified with dibenzocyclooctine groups (DBCO). Probes retain their activity after immobilization, and their density on the surface can be finely tuned.

Beads functionalized with biological probes were used to immobilize DACs, and used to separate EVs from complex media.

Summary/Conclusion : A novel strategy to provide the coating of beads with an antifouling copolymer was developed. The resulting surface, exposing azido groups, can be used to immobilize DBCO‐functionalized biomolecules exploting SPAAC reaction. Additionally, the surface ensures exceptional antifouling properties, essential in reducing non‐specific binding on the surface.

Different biomolecules have been immobilized on the surface and were used to capture DACs. Thanks to DACs, EVs were separated from complex biological media as intact vesicles which can be further analyzed using imaging techniques or even destroyed to analyze cargo composition.

Funding : This work was supported by the European Union Horizon 2020 under Grant 766466: INDEX, Integrated Nanoparticle isolation and Detection system for complete on‐chip analysis of EXosomes.

Characterising extracellular vesicles from individual low volume cerebrospinal fluid samples, isolated by SmartSEC

Yael Hirschberg 1 ; Kurt Boonen 1 ; Karin Schildermans 1 ; Annemieke van Dam 2 ; Isabel Pintelon 3 ; Charysse Vandendriessche 4 ; Yannick Vermeiren 5 ; Inge Mertens 1

1 VITO/UAntwerp, Mol, Belgium; 2 Amsterdam UMC, Amsterdam, Netherlands; 3 Laboratory of Cell Biology & Histology Antwerp Centre for Advanced Microscopy (ACAM) University Antwerp Universiteitsplein 1, Antwerp 2610, Belgium, Antwerpen, Belgium; 4 VIB/Ghent University, Ghent, Belgium; 5 WUR/UAntwerp, Wageningen, Netherlands

Introduction : Extracellular vesicles (EVs) have recently been considered as a potential biomarker source for a variety of diseases, including neurodegenerative disorders. EVs are important mediators of intercellular communication due to their capacity to transfer genetic material, lipids and proteins. By means of their communication role, interesting biomarkers are often enriched in EVs compared to total biofluid. In order to isolate EVs from a biofluid, like cerebrospinal fluid (CSF), a variety of isolation methods are available of which each methods shows different results, and, to date, there is no golden standard. Our study evaluates the use of size exclusion chromatography (SEC) by the SmartSEC HT kit for EV isolation from CSF.

Methods : EVs were obtained from 500μL CSF samples, isolated by SmartSEC HT. SmartSEC traps contaminants, like bulk proteins, into the resin beads and subsequently elutes EVs. To evaluate this isolation method, various characterisation analyses were run. To detect cargo or surface proteïns, analyses on the ExoView R100, Surface Plasmon Resonance imaging (SPRi) and LC‐MS based proteomics analysis were performed. Concentration, and size of the vesicles were evaluated by use of transmission electron microscopy (TEM), and fluorescence nanotracking analysis (Zetaview).

Results : By means of the ExoView, tetraspanins such as CD81 and CD9 were measured on the surface of the EVs. Other EV‐specific proteins, e.g. lactadherin, flotillin‐1, and HSP70, were detected by SPRI. Untargeted protein analysis revealed the cargo proteins. TEM images visualized the presence of EVs. Particle size and concentration was calculated by nanotracking analysis, and more specifically the particles with a lipid membrane, like EVs, with fluorescence nanotracking analysis.

Summary/Conclusion : As EVs hold a lot of promise in the biomarker field, a standard high‐performance isolation method ‐ regarding purity and yield – is essential. From our data, SmartSEC HT is suggested to be an efficient size exclusion chromatography EV isolation method for low volume CSF samples, especially for high throughput, since samples are loaded on a 96‐well plate.

Keywords : Extracellular vesicles ; cerebrospinal fluid ; size exclusion chromatography; low sample volume

Fluorescent‐labelling strategy for phenotyping heterogeneous extracellular vesicles in complex biological matrices

Danilo Mladenović 1 ; Kristjan Orrin 2 ; Andras G. Miklosi 3 ; Saara Laitinen 4 ; Sergei Kopantšuk 5 ; Zoltán Giricz 6 ; Delaram Khamari 7 ; Kairi Koort 2 ; Natasa Zarovni 8

1 Tallinn University / Hansabiomed Life Sciences, Tallinn, Estonia; 2 Tallinn University, Tallinn, Estonia; 3 Oxford Nanoimaging Limited (ONI), Oxford, UK, Oxford, United Kingdom; 4 Finnish Red Cross Blood Service, Helsinki, Finland; 5 University of Tartu, Tartu, Estonia; 6 Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary; Pharmahungary ltd., Szeged, Hungary, Budapest, Hungary; 7 Department of Genetics, Cell‐ and Immunbiology, Semmelweis University, Budapest, Hungary; 8 HansaBioMed Life Sciences Ltd, Mäealuse 2/1, 12618 Tallinn, Estonia, Tallinn, Estonia

Introduction : Single extracellular vesicle (EV) analyses have always been a challenging task, especially for complex biofluids, such is plasma, that contain heterogeneous population of EVs, as well as many lipoproteins (LPs). Fluorescent labelling facilitates EVs analyses to some extent, however, many of the lower expressing markers and underrepresented subpopulations would end up being undetectable due to the lack of sensitivity, or few traceable events in instrument's measuring range.

Here we propose a methodology combining NTA and plate reader for single‐particle and bulk fluorescence analyses, respectively, that provides sufficient data for meta‐analyses of EVs subpopulations and marker abundance.

Methods : Human blood plasma was purified with SEC, EV fractions were pooled, and exact number of particles was stained either with CMG, CFSE, or fluorescent antibodies. After washing, samples were analyzed on NTA and plate reader (bulk fluorescence of EVs). Total number of antibodies was estimated from bulk fluorescence (known amount of soluble antibody in serial dilutions was used as a reference) and normalized to CFSE+ particles.

Purified EVs from different sources, as well as LPs, were used as controls. For comparison, marker abundance per single EV was also assessed using orthogonal methods, such as super‐resolution microscopy.

Results : EVs make up a small portion of total particles in SEC‐purified plasma (< 20%). Dominant marker for EVs was CD9, with ∼100 antibodies bound per single EV, while the other tetraspanins, CD63 and CD81, had more moderate expression (15‐25 antibodies per EV). Markers denoting specific cell origin were spanning from 3‐5 antibodies per particle for CD36, CD42b, and HLA I, and ∼30 for CD41 and CD235a.

Summary/Conclusion : We provide a simple proof of concept that could enable close estimation of marker abundance in heterogeneous EV populations, especially for the events that are not detectable in conventional single‐particle analyses, due to the low epitope density.

Funding : This research has received funding from the Tallinn University Research Fund (project TF7320) and European Union's Horizon 2020 research and innovation programme, under the FET Proactive grant agreement No. 952183.

Keywords : single extracellular vesicle analysis; marker abundance; fluorescent labelling; nanoparticle tracking analysis; super‐resolution microscopy

Effect of triggering strategy using flow cytometry for detecting extracellular vesicles directly in platelet poor plasma samples

Rikke W. Rasmussen 1 ; Jaco Botha 2 ; Mathilde Sanden 3 ; Maiken Mellergaard 1 ; Aase Handberg 1

1 Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark; 2 Department of Clinical Biochemistry, Aalborg University Hospital, Copenhagen, Denmark; 3 Aalborg University Hospital, Department of Clinical Biochemistry, Aalborg, Denmark

Introduction : High‐resolution flow cytometry is a widely used method for characterization of extracellular vesicles (EV). It is, however, important to consider how to collect and analyse data. Using different combinations of light scatter and/or fluorescence triggering threshold strategies is a common way to select for events of interest and subtract background noise. It has previously been reported that higher EV concentrations can be detected using fluorescence triggering compared with light scatter triggering. Here we tested two triggering strategies for rare and abundant EV‐phenotypes in human plasma.

Methods : Platelet poor plasma samples from six anonymous individuals were stained with a mixture of lactadherin, CD41, and CD36, or lactadherin and corresponding isotype controls. Samples were analysed on an Apogee A60 Micro‐PLUS flow cytometer collecting events using light scatter trigger or a combination of light scatter and fluorescence trigger. Gating was performed by using detergent lysis controls for defining lactadherin‐binding (phosphatidylserine positive, PS+) populations and matched isotype controls for CD41 and CD36 on PS+ populations.

Results : Results showed higher concentrations of detected EV populations when using light scatter alone compared with combined light scatter and fluorescence thresholds. This applied to PS+, CD36+, CD41+ as well as double CD36+, CD41+ positive populations.

Summary/Conclusion : We wanted to know how the threshold strategy in general affects EV data analysed on our flow cytometer and hypothesized that fluorescence triggering would detect more of the rare EV phenotypes compared with a light scatter threshold. Surprisingly, we found that we could detect more plasma EVs using light scatter alone. This suggests that fluorescence triggering in this case would fail to collect dim fluorescent EV populations that could be of interest. More studies are needed to further clarify if this is also the case in other designs.

Keywords : Flow cytometry, Threshold, EV quantification

Interaction modes of membrane active peptides with etracellular vesicles (EVs)

Tasvilla Sonallya 1 ; Imola Csilla Szigyártó 2 ; Tünde Juhász 3 ; Zoltán Varga 3 ; Tamas Beke‐Somfai 4

1 Research centre of natural science, Hungary, Budapest, Hungary; 2 Research Centre for Natural Science, Institute of Materials and Environmental Sciences, Budapest, Hungary; 3 Research centre natural science,Hungary, Budapest, Hungary; 4 Research Centre for Natural Science, Budapest, Hungary

Introduction : Membrane active peptides are promising biomaterials with antimicrobial and anticancer applications. They impart their biological applicability either by altering the cell membrane through perturbation or lysis or by enabling targeted drug delivery. The mechanism of interaction of each peptide with the membrane is unique and different models such as carpet, toroidal pore and barrel stave have been used to describe these mechanisms. There are several aspects, from joint presence on infection sites roles to EV cargo loading, where interaction EV – MAP interactions could be relevant, however, our knowledge here is scarce. Based on our initial investigations (1), we have selected 16 antimicrobial peptides and studied the interactions red blood cell derived EV (REV)s to gain an overview on their interaction and action mechanisms.

Methods : Several well‐known peptides i.e. Indolicidin, Aurein 1.2., Dermcidin (DCD‐1), DHVAR 4, Bactenecin, Protegrin, Transportan, Buforin IIb, KLA, Temporin‐La, LL37, FK16, Mellitin, Polybia MPI, Histatin 5 and Penetratin were selected, and their interaction with REV was investigated using several techniques from membrane biophysics.

Results : The biophysical studies demonstrated that that some of the MAP mechanisms were vescicle‐penetrating, while others lytic, or lead to removal of protein corona. More specific, CM15 efficiently removes surface proteins, Melittin has strong membrane disrupting effect whereas, Tempo‐La, Polybia MPI and Bactenecin acts with a different mechanism which has lower disrupting affinity on the original REV composition.

Summary/Conclusion : These results give an insight into an overview of the surface interactions of membrane active peptides with the REVs help us to attain a broad perspective on the molecular level interactions which could, in turn, provide vital information on engineering the surface and the interior of Evs with short MAPs.

Funding : This work was funded by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021‐EGA‐31 and the 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021 funding schemes. Support from Eötvös Loránd Research Network, grant n.o. SA‐87/2021, is also acknowledged.

Keywords : red blood cell, extracellular vesicle, membrane active peptide, light spectroscopy, protein corona

EV isolation kit comparison for stroke biomarker discovery

Olga Neustroeva 1 ; Cristiana Pistono 1 ; Jacopo Zini 2 ; Jere Kekkonen 2 ; Marjo Yliperttula 2 ; Tiia Turunen 1 ; Nea Korvenlaita 1 ; Tarja Malm 1

1 A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland, Kuopio, Finland; 2 Division of Pharmaceutical Biosciences and Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, Helsinki, Finland

Introduction : Ischemic stroke (IS) affects around 15 million people annually. Survived patients suffer from long‐term consequences and the outcome for those cannot be predicted due to lack of reliable biomarkers. Brain‐derived extracellular vesicles (EVs) secreted to the blood stream may carry molecules indicative of brain status of stroke‐exposed patients. As blood is enriched with liposomes, platelet‐derived EVs and a high diversity of proteins that can complicate EV isolation in the blood stream, there is need to standardize the isolation methods and carry out in depth evaluation of the EV composition to ensure reliable biomarker discovery

Methods : There are a number of methods for isolation of EVs including ultracentrifugation, polymer‐based precipitation, centrifugation in a density gradient and size exclusion chromatography (SEC). Each of the methods faces challenges as some of them are time‐consuming and some give a poor yield or poor quality of EVs. The most appropriate method should be chosen depending on the specific aim. In biomarker discovery it is important to choose a method that allows us to work with a high number of samples with a sufficient yield for several type of analysis and quality controls. We carried out in depth characterization of three different isolation methods: lectin‐based purification, precipitation, and SEC. We used nanoparticle tracking analysis (NTA) to determine the efficiency of isolation and to measure EV quantity.

Results : Our data demonstrate that precipitation gives the highest number of particles compared to lectin‐based purification or SEC. However, calculation of particles/protein ratio revealed that SEC results in the purest preparations. Cryo‐EM analysis showed that EV samples obtained by precipitation are enriched with lipoproteins and cell debris. Instead, SEC yielded samples were enriched in EVs and contained less contaminating particles. Western blot (WB) confirmed that purity of SEC fractions was higher compared to other two methods. Raman spectroscopy analysis revealed that fractions 2 and 3 clustered together.

Summary/Conclusion : Considering that WB and Cryo‐EM analysis showed that these fractions are abundant in EV‐associated markers and less contaminated with liposomes, our data supports that isolation of fractions 2 and 3 by SEC as the method of choice for isolating EVs from plasma samples.

Funding : Academy of Finland and Business Finland

Keywords : biomarkers, ischemic stroke, size exclusion chromatography, lectin‐based purification, precepitation, EV characterisation

An integrated fluidic‐plasmonic lab‐on‐chip for the label‐free characterization of extracellular vesicles for liquid biopsy applications

Sabrina Romanò 1 ; Flavio Di Giacinto 1 ; Maria Eleonora Temperini 2 ; Leonetta Baldassarre 2 ; Valeria Giliberti 2 ; Riccardo Di Santo 1 ; Massimiliano Papi 1 ; Alberto Augello 1 ; Marco De Spitiro 1 ; Michele Ortolani 3 ; Gabriele Ciasca 1

1 Fondazione Policlinico Universitario A. Gemelli IRCCS & Università Cattolica del Sacro Cuore, Roma, Italy, Rome, Italy; 2 Dipartimento di Fisica, Sapienza University of Rome & Istituto Italiano di Tecnologia, Center for Life Nanoscience, Rome, Italy; 3 Dipartimento di Fisica, Sapienza University of Rome & Istituto Italiano di Tecnologia, Center for Life Nanoscience, ROme, Italy

Introduction : EVs are considered a promising source of cancer biomarkers. Despite this potential, the EV translational process in diagnostics is still at its birth, and the development of novel medical devices is highly demanded. Here we present a lab‐on‐chip platform for EV characterization with application in cancer diagnostics. The device combines three cutting‐edge technologies: i) a SEIRA metasurface for the biochemical characterization of EVs, ii) an SPR sensor for mass quantification, and iii) a hydrophobicity‐driven wet sample holder that enables IR measurements in liquid.

Methods : The metasurface was fabricated in gold on CaF2 using EBL and, subsequently, functionalized with Anti‐CD63 for EV immunocapture. The fluidic holder was realized in PLA with 3D printing. EVs extracted from human cancer cells were used as a model system, after extensive ultrastructural characterization. IR measurements were carried out with an FTIR spectro‐microscope.

Results : The proposed sensor is endowed with label‐free chemical sensitivity, being tunable on the specific absorption bands of biomolecules within EVs, namely the Amide I‐II (1700‐1500 cm‐1) and lipid (3000‐2750 cm‐1) bands. The 3D‐printed holder enables IR measurements in liquid using a 10 μl droplet. The device allows real‐time monitoring of the metasurface functionalization with antibodies and the subsequent EV immunocapture. A protein sensitivity down to the zeptomoles range is demonstrated. Most importantly, the sensor allows for measuring the specific IR spectral fingerprint of EVs in the Amide‐I and II regions.

Summary/Conclusion : Thanks to the high protein sensitivity and the possibility to work with small sample volumes ‐ two key features for ultrasensitive EV detection ‐ our lab‐on‐chip can positively impact the development of novel liquid biopsy approaches for cancer diagnosis based on the label‐free characterization of EVs.

Funding : The Italian Ministry of Health is gratefully acknowledged (grant n. GR201602363310)

Keywords : Microdevice, Liquid Biopsy, Plasmonics

Extracellular Vesicles (EVs) as targetable drug delivery vehicles

Maria Chiara Ciferri 1 ; Daniele Reverberi 2 ; Silvia Bruno 3 ; Cansu Gorgun 3 ; Chiara Gentili 3 ; Rodolfo Quarto 4 ; Roberta Tasso 3

1 University of Genova, Genova, Italy; 2 IRCCS Ospedale Policlinico San Martino, Genova, Genova, Italy; 3 Department of Experimental Medicine, University of Genova, Genova, Italy; 4 Department of Experimental Medicine, University of Genova and IRCCS Ospedale Policlinico San Martino, Genova, Genova, Italy

Introduction : EVs have been recently considered as valid delivery vehicles thanks to their activity as short and long‐distance mediators of intercellular communication. In this study we propose a two‐step EV‐engineering protocol to obtain a tissue‐specific drug delivery system, based on: (i) loading with a medicinal cargo, and (ii) surface functionalization to be used as a platform to bind molecules.

Methods : Plasma‐derived EVs have been isolated from healthy donors by size exclusion chromatography coupled with iodixanol density cushion and characterized by Nanoparticles Tracking Analysis (NTA), non‐conventional flow cytometry (FCM) and western blot. Drug (Paclitaxel ‐ PTX) loading has been achieved by sonication using a probe sonicator (30 s on/30 s off, 20% W amplitude at RT, 2 min cooling × 6 cycles), while membrane functionalization has been obtained through a copper‐free click chemistry reaction (alkyne‐azide Huisgen 1,3‐dipolar cycloaddition). As a proof of concept, a fluorescent azide (AF647 azide) and a fluorescent Paclitaxel (Pacific Blue Paclitaxel) have been used to easily verify both PTX internalization and the conjugation of the fluorescent azide to the EV membrane, by flow cytometry and fluorescent NTA. The uptake of engineered EVs by responder tumor cells (MDA‐MB‐231, a human breast cancer cell line) has been evaluated in vitro and analyzed by flow cytometry and confocal microscopy

Results : Both copper‐free click chemistry and sonication turned out to be a valid surface engineering technique, since about 50% of EVs were AF647‐positive and Pacific Blue‐positive. Engineered EVs are efficiently internalized by MDA‐MB‐231 cells, inducing significant cytotoxic effects.

Summary/Conclusion : Our EV membrane functionalizing system can be used to bind to the EV surface azido‐modified antibodies/molecules, making the engineered EVs (loaded also with a therapeutic cargo) valuable natural drug nanocarriers able to be directed to a specific antigen/target and so potentially applied in various pathological conditions.

Keywords : Extracellular vesicles, drug delivery, click‐chemistry

High‐resolution Atomic Force Microscopy as a tool for topographical mapping of surface budding

Carolina Sbarigia 1 ; Stefano Tacconi 2 ; Francesco Mura 3 ; Marco Rossi 4 ; Luciana Dini 2 ; Simone Dinarelli 5

1 Department of Biology and Biotechnology "C.Darwin", Sapienza University of Rome, Roma, Italy; 2 Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy; 3 Research Center of Nanotechnologies for Engineering (CNIS), Sapienza University of Rome, Rome, Italy; 4 Research Center of Nanotechnologies for Engineering (CNIS), Sapienza University of Rome, Roma, Italy; 5 Institute for the Structure of Matter (ISM), National Research Council (CNR), Rome, Italy

Introduction : Development of a new methodology to study extracellular vesicles (EVs) morphological features and spatial distribution over the cell surface and their alterations under different physiological conditions, evaluating the efficacy of an Atomic Force Microscopy (AFM) system compared to a conventional Electron Microscopy (EM) method.

Methods : Control and H2O2‐treated SH‐SY5Y neuroblastoma cells were analyzed by SEM and AFM. Large vesicles‐enriched fractions were concentrated by ultracentrifugation from both cells and their morphology, quantity and size distribution were quantified by TEM and AFM. In AFM analysis, defined square areas on the cell nuclear and peripheral regions were selected to map the spatial distribution of the surface budding.

Results : The SEM‐AFM comparative analysis revealed that AFM detected a rougher surface of both control and H2O2‐treated cells, being also capable to identify the presence of vesicles in their terminal portions with respect to the SEM. The buds size distribution and mean diameter were comparable within the two methods. The TEM and AFM analysis of isolated vesicles from control and H2O2‐treated cells displayed the same morphological appearance and range diameter, compatible with large vesicles, further confirmed by the presence of the specific marker annexin A1 by western blotting. We demonstrate how AFM allowed to visualize the 3D structure of isolated vesicles and measured a higher mean diameter of both the populations. Cell roughness and waviness measurements in the cell nuclear region and periphery, according to AFM imaging, showed an increased budding in the periphery of H2O2‐treated cells with respect to the nuclear region and to control cells.

Summary/Conclusion : The results obtained from this comparative study may provide interesting perspectives for the use of AFM for morphological characterization of large vesicles and mapping of surface budding, validating a morphometric approach to understand the pathophysiological state of the cell.

Funding : No external funding

Keywords : Atomic Force Microscopy; surface budding; topographical mapping; roughness

Assessment of extracellular vesicles in pancreatic cancer patient samples for the detection of tumor specific biomarkers

ramsha Masood 1 ; Jerome Nouvel 2 ; George Daaboul 3 ; Uwe Wittel 4 ; Gerhard Puetz 5 ; Irina Nazarenko 6

1 exosomes and tumor biology group university medical center freiburg, Freiburg im breisgau, Germany; 2 1 Institute for Infection Prevention and Hospital Epidemiology; Medical Center ‐ University of Freiburg, Faculty of Medicine, University of Freiburg, freiburg, Germany; 3 NanoView Biosciences, Boston, USA; 4 Department of General and Visceral Surgery, Medical Center ‐ University of Freiburg, Faculty of Medicine, University of Freiburg, freiburg, Germany; 5 2 Institute of Clinical Chemistry and Laboratory Medicine, Medical Center ‐ University of Freiburg, Faculty of Medicine, University of Freiburg, freiburg, Germany; 6 Institute for Infection Prevention and Hospital Epidemiology, Faculty of Medicine, University Medical Center Freiburg, Freiburg, Germany, Freiburg, Germany

Introduction : Pancreatic cancer (PDAC) is considered as one of the deadliest cancers in the world. It has the lowest survival rate of all solid cancers. The poor prognosis of pancreatic cancer is mainly because it is asymptomatic due to which, it cannot be detected at early stages. One strikingly promising source of biomarkers for early detection and patients stratification are the Extracellular Vesicles (EVs). However, the methods allowing EV baseline purification from complex biofluids, such as cell‐free plasma or serum are limited. There are two main challenges to solve: i. non‐EV components, especially lipoproteins, have similar size and density as EVs; ii. concentration of lipoproteins, such as VLDL and LDL is highly variable and exceeds several orders of magnitude the concentration of EVs

Methods : Firstly, potential impact of the pre‐analytic routinely used by the biobank for the storage of cell‐free plasma and serum was controlled by examining haemolysis, amount of remaining platelets, and particle number. Addressing the robustness of quantification of disease‐associated biomarkers, we estimated the presence of different lipoproteins, EV‐, and tumor‐biomarkers in EVs derived from the serum samples of PDAC patients. Comprehensive characterization of biomarker distribution was performed for each of the SEC fractions using either bulk methods, such as ELISA and beads‐assisted flow cytometry with latex‐beads and MACSPlex; or single particle‐ analysis, using fluorescent NTA and interferometric imaging (SP‐IRIS).

Results : We observed a very good consistency of data obtained from different patients regarding the amount and distribution of remaining lipoproteins and tumor‐specific biomarkers in EVs, supported feasibility of EV usage for liquid biopsy. Despite of a certain amount of lipoproteins remained in EV‐enriched fractions, bulk EV analysis, such as ELISA and beads‐assisted flow cytometry showed highly consistent results, which was further confirmed by the single‐EV analysis. While EVs derived from the platelets and harbouring the platelets‐specific surface protein CD41, and EV‐specific biomarkers CD9 and CD63 were mostly concentrated in the fraction F2, the fractions F3 contained majority of EVs positive for EpCAM, Tspan8, and the integrin beta6 pancreatic cancer EV biomarkers.

Summary/Conclusion : Our data indicate that if for the analysis of EV lipidomics, generation of pure EV fractions should be mandatory, for the detection of disease‐specific EV proteins, a certain amount of non‐EV components in EV fractions may be tolerated. Robustness of data across a number of PDAC patients confirmed EV surface signature as promising biomarker. Separation of platelets and cancer‐derived EVs in different fractions suggested that even a short path length of a column may allow to differentiate between EVs of different size and different origin.

Friday 27 May 2022

PF09: Late‐Breaking Poster Session 3: Technologies and Methods II

Chair: Marta Garcia‐Contreras – University of Miami, Diabetes Research Institute

Chair: Malene Møller Jørgensen

Label‐free, real‐time detection of extracellular vesicles expressing CXCR4 using surface plasmon resonance

Arnaud Boonen; Sam Noppen; Yagmur Yildizhan; Dragana Spasic; Cindy Heens; Frank Vanderhoydonc; Tom Van Loy; Johan Swinnen; Jeroen Lammertyn; Christophe Pannecouque; Dominique Schols

KU Leuven, Leuven, Belgium

Introduction : Analysis of extracellular vesicles (EVs) is getting more attention as their role showed to be more important than previously thought. EVs entrap nucleic acids, cellular proteins and metabolites in order to act as carrier vesicles between cells. CXCR4 is a G protein‐coupled receptor known to be expressed on the membrane of EVs. Aside from its natural physiological functions, this protein plays an important role in several pathologies including cancer. CXCR4 is found to be overexpressed on various types of tumor cells. EVs derived from these tumor cells can carry a high amount of CXCR4. This suggests that the analysis of CXCR4 positive EVs can be an important tool in cancer diagnostics. Therefore, we developed a label‐free, real‐time binding assay to detect CXCR4 positive EVs using surface plasmon resonance (SPR).

Methods : EVs were obtained from HEK293 wild‐type, HEK293 CXCR4‐transfected, MOLT4, MCF7, U87 wild‐type, U87 CXCR4‐transfected and MT4 cells. The isolation protocol was optimized using recombinant EVs. EVs were isolated by ultrafiltration paired with size exclusion chromatography. They were characterized by nanoparticle tracking analysis. SPR was used to identify EV biomarkers (CD81, CD63, CD9) as well as CXCR4. Fiber‐optic SPR (FO‐SPR) was used to detect the EV markers and CXCR4 in non‐concentrated media.

Results : SPR detected a difference in EV markers between the EVs isolated from different host cell types. HEK293 and MOLT4 EVs contained mostly CD81. U87 EVs had more pronounced CD63 expression compared to the other markers. MCF7 EVs depicted a high expression of both CD81 and CD63. MT4 EVs contained mostly CD9. Furthermore, overexpression of recombinant CXCR4 in HEK293 and U87 cells translated to an increased CXCR4 expression on the EVs. Endogenously expressed CXCR4 was detected in EVs from most of the cell types, with MT4 and MOLT4 showing the highest response even surpassing their EV biomarker responses. FO‐SPR allowed us to detect colocalized EV markers and CXCR4 in non‐concentrated media.

Summary/Conclusion : Our assay was able to distinguish between endogenously expressed‐ and overexpressed CXCR4 and enabled the detection of cancer cell derived EVs expressing a high amount of CXCR4. In addition, CXCR4 was detected endogenously in EVs from most of the cell types, indicating it could represent a new EV biomarker.

Membrane labelling of extracellular vesicles for high‐resolution flow cytometry

Maiken Mellergaard; Rikke W. Rasmussen; Anders Askeland; Morten Hjuler Nielsen; Aase Handberg

Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark

Introduction : Flow cytometry is a powerful tool for characterizing extracellular vesicle (EV) phenotypes. However, proper EV labelling and specificity of such is challenging and continuously debated. In example, studies have shown superiority of using lactadherin staining compared with other non‐antibody‐based labels such as carboxyfluorescein succinimidyl ester (CFSE), while CFSE labelling was seemingly superior to CD9 and CD81 staining of purified EVs. Previous data from our group showed that lactadherin and Annexin V to some extent also label ApoB‐containing lipoproteins. Finally, findings that a corona of proteins including ApoB and other common markers can form on the EV surface further complicates this issue.

Methods : In this preliminary study, we used high‐resolution flow cytometry (adhering to MIFlowCyt‐EV guidelines) to investigate EV labelling directly in human plasma, to compare and optimize EV staining in terms of incubation time and temperature. We used a cocktail of CD9, CD63, and CD81 (CD9/CD63/CD81) and compared with lactadherin staining for 30 minutes, 2 hours, or overnight (ON) at 4°C or room temperature (RT).

Results : Our preliminary results suggest that maximal lactadherin staining is reached after 2 hours at RT, while CD9/CD63/CD81 labelling, on the contrary increases continuously over time with highest labelling after ON incubation, especially at 4°C.

Summary/Conclusion : In conclusion, more studies are needed to verify and clarify our findings. However, these early data could suggest that lactadherin staining in plasma is saturated faster than labelling using a CD9/CD63/CD81 antibody cocktail, underlining the necessity for specifically optimized staining protocols.

NEXOS: A n electro‐optical bead‐nanochip technology for the ultrasensitive and multi‐dimensional detection of small extracellular vesicles and their markers

Tomas Dias 1 ; Ricardo Figueiras 1 ; Susana Vagueiro 1 ; Renato Domingues 1 ; Yu‐Hsien Hung 1 ; Elnaz Persia 1 ; Pierre Arsene 2

1 Mursla, Cambridge, United Kingdom; 2 Mursla, London, United Kingdom

Introduction : Small extracellular vesicles (sEVs), including exosomes, are enriched in multiomics information mirroring their parental cells. They have been investigated in health and disease and utilised in several applications from drug discovery to diagnostics. In disease diagnostics, sEVs can be sampled via a blood draw, enabling the convenient liquid biopsy of the tissue they originate from. However, few applications with sEVs have been translated into clinical practice. We hypothesize this is due to various factors including the relative limited sensitivity and limited information obtained with the gold standard sEV detection techniques.

Methods : We developed a novel technology, named Nanoparticle EXOsome Sensing (NEXOS), for the ultrasensitive and multi‐dimensional detection of sEVs. NEXOS comprises two methods, Electro‐NEXOS (E‐NEXOS) and Optical‐NEXOS (O‐NEXOS). E‐NEXOS is a novel nanoelectronics method which uses nanochips and O‐NEXOS is a high‐throughput optical method. Yet, they share the same steps for immunocapture and labelling of sEVs and can be combined to derive differentiated measured parameters.

Results : We present the working principle of the technologies and, as proof‐of‐principle, show the linear detection and sensitivity of the methods in detecting target CD9+CD81+ and CD9+HER2+ sEVs, diluted in PBS or spiked in processed plasma, originated from breast cancer cell cultures. We also provide an approach for the determination of new dimensions, unseen with the gold standards or with emerging technologies, namely the detection of target sEVs (TEVs), target epitopes in sEVs (TEPs) and epitopes per target sEV.

Additionally, we demonstrate the higher sensitivity of O‐NEXOS when compared to the gold standards and demonstrate that E‐NEXOS is, at least, on par with those, albeit only powered by 36 nanogap sensors.

Finally, we lay the groundwork for the scaling and electronics miniaturization of E‐NEXOS with millions of nanogap sensors for the translation of NEXOS into routine sEVs analysis.

Summary/Conclusion : To conclude, we presented a novel technology for the ultrasensitive detection of sEVs which also determines new dimensions such as the detection of TEVs, TEPs and number of target epitopes per sEV.

At its present state, O‐NEXOS demonstrates higher sensitivity than the tested gold standard sEV detection technologies while E‐NEXOS, with the indicated scaling and electronics miniaturisation from 36 to 2,000,000 sensors, has the potential to achieve extremely high sensitivity and translate sEV‐based liquid biopsies into routine clinical care practices.

Unbiased and UMI‐informed sequencing of plasma EV‐associated miRNAs at single‐nucleotide resolution

M.A.J. van Eijndhoven 1 ; E. Aparicio‐Puerta 2 ; Cristina Gómez‐Martín 3 ; J.M. Medina 2 ; E.E.E. Drees 4 ; E.J. Bradley 3 ; Leontien Bosch 1 ; C. Scheepbouwer 3 ; M. Hackenberg 2 ; Dirk M. Pegtel 1

1 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, Amsterdam, Netherlands; 2 University of Granada, Granada, Spain; 3 Amsterdam University Medical Centers, Amsterdam, Netherlands; 4 amsterdam university medical centers, amsterdam, Netherlands

Introduction : Recent advances show that apart from 21nt mature miRNAs, miRNAs with non‐templated additions (NTAs), called isomiRs have biological relevance that can dramatically change their pre‐processing, stability and targetome. Deconvolution of these posttranscriptional modifications is challenging in particular for low‐input samples, e.g. plasma extracellular vesicles (pEV) that are considered as a source for minimally‐invasive diagnostics.

Both for biological studies as well as for diagnostic purposes accurate detection and distinction of mature miRNAs and isomiRs are essential.

Methods : RNA sequencing is currently the only reliable method that can distinguish mature miRNAs from isomiRs with single‐nucleotide resolution and that can truthfully reveal isomiR diversity in biological samples. Improvements aside, current small RNA sequencing strategies remain imprecise. We developed “IsoSeek” that diverges from these methods by making use of randomized 5’‐ and 3’‐adapters combined with a 10N unique molecular identifier (UMI).

Results : Using synthetic mature miRNA and isomiR spike‐ins in multiple optimization rounds, we show that IsoSeek has reduced ligation and PCR amplification bias leading to improved accuracy in the detection of isomiRs.

To investigate the detection of isomiRs in a biological background, we sequenced HEK293T cells in which the terminal uridylyl transferases TUT4 and TUT7 were ablated by CRISPR/Cas9 (TUT4/7 DKO). The overall reduction in uridylation of miRNAs is much more pronounced when using IsoSeek compared to commercial protocols. Looking at individual miRNAs, we characterized the terminal uridylase dependent miRNA uridylome and discovered extensive uridylation of disease‐associated miRNAs.

When sequencing pEV samples we show that IsoSeek captures the full complexity of isomiRs in comparison with commercial protocols. Finally we tested our IsoSeek protocol by preparing libraries from 188 DLBCL patient pEV samples. Hierarchical clustering of the sequence results before and after UMI correction shows a vast difference. This suggests that when analyzing low‐input clinical liquid biopsy samples UMI correction can reduce PCR bias based clustering.

Summary/Conclusion : IsoSeek is optimized for qualitative and quantitative miRNA profiling at single‐nucleotide resolution for functional and target prediction studies in vitro as well as in ultra low‐input biological samples.

A‐TEEM spectroscopic characterization of exosome standards and their mixtures

Linda H. Kidder 1 ; Adam M. Gilmore 2

1 HORIBA Scientific, Elliott City, USA; 2 HORIBA Scientific, Piscataway, USA

Introduction : Absorbance‐Transmission Excitation Emission Matrix spectroscopy (A‐TEEM) is sensitive to protein composition, rapid (< 60 sec data acquisition times), and non‐destructive. The purpose of this study is to test the feasibility of using A‐TEEM to quickly and sensitively differentiate exosomes in solution. With access limited to only commercially available samples, the study was limited in scope, but shows promise in rapid qualitative and quantitative characterization of exosomes.

Methods : Lyophilized Exosome Standards PC‐3 (ab239689) and MCF7 (ab239691) were purchased (Abcam, Cambridge, MA). All sample measurements were made on the Aqualog UV‐800 (HORIBA Scientific, Piscataway, NJ), with excitation from 220‐450 nm. A blank measurement was collected with PBS solvent. PC‐3 and MCF7 were reconstituted to 1 ug/ul with PBS, and then diluted an additional 200X and placed in a standard UV/Vis 1 cm path length cuvette for measurement. The PC‐3/PBS solution was measured, and mixtures with MCF7/PBS solution in the following proportions were measured: 75%, 50%, 25%. Pure MCF7/PBS solution was also measured. Data analysis employed multivariate methods. PARAFAC (Parallel Factor Analysis) was used for qualitative purposes, differentiate between the two exosome types. A PLS (Partial Least Squares) approach was used to predict the amount of the two in a mixture.

Results : The fluorescence EEMs profiles of these samples indicate that there are measurable differences in protein signatures between the samples. The PARAFAC model was clearly able to distinguish between the exosomes samples as well as each mixture. The PLS model was predictive of concentration, with an r‐squared value of > 0.99, and SD of ∼2%.

Summary/Conclusion : This feasibility study was undertaken with a limited scope to test the potential for this approach for the characterization of exosomes in solution. A‐TEEM was readily able to distinguish between the two exosomes, as well as their mixtures, and to predict the concentrations of the two components in the mixtures.

Funding : This research was fully funded by HORIBA Scientific, a for‐profit company that develops and sells analytical instrumentation.

Keywords : A‐TEEM, Fluorescence EEMs

Microbead‐based isolation of EVs from urine provides high specificity and effective yield with minimal co‐isolation of contaminants

Andres Lõhmus 1 ; Riccardo Vago 2 ; Natasa Zarovni 1

1 HansaBioMed Life Sciences Ltd, Mäealuse 2/1, 12618 Tallinn, Estonia, Tallinn, Estonia; 2 UniSR Università Vita‐Salute San Raffaele, Milan, Italy

Introduction : Urine features an ideal source of non‐invasive diagnostic markers for diverse clinical conditions. Urine EVs (uEVs) originate from different tissues, with the expected enrichment from the urogenital route. Many uEV isolation protocols for proteomic and transcriptomic analysis exist, likely to isolate different subpopulations or at least enable different uEV yield and purity, appropriate for different clinical questions. Out of these, affinity pull‐down protocols provide scalability and single‐step EV isolation, allowing the processing and analysis of both routinely harvested and biobanked urine samples. Such protocols are amenable and user friendly, can be automated and serve different analytical platforms, allowing for integration to end‐to‐end diagnostic assays. However, they typically trade‐off yield for purity, while a delicate balance of both is needed for reliable biomarker detection.

Methods : Micro‐beads of different size and material are compared to peptide based isolation kits in the purification of model EVs, both HEK293 and human urine‐derived particles. Pre‐labelled and recombinant fluorescent EVs are purified from buffers and human urine. The recovery of particles and common urine contaminants such as uromodulin is studied by F‐NTA, FIA, qPCR and WB.The specificity of immunoaffinity pull‐down is assessed by isotype antibodies and non‐human derived EV controls.

Results : We show that pre‐labelled and recombinant fluorescent EVs can be a very sensitive tool in assessing particle recovery from buffers and human urine. Using these tools we compare EV affinity‐isolation methods and show that magnetic immuno‐beads provide high effective yield and minimal co‐purification of contaminants compared to precipitation and centrifugation‐based isolation methods. In addition, we assess the impact of urine preanalytical variables, such as pH and variations in salts, and operative features such as cost‐effectiveness and time consumption of the affinity isolation protocols.

Summary/Conclusion : Affinity‐isolation of EVs with magnetic immunobeads provides high effective yield and is superior in specificity and purity compared to precipitation and centrifugation‐based isolation methods.

Funding : Funding: MARVEL Project ID 951768.

Keywords : immuno‐affinity, microbeads, magnetic beads, urine

Investigating the capture and fractionation of extracellular vesicles using heparin

Benjamin Barnes 1 ; Thomas Caws 2 ; Samantha Thomas 2 ; Alex P. Shephard 3 ; Randolph Corteling 2 ; Paul Hole 2 ; Daniel Bracewell 1

1 University College London, London, United Kingdom; 2 ReNeuron, Bridgend, United Kingdom; 3 NanoView Biosciences, Malvern, United Kingdom

Introduction : Despite the rapidly growing commercial interest in extracellular vesicles (EVs), manufacturing processes for EV production are still in the development phase. The recovery and purification of EVs remains a major hurdle in the progression of fundamental research and the application of EV‐based products.

Methods : The use of the glycosaminoglycan, heparin, to capture EVs is a relatively novel and unexplored method of EV purification. Heparin affinity chromatography (HAC) is a highly scalable and reproducible form of pseudo‐affinity chromatography. In this study, we demonstrated the use of a HAC protocol to fractionate EVs based on their affinity to heparin.

Results : This protocol fractionated EVs into two discrete eluted populations that exhibited statistically significant differences in tetraspanin immunophenotypes, variable expression of the exosome‐associated protein TSG101 and differences in purity. On average, the removal of 98.8% of residual protein and 99.0% of DNA was achieved using this chromatography protocol.

Summary/Conclusion : In summary, HAC shows promise as a method to fractionate EVs and with further development may be capable of isolating EV subpopulations with more desirable properties. However, a more detailed proteomic analysis of the eluted EV populations, with a focus on known heparin‐binding proteins, is needed to establish whether this protocol is truly isolating EV subpopulations with differing molecular profiles, or the observed differences are a result of heterogeneity in marker expression.

Funding : This work was supported by a 4‐year EPSRC EngD programme at University College London that was sponsored by ReNeuron (grant code: EP/L01520X/1) and by an Industrial Fellowship from the Royal Commission for the Exhibition of 1851.

Keywords : Extracellular vesicles, Heparin, Purification, Isolation

Identification and surface characterization of individual HIV‐1 virions amongst an extracellular vesicle background using single‐particle interferometric reflectance imaging sensing (SP‐IRIS)

Zach A. Troyer 1 ; Olesia Gololobova 2 ; Zhaohao Liao 3 ; Kenneth W. Witwer 2

1 Johns Hopkins University, Baltimore, USA; 2 Johns Hopkins University School of Medicine, Department of Molecular and Comparative Pathobiology, Baltimore, USA; 3 Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA, Baltimore, USA

Introduction : Enveloped viruses can be considered a type of extracellular vesicle that is influenced by a viral genomic program. Both host and viral EVs are nanoscale, lipid bilayer particles that share common egress pathways. Much effort has been spent attempting to separate true enveloped virions, such as those of HIV, from non‐viral EVs. The gold standard, density gradient ultracentrifugation, is costly and time‐consuming. Recently, the idea of using the EV marker tetraspanins CD9, CD63, and CD81 to immunopurify EVs away from HIV has been suggested. However, HIV virions also carry tetraspanins. Single‐particle surface characterization of HIV would be beneficial to the development of immunocapture separation methods. Here, we demonstrate that single HIV‐1 virions can be analyzed for surface markers using single‐particle interferometric reflectance imaging sensing (SP‐IRIS).

Methods : HIV‐1 (BaL strain) was concentrated from the conditioned medium of chronically‐infected PM1 cells. Uninfected PM1 cells provided an EV control. Conditioned media was processed via differential ultracentrifugation to enrich an HIV/EV or EV‐containing pellet. Samples were UV‐inactivated according to various protocols before incubation on SP‐IRIS chips. An AF488‐conjugated anti‐gp120 antibody was used as a fluorescent probe. SP‐IRIS was done with the NanoView ExoView R100 and tetraspanins capture chips.

Results : SP‐IRIS detected a distinct and relatively heterogeneous population of presumed HIV virions against a background of EVs. HIV particles were mostly C63+/CD81+/gp120+ and were approximately 100 nm in diameter. This distinct size population did not appear in the uninfected EV control.

Summary/Conclusion : The detected virus population likely represents a mixture of mature and immature HIV virions. In ongoing studies, we will use fluorescence‐linked antibodies to phenotype viruses for additional surface proteins. Use of a fluorescent probe targeting HIV gRNA in the presence of permeabilizing agents may also identify true infectious virions. This approach, including the inactivation protocols we optimized, can be used to study enveloped viruses outside of containment laboratories.

Keywords : HIV, EVs, SP‐IRIS, surface, characterization, separation

Advanced In‐situ Liquid Phase Transmission Electron Microscopy: A Revolutionary Technique for Extracellular Vesicle Research

Hugo Perez 1 ; Hongyu Sun 2 ; Vasilis Papadimitriou 2 ; Eva Bladt 2

1 DENSsolutions B.V., Delft, Netherlands; 2 DENSsolutions, Delft, Netherlands

Introduction : EV imaging by cryo‐Transmission Electron Microscopy allows for the observation of EVs in their near‐native state, achieving a detailed understanding of the morphology, size distribution, and phenotypes. However, the enumeration of EVs by this approach is affected by a systematic error, and the process prevents the possibility of enabling high‐throughput studies.

Methods : We introduce our technology for in‐situ Liquid Phase Transmission Electron Microscope (LPTEM) studies, which is an add‐on and a plug‐and‐play system. The latter relies on a Micro Electro‐Mechanical System (MEMS)‐based device as a smart sample carrier, referred to as the Nano‐Cell, which contains an integrated set of biasing electrodes or an integrated microheater. These capabilities enable comprehensive in‐situ studies on extracellular vesicles and pharmaceutical research as a function of temperature/biasing/pressure/flow rate. Similarly, the device contains chemical scavenging capabilities to mitigate the effect of the chemical radicals (i.e. radiation damage).

Results : The system enables high level of control over the microfluidics and high throughput studies. As part of the workflow, the user can: a) flow the liquid containing the EVs, b) enable static analysis of the batch (e.g. count the amount of EVs in the field of view, analyze their area and size distribution, or even study the particle trajectory), c) flush the batch and flow in a new batch, d) repeat the analysis. The waste can be collected to do further analysis. We have reported single particle analysis of proteins with 4.5 angstrom resolution while being in liquid. Additionally, we can use Brownian motion of the particle to resolve its 3D structure while being in liquid. Furthermore, the user can avoid this liquid‐air interface. Additionally, the system allows to control the mass transport, enabling the user to optimize the biochemistry on‐the‐fly. We have shown that these capabilities allow to visualize in‐situ the synthesis of vesicles (e.g. for drug delivery purposes). To fight radiation damage, we have successfully integrated Graphene onto our Nano‐Cell, providing chemical scavenging. We have studied micelles, where we can clearly see that without Graphene the particle starts getting destroyed with a dose rate of 0.3 electrons/square Angstrom, 1s/frame, whereas with Graphene we can successfully increase the dose by 3.5 times without attacking the sample.

Summary/Conclusion : Our development provides the unique possibility to visualize biological processes in real time with angstrom resolutions, while the biosample is in its native environment (as a function of different stimuli). This opens up several possibilities such as in‐vivo studies of protein‐protein interactions, biomedical/pharmaceutical applications of EVs (e.g. disease biomarkers, drug delivery vehicles or vaccines) and fundamental studies in biophysics and biochemistry.

Extracellular vesicle‐free foetal calf serum and human platelet lysate to sustain high extracellular vesicle production from human cells

Bileyle Lorenzini 1 ; Chloé Lézin 1 ; Sylvie Goulinet 2 ; Sabrina Soave 1 ; Philippe Mauduit 2

1 INSERM UMR‐MD 1197, Villejuif, France; 2 Inserm UMR‐MD 1197, Villejuif, France

Introduction : Therapies using human Mesenchymal Stromal Cells (MSCs) or Endothelial Progenitor Cells (EPCs) are getting a growing interest in a wide range of indications. A substantial part of their therapeutic effects relies on cell‐secreted factors and the EV are proposed as a cell‐free surrogate for therapy. Commonly used culture additives such as Fetal Calf Serum (FCS) or Human Platelet Lysate (HPL) contains large amounts of EV. Purification and characterization of cell secreted EV therefore needs either starvation conditions or the prior elimination of contaminant EV from these additives. Starving to produce EV often limit cell survival thus EV production. Hence, we compare the effect of starvation to EV‐free FCS or HPL addition on cell survival and EV production from both human adherent primary cells: MSC and EPC and non‐adherent human cell lines: JURKAT and THP1.

Methods : EV are removed from FCS/HPL by tangential‐flow‐filtration (TFF). After an amplification phase in complete media MSCs, EPCs, JURKATs and THP1 were placed in medium with or without EV‐free FCS/HPL. After 72h, the media were collected for EV quantification by NTA and replaced by fresh media for a new 72h production cycle. Cell number, morphology and EV production were followed at each step and in each incubation condition.

Results : EV‐free FCS or HPL allowed survival of all 4 different cell types for at least 3 cycles of 72 hours. On contrary, starvation did not allow to maintain EPCs, JURKATs and THP1 survival for more than one 72h cycle. MSC survival was less impaired but also clearly limited with cell showing phenotypical sign of stress. In any case, EV production was fully correlated to cell survival.

Summary/Conclusion : EV‐free FCS/HPL containing medium and not starvation could be used to sustain the EV production from MSCs, EPCs, JURKAT and THP1 cells. EV‐free HPL will allow large volumes of xenobiotic‐free conditioned media such as in bioreactors and large‐scale production of therapeutic EV.

A comparison of methods for the enrichment and separation of human salivary extracellular vesicles

Zhaohao Liao 1 ; Olesia Gololobova 2 ; Bonita H. Powell 3 ; Gabriela Lima 3 ; Kenneth W. Witwer 2

1 Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA, Baltimore, USA; 2 Johns Hopkins University School of Medicine, Department of Molecular and Comparative Pathobiology, Baltimore, USA; 3 Johns Hopkins University School of Medicine, Baltimore, USA

Introduction : Saliva is a non‐invasively accessed biofluid for biomarker discovery. Sample acquisition is easier and safer than blood collection and can be done in‐home, convenient for the elderly and small children. Salivary extracellular vesicles (EVs) can be used as potential biomarkers for certain oral cancers and dental diseases and have recently attracted much attention. However, most biomarker studies use polymer‐based precipitation and do not include rigorous EV characterization per the Minimal Information for Studies of EVs (MISEV). Here, we present a comparison study of salivary extracellular vesicles separation techniques, including ultracentrifugation (UC), several variations of size exclusion chromatography (SEC)/bind‐elute chromatography, and a combination of sizing and affinity capture.

Methods : Saliva was collected by passive drool from healthy donors directly into 2 ml protein low‐binding tubes. Cells and debris were removed by centrifuging the samples at 12,500xg for 10 min. SEC/bind‐elute separation was performed using IZON qEV original 70nm columns, SmartSEC HT plates, Exo‐spin 96 plates, and Superdex 200 Increase 10/300GL (GE Healthcare, IL, USA) column capped with fast performance liquid chromatography system. UC pellet was obtained by centrifugation of the samples at 120,000xg for 90 min at 4°C. Sizing/affinity capture was performed with EXO‐NET® beads. The final product was characterized using Transmission Electron Microscopy (TEM), Nanoflow Cytometry Analyzer (NanoFCM), Western blot (WB) for EV markers (CD9, CD63, TSG101, and Syntenin‐1), and co‐isolate markers (Albumin, ApoB100, ApoE, Calnexin, and GM130), and single‐particle tetraspanin assays (SP‐IRIS: ExoView).

Results : All presented methods allowed us to obtain EV‐rich fractions that were positive for EV markers and of similar size distribution, mostly within a detected size range of 50 to 120 nm. SP‐IRIS indicated a tetraspanin profile with higher expression of CD9 and CD63 than CD81. However, WB characterization of the UC pellet and fractions obtained by some of the SEC approaches (qEV, Exo‐spin 96) showed presence of co‐isolating material like low‐density lipoproteins and albumin.

Summary/Conclusion : All evaluated methods can be used to enrich salivary EVs. However, additional purification may be required for UC and some SEC/bind‐elute methods if highly pure EVs are needed for further analysis.

Keywords : Saliva, Extracellular vesicles, Size Exclusion Chromatography, EV separation

Detection of extracellular vesicles using the BD FACSymphony™ A1 Cell Analyzer

Tina Van Den Broeck 1 ; Estefanía Lozano‐Andrés 2 ; Mark Dessing 3 ; Annette Meyer 4 ; Tim Schenkel 4 ; Xiaoshan Shi 5 ; Ludovic Monheim 1 ; Stephanie Widmann 6 ; Marca H.M Wauben 2 ; Aaron Tyznik 6

1 Becton Dickinson, BD Biosciences, Europe, Erembodegem, Belgium; 2 Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, Utrecht, Netherlands; 3 Becton Dickinson, BD Biosciences, Europe, Allschwil, Switzerland; 4 Becton Dickinson, BD Biosciences, Europe, Heidelberg, Germany; 5 Becton Dickinson, BD Biosciences, CA, United States, San Jose, USA; 6 Becton Dickinson, BD Biosciences, CA, United States, San Diego, USA

Introduction : Extracellular vesicles (EVs) are small lipid‐based vesicles secreted from cells. Flow cytometry provides an attractive analytical approach as the heterogeneous EV populations can be studied with high‐throughput, single‐vesicle, multiparameter analysis. Conventional flow cytometry instruments and methods optimized for cell analysis are not well suited for the measurement of EVs as the small size and low refractive index of EVs make it challenging to resolve them from noise. Improvement in instrument performance, method robustness, experiment design, data analysis and cross‐measurement standardization are necessary to support analysis of EVs using flow cytometry.

Methods : In this study, we evaluated the BD FACSymphony™ A1 Cell Analyzer with BD® Small Particle Detector Option for analysis of small particles including EVs and sub‐micron beads. Following the MIFlowCyt‐EV framework for publication, we stained and examined EVs purified from the human MCF7 breast cancer cell line, along with necessary controls, using a novel small particle side scatter channel (SP‐SSC). We also evaluated the sensitivity, size resolution, noise levels and dynamic range using various sub‐micron beads. Replicate experiments were performed on multiple instruments and over different days.

Results : The BD FACSymphony™ A1 Cell Analyzer with BD® Small Particle Detector Option was set up in a standardized fashion. Using this methodology, we show that multiple instruments can consistently resolve 90‐nm NIST‐traceable polystyrene particles from noise and correctly determine their concentration as low as 1E+06 beads per mL on SP‐SSC threshold, with an R‐square value >0.99 upon 2‐fold serial dilutions, proving single small particle detection over a significant dynamic range. Furthermore, upon staining with a membrane dye and a fluorochrome‐labeled tetraspanin CD9 antibody, we showed that purified EVs from MCF7 cells can be detected and that a combined “OR” thresholding (SP‐SSC “OR” fluorescence) can add value over SP‐SSC or fluorescence only.

Summary/Conclusion : EVs and sub‐micron particles can be detected using BD FACSymphony™ A1 Cell Analyzer. Standardized instrument setup, calibration, proper experimental controls and “OR” thresholding can help improve EV flow cytometry measurements.

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Funding : European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No [722148]

BD Biosciences is a partner organization in TRAIN‐EV and offered training and secondment to a PhD student within the consortium.

Revealing unique surface profile of cell‐derived vesicles by combining proteome analysis and nanoparticle flow cytometry

Hui‐Chong Lau 1 ; Ilaria Passalacqua 2 ; Jik Han Jung 1 ; Yerim Kwon 1 ; Jeong Eun Park 1 ; Sung‐Soo Park 1 ; Davide Zocco 2 ; Senthil Ramaswamy 3 ; Seung Wook Oh 1

1 BioDrone® Research Institute, MDimune Inc., Seoul, Republic of Korea; 2 Lonza Siena Srl, Toscana, Italy; 3 Lonza Walkersville Inc., Rockville, USA

Introduction : The cell‐derived vesicles (CDVs) are obtained from virtually any cell by applying MDimune's proprietary extrusion technology. With superior productivity and versatility, this technology has garnered increasing attention among drug developers, especially those who wish to use them as a drug delivery vehicle. We previously described the similarity and differences between CDVs and exosomes. Particularly, among well‐known exosome markers, CD9 and CD81 are less represented in CDVs compared to exosomes while CD63 is more prominent in CDVs. In an early survey of CDV markers at the single‐particle level, we also found that three tetraspanin markers are differentially expressed between CDVs and exosomes. Therefore, a more systematic survey of marker expression profiles was necessary to better understand CDVs and their therapeutic potentials.

Methods : Multiple batches of CDVs were produced from Lonza's HEK293 cells by extruding cells serially through membrane filters (10, 3, and 0.4 μm). Exosomes were obtained from the culture medium of the same cell sources. Then, both CDVs and exosomes were purified using the same purification processes to minimize artifacts associated with purification methods. Then, CDVs and exosomes were subjected to proteome analysis and nanoparticle flow cytometry using the NanoAnalyzer (NanoFCM Co).

Results : Here, we present the proteome analysis results of CDVs derived from HEK293 cells in comparison with exosomes or source cells. We first identify membrane proteins uniquely enriched in CDVs compared to exosomes or source cells. Then, we examine their expression patterns at the individual nanoparticle level as well as confirm them using FACS and western blot. The characterization results of both nanoparticle flow cytometry and other conventional biochemical means coherently reveal a unique surface profile of CDVs.

Summary/Conclusion : This study broadens our understanding of this intriguing technology to the particle level. The current findings provide essential insights into the biogenesis of CDVs as well as facilitate the further development of CDVs in diverse therapeutic applications.

Keywords : Cell‐derived vesicles, Single‐particle analysis,

PF10: Late‐Breaking Poster Session 4: Fundamental Biology

Chair: My Mahoney, n/a – TJU

Chair: Valentina R. Minciacchi – Cedars Sinai Medical Center

Extracellular vesicles in the peptide‐membrane interaction landscape

Tamás Beke‐Somfai 1 ; Imola Szigyártó 2 ; Zoltán Varga 3 ; Anikó Gaál 3 ; Judith Mihäly 2 ; Diana Kitka 2 ; Lilla Turiák 4 ; László Drahos 2 ; Mayra Queme Pena 5 ; Lívia Fülöp 6 ; Priyanka Singh 5 ; Sonallya Tasvilla 2

1 Research Centre for Natural Sciences, Budapest, Budapest, Hungary; 2 Research Centre for Natural Sciences, Budapest, Hungary; 3 Biological Nanochemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest, Hungary; 4 Research Centre for Natural Sciences, Institute of Organic Chemistry, Budapest, Hungary, Budapest, Hungary; 5 Research Centre for Natural Science, Budapest, Hungary; 6 Department of Medical Chemistry, University of Szeged, Szeged, Hungary

Introduction : Host defense membrane active peptides (HDPs) are most often cationic species exerting not only antimicrobial, antiviral, antifungal and anticancer effects, but also a diverse set of roles in immunomodulation. In this respect, extracellular vesicles (EVs), are highly mobile lipid bilayer bodies, and can have widespread important roles both for human host and invasive bacterial cells. However, we know comparatively very little about the potential EV‐HDP interactions, and their relevance in blocking, distritbuting, or assembling EV cargo and associated biomolecular compounds at important locations such as infectious wound sites. In this presentation, we aim to address the various aspects of these interactions and demonstrate on selected studies how Nature could use HDPs to manipulate EVs and their content.

Methods : The action mechanism of HDPs on red blood cell‐derived extracellular vesicles (REV) were tested using flow‐linear dichroism, microscale resistive pulse sensing and microscale thermophoresis. In addition standard biophysical techniques were also employed (e.g. DLS, TEM, IR, CD spectroscopy)

Results : We have sown [1,2] that the selected HDPs can remove and redistribute REV elements efficiently, by both removing the surface proteins of vesicles at lower concentrations, but also by disrupting the vesicles and forming larger, lamellar bilayer structures, that can be beneficial during tissue repair, or in separating protein and lipid components.

Reference

[1] Szigyártó, I. C.; Deák, R.; Mihály, J.; Rocha, S.; Zsila, F.; Varga, Z.; Beke‐Somfai, T. Flow Alignment of Extracellular Vesicles: Structure and Orientation of Membrane‐Associated Bio‐Macromolecules Studied with Polarized Light. ChemBioChem 2018, 19 (6), 545–551. https://doi.org/10.1002/cbic.201700378 .

[2] Singh, P.; Szigyártó, I. C.; Ricci, M.; Zsila, F.; Juhász, T.; Mihály, J.; Bősze, S.; Bulyáki, É.; Kardos, J.; Kitka, D.; Varga, Z.; Beke‐Somfai, T. Membrane Active Peptides Remove Surface Adsorbed Protein Corona From Extracellular Vesicles of Red Blood Cells. Front. Chem. 2020, 8. https://doi.org/10.3389/fchem.2020.00703 .

Summary/Conclusion : The studies provided molecular level insight to EV‐HDP processes which may also offer therapeutic strategies for related areas.

Funding : This work was funded by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021‐EGA‐31 and the 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021 funding schemes. Support from Eötvös Loránd Research Network, grant n.o. SA‐87/2021, is also acknowledged.

Keywords : Red blood cell, extracellular vesicle, membrane active peptide, light spectroscopy, protein corona.

PC‐3 prostate cancer cells constitutively release beta‐2 adrenergic receptor via extracellular vesicles

Krizia Sagini 1 ; Ilaria Guerriero 2 ; Alicia Llorente 3

1 Oslo University Hospital Oslo, Department of Molecular Cell Biology, Institute for Cancer Research, Oslo, Norway; 2 Biogem, Biology and Molecular Genetics Institute, Ariano Irpino, Italy; 3 Oslo University Hospital Oslo, Department of Molecular Cell Biology, Institute for Cancer Research, oslo, Norway

Introduction : Beta‐2 adrenergic receptor (ADRB2) belongs to the G protein‐coupled receptor (GPCR) family. Evidence suggests that GPCRs can be secreted in extracellular vesicles (EVs), which may result in the horizontal transfer of functional receptors. In advanced prostate cancer (PCa), ADRB2 is considered a molecular switch for neuroendocrine differentiation, which is associated with poor prognosis. Moreover, ADRB2 level correlates with tumor progression. The aim of this study is to investigate whether PCa cells release ADRB2 via EVs and how this affects the tumor microenvironment.

Methods : EVs were isolated by differential centrifugation (large‐medium EVs at 10,000g and small EVs (sEVs) at 100,000g) from the conditioned media of the highly metastatic cell line PC‐3. In selected experiments, EVs were further purified by high resolution density gradient. EVs were characterized by immunoblotting, radioligand binding assay and nanoparticle tracking analysis (NTA). In some experiments, cells were treated with the ADRB2 agonist isoproterenol and/or the antagonist propranolol.

Results : Our results show that PC‐3 cells constitutively secrete ADRB2 in annexin‐positive sEVs. Treatment with the agonist reduced the level of ADRB2 in released EVs, whereas the antagonist prevented this effect. NTA showed that the lower ADRB2 level in EVs was not due to a general inhibition of EVs secretion by isoproterenol.

Summary/Conclusion : Our results suggest that ADRB2 is released by PC‐3 cells via sEVs budding from the plasma membrane, as these vesicles contain annexins. Indeed, treatments modifying ADRB2 localization (activation) affected its extracellular release. Finally, we have generated a stable cell line expressing fluorescent ADRB2‐neonGreen will allow to monitor the release of ADRB2 and to track its transfer to recipient cells thus providing further insight into the role of tumor EVs in the horizontal transfer of ADRB2.

Funding : The Norwegian Cancer Society and the South‐Eastern Norway Regional Health Authority

Profiling the small RNA cargo in extracellular vesicles derived from oxidised low‐density lipoprotein‐stimulated vascular smooth muscle cells

Eleni Charla 1 ; Stuart A. Nicklin 2 ; Pasquale Maffia 3

1 University of Glasgow, Glasgow, United Kingdom; 2 Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, UK, Glasgow, United Kingdom; 3 Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK, Glasgow, United Kingdom

Introduction : Oxidised low‐density lipoprotein (oxLDL) is a pro‐atherogenic factor that induces signaling responses in endothelial cells (ECs) and vascular smooth muscle cells (VSMC). Extracellular vesicles (EVs) are secreted from cells and are implicated in intercellular communication predominantly via microRNA transfer. Here the effect of oxLDL stimulation on EVs isolated from VSMC was investigated.

Methods : Primary human coronary artery SMC (HCASMC) were treated ± 10μg/mL oxLDL for 24h prior to EV isolation from the media by Size‐exclusion chromatography (SEC). Small RNAs were extracted from HCASMC‐derived EVs, and small RNA sequencing was performed. 12 miRNA‐mRNA prediction algorithms were used to find predicted gene targets for small RNAs identified by RNASeq. Validation of RNASeq in SMC‐derived EVs was performed using RT‐qPCR. HCAEC were treated with HCASMC‐derived Control and oxLDL‐EVs labeled with PKH67 and uptake and EV trafficking were assessed.

Results : OxLDL treatment of HCASMC did not alter the concentration of secreted EVs compared to control HCASMC. RNASeq HCASMC‐EVs identified 475 small non‐coding RNAs (112 miRNAs, 206 snRNAs, 145 small nucleolar RNAs (snoRNAs), and 12 small Cajal‐body specific RNAs). Differential expression analysis showed that oxLDL treatment resulted in 4.5‐fold downregulation of miR‐125b‐5p (p< 0.05) and 4.5‐fold upregulation of 3 snRNAs, RNU2‐7p, RNU2‐29p, RNU2‐57p (p< 0.05). Bioinformatic analysis revealed 303 predicted and validated gene targets for miR‐125b‐5p. Validation of miR‐125b‐5p and snRNAs in HCASMC‐EVs was successful using RT‐qPCR (p< 0.05). Assessing PKH67‐labelled EV uptake into HCAEC revealed that the level of oxLDL‐EV uptake was significantly lower than control EV uptake (p< 0.05).

Summary/Conclusion : These data suggest that oxLDL treatment alters the small RNA EV cargo in SMC‐derived EVs. Future work will investigate whether miR‐125b‐5p‐EV transfer to HCASMC and HCAEC could alter cell migration or proliferation of recipient cells.

Funding : British Heart Foundation and the University of Glasgow.

Keywords : Extracellular vesicles, MicroRNAs, atherosclerosis

Yeast Mitochondrial Derived Vesicles (MDVs) have membrane potential and contain functional ATP synthase

‏ 1 Reut Hazan; ophry Pines 2 ; Neta Regev‐Rudzki 3 ; Dvora Lintzer 4

1 Hebrew university, Hadassa medical school, Jerusalem, Jerusalem, Israel; 2 Hebrew university, Hadassa medical school, jerusalem, Jerusalem, Israel; 3 Weizmann Institute of Science, Rehovot, Israel; 4 Hebrew university, Jerusalem, Israel

Introduction : Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Interestingly, while the import machinery of proteins into mitochondria is thoroughly understood, there is currently no known protein export mechanism from mitochondria. Vesicular transport is a means of inter‐organellar communication, and in fact cells release vesicles in order to shuttle lipids, proteins, RNA and DNA between one another.

Methods : Vesicles isolation methodes, Microscopy, Biochemistry, Masspec

Results : Here we show for the first time that functional mitochondria, isolated from Saccharomyces cerevisiae can release vesicles, independent of the fission machinery. Mitochondrial secreted vesicles are relatively uniform in size, of about 100nm and carry a selection of cargo; only certain proteins are carried by these vesicles, of which some are matrix soluble and some are membrane proteins. We further demonstrate that yeast MDVs harbor a membrane potential, a functional ATP synthase complex, produce ATP, and fuse with mitochondria.

Summary/Conclusion : We suggest, that Yeast mitochondria generate MDVs which may function in organellar protein export, recovery and communication.

Keywords : Mitochondria, Mitochondrial derives vesicles

Exosome Mediated Translational Regulation in Ovarian Cancer

Sonam Mittal 1 ; Pradeep Chaluvally Raghavan 2 ; Sunila Pradeep 3

1 Medical College of Wisconsin, Milwaukee, USA; 2 Megical college of Wisconsin, Milwaukee, USA; 3 OB‐GYN, Medoical college of wisconsin, Milwaukee, USA

Introduction : Dysfunctional translational regulation often leads to uncontrolled cell growth and proliferation and is a key attribute cancer. Misexpression of the eukaryotic translation initiation factor eIF4E, an mRNA 5' cap‐binding protein, promotes tumorigenesis and has been implicated in cancer development and progression. Aberrant eIF4E function also disrupts key pathways important for anti‐tumor immune responsiveness in ovarian cancer, offering a possible explanation to the minimal success of an immunotherapy approach for this disease setting. We unexpectedly found that eIF4E co‐localizes and associates with extracellular vesicle (EV) proteins in ovarian cancer cells. Furthermore, our data suggests increased uptake of eIF4E packaged EVs (eIF4E‐EVs) leads to enhanced translational activity and activation of PD‐L1 expression. Unfortunately, a coherent model that explains how eIF4E‐packaged EVs impair immune response in the tumor microenvironment remains undeveloped and will be necessary to develop novel immunotherapy strategies that encourage immune cells to recognize and attack cancer cells.

Methods : We used polysome fractionation to determine whether eIF4E‐EVs affect global translation. We used multiple in vivo models by altering eIF4E expression in Rab27a KO using CRISPR/Cas9 model to study the effect of EV packaged eIF4E in tumor progression and its effect on immunotherapy. Furthermore, we also determined the effect of eIF4E packaged EVs on both protein and mRNA stability of PD‐L1 by directly measuring decay rates of endogenous mRNAs using kinetic labeling techniques and transcriptional inhibitors.

Results : This study shows that tumor cells employ EVs to deliver eIF4E to the tumor microenvironment, facilitating translational adaptation, and PD‐L1 expression to support tumor growth. In addition, our data shows eIF4E‐EVs enhances protein synthesis and eIF4E‐EVs promote tumor growth by increasing PD‐L1 expression. Our study uncovers a novel mechanism that details how eIF4E‐EVs promote translational adaptation in high‐grade serous ovarian cancer cells and facilitate to fuel tumor progression.

Summary/Conclusion : Our findings shows that inhibiting eIF4E synergizes with PD‐1 blockade, providing much needed evidence that supports the possibility of an immunotherapy‐based treatment regimen for ovarian cancer.

Funding : This work was supported in part to S.P. by the Ovarian Cancer Research Fund Alliance (OCRFA 457466, 657713), the Women's Health Research Program (WHRP), MCW Cancer Center Institutional Research Grants from the American Cancer Society (ACS‐IRG 19‐138‐34), and the Department of Defense (DoD W81XWH‐21‐1‐0361, W81XWH2110413).

Keywords : Extracellular vesicles, Translational Regulation, Ovarian cancer

The role of executioner caspases in extracellular vesicle biogenesis

Christina Nedeva; Suresh Mathivanan; Sai V. Chitti

La Trobe University, Melbourne, Australia

Introduction : Almost 30 years ago the first executioner caspase was identified in C. elegans, which was proposed to have apoptotic functions. This ground‐breaking discovery paved the way for an explosion of studies investigating the roles of caspases in apoptosis, establishing their pro‐death role in effectively all metazoan species. More recently, nonlethal roles for caspases have been described including activity in cellular remodeling, differentiation and even survival. Moreover, studies have described non‐apoptotic activity of the protease caspase‐3 in chick auditory brain development. Further investigation revealed an enrichment of caspase‐substrate proteins in auditory brainstem‐derived extracellular vesicles (EVs). EVs are small membrane‐bound structures released by virtually all cells that function in inter‐cellular communication. However, the role of caspases in EV biogenesis is yet to be investigated.

Methods : We have recently performed whole proteomic analysis of EV's isolated from established TK6 cell lines deficient in executioner caspases 3 and 7. Analysis of these cells and their respective EV's has allowed identification of caspase substrates that participate in the EV biogenesis process. These substrates have been validated using western blotting and rescue experiments.

Results : Our results showed that cells lacking caspase 3 or 7 produced EVs that were smaller and fewer in number compared to WT‐derived EVs. Quantitative proteomic analysis highlighted that loss of caspases modified the protein cargo of small and large extracellular vesicles. Functional annotation analysis deduced from proteomics data revealed that several proteins involved in cell adhesion, exocytosis and in ESCRT III assembly are down‐regulated in the absence of caspases 3 and/ or 7. These protein candidates include VPS4 and CHMP proteins.

Summary/Conclusion : Our early findings suggest a novel mechanism for executioner caspases in regulating EV biogenesis via the regulation of known mediators of this process. These findings allow us to better understand how these processes work and how they may regulate pathology associated with their dis regulation.

A role for integrin beta 1 in extracellular vesicle‐mediated functional RNA delivery

Omnia M. Elsharkasy 1 ; Olivier G de Jong 2 ; Leanne van der Werff 1 ; Raymond M. Schiffelers 3 ; Pieter Vader 4

1 CDL Research, University Medical Center Utrecht, Utrecht, The Netherlands, Utrecht, Netherlands; 2 Utrecht University, Utrecht, The Netherlands, Utrecht, Netherlands; 3 University Medical Center Utrecht, Utrecht, The Netherlands, Utrecht, Netherlands; 4 CDL research, University Medical Center Utrecht, Utrecht, The Netherlands, Utrecht, Netherlands

Introduction : While various uptake mechanisms for extracellular vesicles (EVs) have been widely explored, more work is required to fully unravel the underlying molecular mechanisms by which EVs are able to provide functional RNA transfer to the recipient cells. Here, we demonstrate a role for integrin beta 1 (ITGB1) in recipient cells in EV‐mediated uptake and RNA delivery.

Methods : To study RNA transfer we used a fluorescent stoplight reporter system which is activated by Cas9 protein upon functional sgRNA delivery. HEK293T reporter cells were transfected with ITGB1 siRNA or non‐coding siRNA as a control. ITGB1 knockdown efficiency was determined using RT‐qPCR. EVs from MDA‐MB‐231 cells expressing targeting sgRNA were isolated by tangential flow filtration followed by size exclusion chromatography and were then added to HEK293T reporter cells. Functional RNA transfer was quantified by flow cytometry. For uptake experiments, EVs were labelled using MemGlow640.

Results : ITGB1 mRNA knockdown efficiency in recipient cells was ∼ 85%. EVs containing targeting sgRNA showed functional RNA delivery in cells transfected with non‐coding siRNA but not in cells transfected with ITGB1 siRNA, both in a co culture setting and using isolated EVs. Complementarily, knockdown of ITGB1 or blockage using neutralizing antibodies resulted in a significant decrease in EV uptake in the recipient cells.

Summary/Conclusion : MDA‐MB‐231‐EV uptake and EV‐mediated functional RNA transfer into HEK293T recipient cells is mediated by ITGB1. This demonstrates a role for ITGB1 in EV‐mediated RNA transfer, which could contribute to achieving more efficacious EV‐mediated RNA delivery and improved RNA therapeutics.

Funding : The work of O.M.E, L.v.d.W, R.M.S. and P.V. is supported by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 825828. O.G.d.J. is supported by a VENI Fellowship from the Netherlands Organisation for Scientific Research (NWO) VI.Veni.192.174.

Keywords : RNA delivery, EV uptake, drug delivery

A novel role for Caveolin‐1 in extracellular vesicles (EV) production and dynamics under mechanical stress

Cristian Saquel 1 ; Christine Viaris de Lesegno 2 ; Cedric Blouin 2 ; Christophe Lamaze 2

1 Institut Curie, Paris, France., Paris, France; 2 Institut Curie, Paris, France

Introduction : Caveolae, along with their main constituents Caveolin‐1 (Cav1) and Cavin1, have been established as new mechanosensors and mechanotransducers by our laboratory. Thus, caveolae can flatten in response to different types of mechanical stress, which in turn protects the cell by providing extra membrane that was stored in the caveolar invagination, acting thereby as a membrane tension buffer. Interestingly, Cav1 has also been found on extracellular vesicles (EV) derived from cancer cells that have been observed to promote malignancy traits in recipient cells including enhanced invasion and migration. My aim is to analyze the role of caveolae, Cav1 and Cavin1 in EV biology, including composition, cargo, dynamics i.e. release and uptake by cells, in different models of cancer cells under mechanical stress.

Methods : I used 2D hypo‐osmotic shock and 3D dextran compression as mechanical stress models, I purified EVs from these conditions and characterised the samples by electronic microscopy and western blot analysis for EV markers. To measure EV release I used a pH‐sensitive dye (pHluorin‐CD63) that allowed me to visualise in real time MVB‐PM fusion events. For the analysis of the uptake of EVs I used a nanoluciferase‐HSP70 construct and GFP‐HSP70, that allowed me to visualise the uptake by luminescence and by fluorescence in recipient cells.

Results : I observed that mechanical stress led to a significant increase of released EVs with typical morphology and markers of small EVs; I was able to visualise this effect in live cells using live microscopy. In both cases, the depletion of Cav1 or Cavin1 inhibited this increased release of EVs from donor cells.

When I analysed the effect of the absence or overexpression of caveolar components in the uptake of EVs I observed that the overexpression of Cav1 showed an increase uptake of EVs. Interestingly, the uptake was shown to be Cav1 dependent but not Caveolae dependent as shown on knock out experiments were the expression of only Cav1 is enough to observe the increased uptake phenotype.

Summary/Conclusion : Taken together, these results open a new and interesting link between caveolae and EV dynamics including release and uptake by cells under mechanical stress. For the remainder of my project I will delve deeper into the molecular mechanisms underlying these changes and further validate my in vitro results with a physiological model more relevant to cancer biology in vivo.

Funding : Fonds France‐Canada pour la Recherche (FFCR)

Keywords : Caveolae, Caveolin‐1, Mechanical stress, EV secretion, EV uptake

PLD2‐MVB12B support small extracellular vesicle production

Antonio L. Egea‐Jimenez 1 ; Lukas Hyka 2 ; Stéphane Audebert 3 ; Monica Castro‐Cruz 4 ; Rania Ghossoub 5 ; Jean‐Paul Borg 6 ; Guido David 7 ; Luc Camoin 3 ; Pascale Zimmermann 8

1 INSERM, Marseille, France; 2 KU Leuven, Leuven, Belgium; 3 Centre de Recherche en Cancérologie de Marseille (CRCM), Aix‐Marseille Université, Marseille, France; 4 KU leuven, Leuven, Belgium; 5 Cancer Research of Marseille ‐ Aix‐Marseille University, Marseille, France; 6 CRCM, Marseille, France; 7 Centre de Recherche en Cancérologie de Marseille, Marseille, France; 8 KU Leuven, MARSEILLE 09, France

Introduction : Phospholipase D2 (PLD2) is a multidomain protein converting phosphatidylcholine into phosphatidic acid (PA) and choline. We previously reported that PLD2 works downstream of the small GTPase ARF6 to stimulate endosomal budding (EB) and the production of syntenin and CD63‐positive exosomes (Ghossoub, 2014). Here we further investigated the role of PLD2 in the production of extracellular vesicles (EV).

Methods : We prepared mutants by site‐directed mutagenesis; silenced protein expression by siRNA treatment; inhibited PLD2 activity with VU0364739; purified EV by differential centrifugation, characterized protein composition by western blot and differential proteomics; studied endosomal budding by electron & fluorescence microscopy; studied protein‐protein interactions by co‐immunoprecipitation; characterized protein‐lipid interactions by surface plasmon resonance.

Results : We show that PLD2 effects on EVs go beyond syntenin and CD63 exosomes in MCF‐7 cells, impacting on the secretion of CD9, CD81 and flotillin positive small EV (sEV), but not on large EVs. Inhibition of PLD2 activity or PLD2 silencing reduce sEV number by 60%, while its overexpression increases the overall sEV number. Effects rely on several PLD2 domains. Yet, PLD2‐dependent EB strictly depends on the lipase activity. We reveal that PLD2 recruits the ESCRTI component MVB12B to late endosomes, and that MVB12B binds to PA. We found that MVB12B loss‐of‐function phenocopies the effect of PLD2 loss‐of‐function on EB, and also the general effects on sEV number and composition.

Summary/Conclusion : We propose that PLD2 works in sEV biogenesis by producing PA on late endosomes, recruiting MVB12B to drive EB for the production of exosomes. Since MVB12B has broad effects, PLD2‐MVB12B might also work at the plasma membrane or alternatively sEV production is restricted to endosomes in MCF‐7 cells.

Funding : FWO‐G0C5718N, ANR‐18‐CE13‐0017, Ligue contre le cancer France, EU Horizon 2020 Marie Sklodowska‐Curie.

Effect of Atg8 knockdown in HeLa cell lines on secretion of CD81+ extracellular vesicles

Berglind E. Benediktsdóttir 1 ; Ingibjorg Steinunn Sigurbjörnsdottir 2 ; Þengill Fannar Jónsson 3 ; Margrét Helga Ögmundsdóttir 4

1 Faculty of Pharmaceutical Science, School of Health Sciences, University of Iceland, Reykjavík, Iceland; 2 Faculty of Pharmaceutical Science, School of Health Sciences, University of Iceland, Akranesi, Iceland; 3 Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavík, Iceland; 4 Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland

Introduction : Autophagy is a degradation process important for cellular homeostasis and is impaired during various disease progression such as cancer. Various authophagy related genes (ATGs) encode for proteins participating in the autophagosomal membrane formation, including the ATG8 protein family (LC3s and GABARAPs). The possible interplay between extracellular vesicles (EVs) and autophagy proteins is becoming increasingly clear. ATGs have been implicated in cellular secretion of EVs in a function distinct from their role in the classical autophagy pathway. The aim of this study was to determine if knockouts (KOs) of ATG8 genes would affect EV release and content.

Methods : The HeLa cell lines: wild‐type (WT), HexaKO (LC3 and GABARAP), triple KO (TKO) of LC3 and TKO of GABARAP were cultured in serum free medium (+antibiotics) 48h (cell viability 95‐98%) before EV isolation with CD81 Fab‐TACS affinity capture (IBA) according to manufacturers protocol. NanoSight was used for particle concentration and size determination: camera level 15, 60 sec capture, four repeats. Concentrated samples were analyzed with capillary western blotting (WB; anti‐CD9 1:40, Cell Signalling, 13403) and TEM. Statistical tests include students paired t‐test, p < 0.05 regarded as statistically significant.

Results : WT isolates contained significantly higher number of particles compared to HexaKO isolate (1.56*107 compared to 0.83*107 particles/ml/million cells) (n = 4). Early results on the TKO cell lines (n=2) indicate reduced particle release for those cell lines as well. EVs from the different HeLa cell lines all had similar mean size (94.8‐104.9 nm) and mode size (74.2‐80.4 nm). Cell lysates (CL) were positive for CD9 (32 kDa) whilst EV samples had WB bands appearing at ∼144kDa. TEM images revealed vesicles in the size range of 80‐150 nm. This suggests positive isolation of EVs.

Summary/Conclusion : KO of all ATG8 specific genes reduced the number of secreted EVs, suggesting that ATG proteins participate in CD81+ EVs release and possibly their biogenesis. The mechanism by which the ATG8 proteins participate in the EV process remains somewhat unclear. Dissecting this interplay at a molecular level will be important for better understanding the role of autophagy and EVs in health and disease.

Funding : Icelandic Research Fund 228586‐051 (MHO).

Keywords : Autophagy,ATG8

Investigation of extracellular vesicles derived from cardiac cell lines under normoxia and hypoxia

Anna Koncz 1 ; Krisztina Németh 1 ; Lilla Turiák 2 ; Dorina Lenzinger 1 ; Krisztina V. Vukman 1 ; Péter Lőrincz 3 ; Helga Zelenyánszki 4 ; Edit I. Buzás 5 ; Tamás Visnovitz 1

1 Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary, Budapest, Hungary; 2 Research Centre for Natural Sciences, Institute of Organic Chemistry, Budapest, Hungary, Budapest, Hungary; 3 Eötvös Loránd University, Department of Anatomy, Cell and Developmental Biology, Budapest, Hungary, Budapest, Hungary; 4 Eötvös Loránd University, Department of Plant Physiology and Molecular Plant Biology, Budapest, Hungary, Budapest, Hungary; 5 Semmelweis University Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary

Introduction : Myocardial dysfunctions are one of the leading cause of death in developed countries. Extracellular vesicles (EVs), released by these cells may help in early diagnostics and therapy. Cardiac cell lines (such as H9c2, AC16 and HL1) are widely used in cardiovascular EV research as in vitro models. Increasing number of studies are using these cell lines, although they have significant limitations. We have decided to perform comparative characterization of EVs released by differentiated H9c2, AC16 and HL1 cells. The aim of the recent project was to compare cardiac cells derived EVs under normal and hypoxic conditions.

Methods : Cell lines, AC16 (human), HL1 (mouse) and H9c2 (rat) cardiomyoblast cells were used in this study. Following differentiation protocols, small‐ medium‐ and large‐sized EVs (sEV, mEV, lEVs) were collected from conditioned medium of normal and hypoxia treated cells. EVs separated by a combination of gravity filtration, differential centrifugation and tangential flow filtration (TFF). EVs were characterized according to MISEV 2018 by MicroBCA, SPV‐Lipid assay, Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). The protein profile of sEV and mEV were determined by mass spectrometry. Some of the proteomic hits were validated by Western blotting, flow cytometry and immunogold TEM. EV release was examined by fluorescent live‐cell imaging using GFP‐fusion protein transfected cells.

Results : One of our most relevant proteomic hits was endoplasmin (ENPL, grp94, gp96). It was specifically present in EVs released by hypoxia‐treated cells. Even though ENPL is an endoplasmic reticulum resident chaperon, its presence in EVs were confirmed by Western blotting, flow cytometry and Immunogold TEM. Secretion of ENPL containing EVs was analyzed by confocal microscopy using fixed and living HL1 cells.

Summary/Conclusion : We provide evidence that ENPL‐containing EVs are released by cardiac cell lines. We suggest that ENPL may take part in cardio‐protection during hypoxia through reducing ER stress.

Funding : This work was funded by the NVKP_16‐1‐2016‐0004 grant of the Hungarian National Research, Development and Innovation Office (NKFIH), Semmelweis Innovation Fund (STIA 2020 KFI), Hungarian Scientific Research Fund (OTKA K120237), VEKOP‐2.3.2‐16‐2016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, Higher Education Excellence Program (FIKP) Therapeutic Thematic Programme.

Participation of Anna Koncz in ISEV2022 is supported by Bioxol Ltd.

Keywords : cardiac extracellular vesicles, endoplasmin, cardiac cell lines

PF11: Late‐Breaking Poster Session 5: Physiology/Pathology I

Chair: Janusz Rak, MD, PhD – Professor

Chair: Houjian Cai, n/a – University of Georgia

Mouse Tumor Tissue‐Derived Extracellular Vesicles Induce Angiogenesis Through Macrophage Infiltration

Seoyoon Bae; Yong Song Gho

POSTECH, Pohang, Republic of Korea

Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed vesicles secreted by all types of cells. As EVs have emerged as mediators of intercellular communication, the roles of EVs in tumor microenvironment are being actively investigated. Angiogenesis, the formation of blood vessels from pre‐existing vasculature, is essential for tumor growth and metastasis. Several previous reports suggested that EVs from in vitro cultured cancer cells have potent angiogenic activities. However, the angiogenic role of in vivo tumor tissue‐derived EVs has never been investigated. In this study, we isolated mouse tumor tissue‐derived EVs (tEVs) with high purity and investigated the role of tEVs in vitro and in vivo angiogenic activities.

Methods : B16BL6 cells were subcutaneously injected into C57BL6 mice. On day 25 after injection, mice were sacrificed and viable regions of tumors were soaked into serum‐free MEM and incubated for 2 h at 4°C. After incubation, tEVs were isolated by the combination of sequential centrifugation, buoyant density gradient ultracentrifugation, and ultracentrifugation. The purified tEVs were characterized according to the MISEV 2018 guideline. Pro‐angiogenic activity of tEVs were evaluated by both in vitro endothelial cell proliferation, migration, and differentiation assay and in vivo Matrigel plug assay.

Results : The purified tEVs had approximately 140 nm in a diameter, spherical lipid bilayer structure, and enriched with EV marker proteins, including CD81 and CD9 while no contamination of subcellular organelles such as Golgi apparatus and nucleus. In vivo Matrigel plug assay showed that the tEVs had similar pro‐angiogenic activity to that of VEGF. However, tEVs did not induce endothelial cell proliferation, migration, and differentiation but induced macrophage migration in vitro. In addition, we observed that tEVs induce the infiltration of macrophage into tumor tissue. Furthermore, depletion of tumor tissue‐infiltrated macrophage by the treatment of clodronate liposome, a macrophage depletion reagent, completely blocked tEVs‐induced in vivo angiogenic activity in Matrigel plug assay.

Summary/Conclusion : Take together with all data, mouse tumor tissue‐derived EVs has strong in vivo angiogenic activity by induction of Macrophage infiltration into tumor tissue. This study provides the first direct evidence on the role of tEVs in tumor angiogenesis.

Funding : This work was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2021R1A2C3005275).

Keywords : Tumor Tissue‐Derived Extracellular Vesicles, Tumor Microenvironment, Angiogenesis, Macrophage

Picornavirus proteins promote the release of virus‐carrying EV subsets via the modulation of host kinases

Kyra A.Y. Defourny 1 ; Xinyi Pei 1 ; Frank J.M. Van Kuppeveld 2 ; Esther N.M Nolte‐'t Hoen 1

1 Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands; 2 Virology Section, Division Infectious Diseases & Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

Introduction : Virus infection can affect the release and composition of extracellular vesicles (EVs) produced by host cells. These EVs can affect disease severity by promoting virus spread or altering antiviral immune responses. Currently, little is known about the type of virus‐host interactions that influence the release of EVs by infected cells. We previously described an important role for the viral Leader protein of the picornavirus EMCV in the induction of EV subsets with distinct molecular and physical properties. These EVs contained mature virus particles and efficiently transmitted the infection. Here, we used recombinant viruses that differentially interact with host signaling pathways as well as various knock‐out cells to further delineate molecular pathways involved in the release of picornavirus‐induced EVs.

Methods : EVs released by infected cells were isolated by UC pelleting/PEG precipitation followed by iodixanol density gradient centrifugation. Quantitative and qualitative analysis of EVs was performed using high‐resolution flow cytometry and western blot analysis. Infectivity of EVs was assessed in CCID50 assays.

Results : Virus‐induced inhibition of the antiviral sensor PKR contributed to the release of virus‐containing EVs, whereas signaling pathways leading to type I interferon production were not involved in EV release. Moreover, we observed that the ability of recombinant viruses to efficiently induce EV release correlated with their ability to induce lytic instead of apoptotic cell death, along with the activation of the stress kinase P38 MAPK.

Summary/Conclusion : Our data highlight several host kinases that are key to inhibiting or promoting EV release during virus infection. These kinases are targeted by specific virus proteins during infection to promote the release of virus‐carrying EVs. Insights into the molecular regulation of EV release during infection can reveal candidate therapeutic targets to counter the effect of EVs in virus‐mediated pathology.

Funding : This work was supported by The Netherlands Organisation for Scientific Research (NWO‐ALW grant number ALWOP.351).

Keywords : virus infection, virus‐host interactions, naked viruses, antiviral signalling

Optimizing isolation and purification of EV‐like nanoparticles from ginger rhizome (Zingiber officinale) by enzyme‐assisted cell wall digestion

Francesca Loria 1 ; Andres Lõhmus 1 ; Danilo Mladenović 1 ; Paolo Guazzi 1 ; Jekaterina Kazantseva 2 ; Raivo Vilu 2 ; Natasa Zarovni 1

1 HansaBioMed Life Sciences Ltd, Mäealuse 2/1, 12618 Tallinn, Estonia, Tallinn, Estonia; 2 Center of Food and Fermentation Technologies, Akadeemia tee 15A, 12618 Tallinn, Estonia, Tallinn, Estonia

Introduction : Extracellular Vesicle (EV)‐like nanoparticles have been recently recognized as bioactive components of ginger (Zingiber officinale), proven to hold health beneficial properties and displaying encouraging efficacy and safety profiles in pre‐clinical settings. Hitherto, most protocols for isolation and purification of ginger‐derived nanoparticles (GDNs) have relied on methods limiting or poorly promoting their extraction from the apoplast. Our objective was to investigate enzyme‐assisted ginger rhizome cell wall digestion for improved downstream GDN isolation and purification efficiency.

Methods : Ginger was washed, peeled and ground using a centrifugal juicer to extract juice and dry residues. Three concentrations of a commercially available multi‐enzyme cocktail containing a wide spectrum of carbohydrases [0%, 0.1% and 0.5% (v/w)] were used to perform overnight pre‐treatment, under constant shaking, at 37◦C and pH 5.5. For GDN isolation and purification, differential centrifugation, filtration and size exclusion chromatography were adopted. GDN characterization was executed by nanoparticle tracking analysis, protein quantification assay, enzymatic activity assays and fluorometric microplate‐based nucleic acid (NA) and lectin assays.

Results : Compared with control isolation and purification workflow, digestion with 0.1% multi‐enzyme cocktail enabled 47% increase in particle recovery, associated with particle size and protein content preservation, as well as only minor changes in particle enzymatic activity, NA content and lectin‐associated glycan expression. Pre‐treatment with 0.5% multi‐enzyme cocktail suggested substantial GDN degradation.

Summary/Conclusion : Enzymatic pre‐treatment of ginger rhizome resulted in optimization of original GDN isolation and purification protocol, as highlighted by the observed process yield improvement in correlation with preservation of tested biocomponents and enzymatic activity. This upgraded workflow, complemented with multiparametric quantity and quality assessment, has high translational potential into GDN functional testing and ultimate proof‐of‐concept clinical and/or industrial application.

Funding : This work is funded by the Horizon 2020 Framework Programme under the grant BOW ID: 952183.

Isolation and characterization of extracellular vesicles secreted by the pathogenic free‐living amoeba Naegleria fowleri

Lissette Retana Moreira 1 ; María Fernanda Steller Espinoza 2 ; Alberto Cornet Gómez 3 ; Natalia Chacón Camacho 2 ; Giovanni Saenz Arce 4 ; Oscar Rojas Carrillo 5 ; Antonio Osuna 3 ; Elizabeth Abrahams Sandí 1

1 Departamento de Parasitología, Facultad de Microbiología, Universidad de Costa Rica; Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiología, Universidad de Costa Rica., San José, Costa Rica; 2 Departamento de Parasitología, Facultad de Microbiología, Universidad de Costa Rica., San José, Costa Rica; 3 Departamento de Parasitología, Grupo de Bioquímica y Parasitología Molecular CTS‐183, Universidad de Granada, Granada, Spain; Instituto de Biotecnología, Universidad de Granada, Granada, Spain., Granada, Spain; 4 Departamento de Física, Universidad Nacional, Heredia, Costa Rica., Heredia, Costa Rica; 5 Laboratorio de Investigación y Tecnología de Polímeros, Escuela de Química, Universidad Nacional, Heredia, Costa Rica., Heredia, Costa Rica

Introduction : Naegleria fowleri is a thermophilic free living amoeba that produces primary amoebic meningoencephalitis (PAM), a severe and fulminant infection of the central nervous system (fatal in more than 95% of cases). Until 2019, more than 440 cases of PAM were reported worldwide.

Due to the characteristics of PAM, rapid and accurate diagnosis and early treatment are key to fight this infection. In this sense, a reliable diagnosis panel using the pathogen‐specific extracellular vesicles (EVs) could probably improve on‐time intervention and prognosis.

To our knowledge, the secretion of EVs by trophozoites of N. fowleri has not been reported yet. In this study, we present a characterization of EVs secreted by two clinic isolates of N. fowleri from Costa Rica.

Methods : EVs of trophozoites were isolated by ultracentrifugation. Characterization analyses performed included transmission electron microscopy, atomic force microscopy, nanoparticle tracking analyisis and dynamic light scattering. Protein profiles of EVs were compared after Coomasie and silver stainings. Protease activity was analyzed by zimography and recognition of EV proteins by polyclonal anti‐N. fowleri antibodies was achieved using Western blot. Preliminary proteomic analyses were also performed.

Results : The production of EVs from both clinic isolates of N. fowleri was confirmed. Protein profiles, protease activity and types of proteases of EVs from both isolates resulted very similar. Western blot analysis revealed differences in protein recognition by polyclonal anti‐N. fowleri antibodies; a higher recognition of EV proteins under 58 KDa in one of the isolates was identified.

Summary/Conclusion : The production of EVs by two clinic isolates of N. fowleri was confirmed. The results obtained in this study are necessary as they precede complete proteomic and functional analyses of EVs secreted by this pathogen, which will include an evaluation of effect of these EVs in pathogenesis and immunomodulation, and to latter explore the possibility of employing EVs for diagnostic purposes.

Funding : This work was funded by the "Vicerrectoría de Investigación" of the "Universidad de Costa Rica".

Research projects related to this work:

‐C‐1061: "Caracterización de antígenos de excreción/secreción y antígenos somáticos en amebas de vida libre mediante empleo de anticuerpos policlonales producidos en roedores".

‐C‐2600: "Secreción de vesículas extracelulares por Naegleria fowleri y evaluación de su potencial rol inmunomodulador en un modelo in vitro".

Keywords : Primary amoebic meningoencefalitis, Naegleria fowleri, trophozoites

Body fluid‐derived EVs exert selective antiviral activity with variable potency

Rüdiger Groß 1 ; Markus Hoffmann 2 ; Mirko Cortese 3 ; Dhanu Gupta 4 ; Stefan Pöhlmann 2 ; Ralf Bartenschlager 3 ; Samir El Andaloussi 5 ; Jan Münch 6 ; Janis A Müller 6

1 Ulm University Medical Center, Institute of Molecular Virology, Ulm, Germany; 2 Infection Biology Unit, German Primate Center, Göttingen, Germany, Göttingen, Germany; 3 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany, Heidelberg, Germany; 4 Department of laboratory medicine, Karolinska Institutet, Stockholm, Sweden; 5 Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, Huddinge, Sweden; 6 Institute of Molecular Virology, Ulm University Medical Centre, Ulm, Germany, Ulm, Germany

Introduction : We have previously shown EVs from seminal plasma (SP) and saliva to inhibit Zika virus (ZIKV) infection in vitro. These findings offer clues on the lack of sexual and oral transmission of ZIKV despite high viral RNA levels in both body fluids. It is unclear, if this activity is common to EVs from all sources and which viruses are affected by it.

Methods : Tangential flow filtration combined with bind‐elute size exclusion chromatography was used to purify EVs from human SP, saliva, urine, breast milk, serum and HEK293T cell culture supernatant. EV preparations were analyzed by western blot and characterized using NTA, DLS, TEM and bead‐assisted flow cytometry to quantify EV membrane proteins. Antiviral activity of these EVs was evaluated against ZIKV, West Nile Virus (WNV), HIV‐1, herpes simplex viruses (HSV) 1/2, SARS‐CoV‐2, and Ebola VLPs.

Results : EV purification was confirmed by enrichment of CD9, Flotillin‐1, Alix and Hsp70 and depletion of matrix proteins. EVs from all sources inhibited ZIKV with variable potency: EVs from cell culture, SP and saliva were most potent (IC50 ∼ 10ˆ10 particles/ml), while EVs from urine, breast milk and serum required 10x more particles. SP‐derived EVs prevented virion attachment and also inhibited infection of ex vivo vaginal explants and foreskin fibroblasts. These EVs further potently inhibited infection by WNV and Ebola VLPs, but not HIV‐1, HSV‐1/2 or SARS‐CoV‐2. MACSplex revealed a positive correlation of CD63 and CD81 levels with potency of anti‐ZIKV activity, in agreement with antiviral activity exerted by classical EVs.

Summary/Conclusion : Anti‐ZIKV activity seems to be a general property of EVs, but variable potencies suggest compositional differences between body fluids. SP‐EVs inhibited ZIKV infection in cells and tissue of the genital tract, supporting their role in restricting sexual ZIKV transmission. Antiviral activity against certain but not all enveloped viruses may offer hints on innate transmission barriers and the underlying mechanism.

Keywords : Body fluids, viruses, zika, ebola, west nile virus, hiv, hsv, immunity, innate immunity, sexual virus transmission

Role of P2X ionotropic receptors in exosomal release from cancer cells and consequences on tumors progression

Thomas Duret 1 ; Stéphanie Chadet 1 ; Roxane lemoine 1 ; Audrey Héraud 1 ; Pierre Besson 1 ; Lili Fan 2 ; Gang Yin 2 ; Christophe Baron 1 ; Sébastien Roger 1

1 University of Tours, Tours, France; 2 Central South University, Changsha, China (People's Republic)

Introduction : Exosomes are a subtype of microvesicules originary from the Multivesicular Bodies (MVBs) compartment, well‐known to be involved in cancer cell aggressiveness. Even though it is known that the release of exosomes is due to the fusion of MVBs to plasma membrane, the molecular process involved is not very clear. In this study, we investigated the role of ionotropic purinoreceptors P2X4 and P2X7 in exosomes release, composition and consequences in cancer aggressiveness.

Methods : We have generated stable clones knocked down for the expression of either P2X4 (4T1‐CR4) or P2X7 (4T1‐CR7) from 4T1 cancer cell line, using CRISPR‐Cas9, and compared to a null‐target CRISPER‐Cas9 cell line (4T1‐CTL). Exosomes from supernatants of theses cell lines were purified by ultracentrifugation, characterized by scanning electron microscopy and nanoparticule tracking assay (NTA). The expression of intracellular CD9, CD63, and CD81 was observed by flow cytometry. Purified exosomes were labeled with PKH67 to evaluate their integrating capacity. We developed co‐culture assays between donor 4T1‐CTL, CR4, CR7, and 4T1‐CTL recipient cells, in presence or not of the exosomes release inhibitor GW4869, to investigate the capacity of exosomes to modulate the invasion of recipient cells.

Results : NTA from 4T1‐CTL supernatants after ultracentrifugation‐based purification indicates the presence of particles of size below 200 nm. These express CD63 and PD‐L1, as identified by immunogold labelling in scanning electron microscopy. Intracellular content of CD9, CD63, and CD81 increases in both 4T1‐CR4 and CR7 cell lines compared to 4T1‐CTL suggesting an intracellular accumulation of exosomes. PKH67‐stained exosomes can integrate into recipient 4T1‐CTL cell line. 4T1‐CTL recipient cells co‐cultured with 4T1‐CR4 donor cells show a reduced invasion to those co‐cultured with 4T1‐CTL donor cells.

Summary/Conclusion : P2X4 seems to modulate the release, or composition, of proinvasive exosomes by 4T1 cancer cells.

Funding : This project is funded by the Région Centre‐Val de Loire (Project CanalEx and PhD grant) and the INCa PLBIO_16110 “PURIN4EXO”.

Distinct EV profiles of rheumatoid arthritis patients in response to methotrexate treatment

Anne Rydland 1 ; Fatima Heinicke 2 ; Siri Flåm 3 ; Anne‐Marie Siebke Trøseid 4 ; Reidun Øvstebø 5 ; Maria Mjaavatten 6 ; Benedicte Lie 7

1 Department of Medical Genetics, Oslo University Hospital, Oslo, Norway. Institute of Clinical Medicine, University of Oslo, Oslo, Norway, OSLO, Norway; 2 Department of Medical Genetics, Oslo University Hospital, Oslo, Norway. Institute of Clinical Medicine, University of Oslo, Oslo, Norway, Oslo, Norway; 3 Department of Medical Genetics, Oslo University Hospital, Oslo, Norway, Oslo, Norway; 4 The Blood Cell Research Group, Department of Biochemistry, Oslo University Hospital Ullevaal, Oslo, Norway, Oslo, Norway; 5 Oslo University Hospital, Oslo, Norway; 6 Division of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway, Oslo, Norway; 7 Department of Medical Genetics, Oslo University Hospital, Oslo, Norway. Institute of Clinical Medicine, University of Oslo, Oslo, Norway. Department of Immunology, Oslo University Hospital, Oslo, Norway, Oslo, Norway

Introduction : Rheumatoid arthritis (RA) is an inflammatory autoimmune disease mainly affecting joints, although it can also lead to systemic alterations. If insufficiently treated RA can cause irreversible damage disabling affected individuals. Methotrexate (MTX) is usually the initial drug prescribed, however a proportion of patients do not respond and must undergo several treatment trials while suffering further damage. Molecular signatures for treatment response are hence warranted.

Methods : In this study, we assessed extracellular vesicle (EV) profiles of 8 RA patients stratified based on MTX treatment response and 5 matched healthy controls. EVs were isolated from 0.5 ml plasma by size exclusion chromatography and fractions 7‐9 was used in downstream analyses. Nanoparticle tracking analysis and transmission electron microscopy was performed on fresh EV material, while EV‐marker (CD9, CD63 and CD81) profiles were assessed in frozen EV aliquots utilizing the ExoView platform (NanoView Biosciences).

Results : We observed an increased concentration of EVs in RA that was driven by the responder group. Colocalization analysis revealed a decreased expression of CD9/CD81 double positive EVs in non‐responders, while responders showed distinct high expression of CD81 single positive EVs.

Summary/Conclusion : Our study revealed clear tetraspanin differences between MTX treated RA patients and controls that may be implicated in RA pathophysiology and patient treatment response.

Funding : This study was funded by the Research Council of Norway.

Keywords : rheumatoid arthritis, methotrexate, EV tetraspanins

Colorectal cancer derived EVs drive invasion and tumour microenvironment remodelling in a 3D organotypic culture model

Sonia Guarnerio 1 ; Rob Tempest 2 ; Christine Le Maitre 1 ; Laura Cole 1 ; Stuart Hunt 3 ; Alex Mirnezami 4 ; Nicholas Peake 1

1 Sheffield Hallam University, Sheffield, United Kingdom; 2 NanoFCM, Nottingham, United Kingdom; 3 University of Sheffield, Sheffield, United Kingdom; 4 University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom

Introduction : Cancer growth and invasion is driven by numerous molecular and cellular factors in the tumour microenvironment (TME). Extracellular vesicles (EVs) have been discovered to play a pivotal role in remodelling the pre‐metastatic niche to support cancer cell invasion. Colorectal cancer (CRC) is the third most common cancer worldwide and it is frequently diagnosed in advanced stages, with a poor prognosis once metastasized. Thus, understanding the mechanisms involved in metastasis has become crucial for the improvement of diagnostic tools and treatments. Here, a 3D model was developed to evaluate the influence of CRC‐derived EVs on cancer invasion and stroma.

Methods : MRC5 fibroblasts were embedded on a mixture of collagen type I and GelTrex on a Transwell® system. SW480 colorectal cancer cells were allowed to invade into the models for 7 days. The 3D cultures were treated with EVs obtained by culturing primary and metastatic patient‐matched CRC cell lines (SW480 and SW620) in bioreactor flasks. CRC‐derived EVs were purified through size‐exclusion chromatography and fully characterised by nanoscale flow cytometry (NanoFCM). Invasive measurements were taken based on histological and immunohistochemical analysis. EV uptake was evaluated through confocal microscopy.

Results : CRC derived EVs were retained within the matrix of the 3D model, as observed through confocal microscopy. Quantitative analysis of the invasive layer highlighted changes in the invasion of cancer cells upon treatment with EVs. Invasive clusters length and size were significantly increased when the 3D culture was treated with SW620 EVs. Stromal cross‐linking activity around the invasive layer was also increased when the model was treated with CRC derived EVs. Interestingly, while inhibition of SW480 proliferation was observed in EV‐treated monocultures, MRC5 proliferation increased, indicating involvement of stromal mechanism surrounding the tumour.

Summary/Conclusion : The results obtained indicated a direct role of metastatic cancer derived EVs in driving cancer invasiveness. Conclusively, the 3D model developed was suitable for the analysis of stromal cancer cells invasion and provides a versatile system for the study of EVs within colorectal cancer metastasis.

Funding : Bowel Research UK, Sheffield Hallam University

Keywords : Cancer EVs, Colorectal cancer, Tumour microenvironment, 3D model

Insulin‐resistant M2‐CD163+ macrophages release extracellular vesicles affecting lipid metabolism and extracellular matrix gene expression in muscle cells

Stefano Tacconi 1 ; Anna Maria Giudetti 2 ; Emmanuelle Meugnier 3 ; Serena Longo 2 ; Federica Angilé 2 ; Francesco Paolo Fanizzi 2 ; Elisa Panzarini 4 ; Francesco Mura 5 ; Marco Rossi 6 ; Marco Fidaleo 1 ; Audrey Jalabert 3 ; Benjamin Gillet 7 ; Sandrine Hughes 3 ; Rienk Nieuwland 8 ; Sophie Rome 3 ; Luciana Dini 1

1 Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy; 2 Department of Biological and Environmental Sciences and Technologies, Lecce, Italy; 3 CarMeN Laboratory (UMR INSERM 1060/INRA 1397), Lyon‐Sud Faculty of Medicine, University of Lyon, Lyon, France; 4 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy; 5 Research Center of Nanotechnologies for Engineering (CNIS), Sapienza University of Rome, Rome, Italy; 6 Research Center of Nanotechnologies for Engineering (CNIS), Sapienza University of Rome, Roma, Italy; 7 Institut de Génomique Fonctionnelle de Lyon (IGFL), Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS UMR 5242, Lyon, France; 8 Department of Clinical Chemistry; Vesicle Observation Center; Amsterdam University Medical Center, University of Amsterdam, Amsterdam, Netherlands

Introduction : Skeletal muscle (SkM) insulin resistance is an early event in obesity‐induced diabetes, characterized by increased numbers of muscle‐resident macrophages. We tested whether muscle‐resident macrophages release extracellular vesicles (EVs) affecting SkM homeostasis.

Methods : Small and large EVs (sEV and lEV) were collected by differential centrifugation from macrophages treated with palmitate + oleate (FFA) with or without 15 mM D‐glucose (FFA/G15). Macrophages were characterized by quantifying polarization markers, lipid composition, and insulin sensitivity. C2C12 myotubes were treated with sEV or lEV to determine their possible effects on muscle lipid composition, gene expression, and insulin‐induced AKT phosphorylation.

Results : FFA‐treated macrophages polarized into M2‐CD163+ which released pro‐/anti‐inflammatory cytokines, accumulated triacylglycerols (TAG), FFA, phospholipids, and had reduced insulin‐sensitivity compared to untreated macrophages. Myotubes treated with lEV‐FFA accumulated TAG and FFA and had reduced insulin‐sensitivity compared to untreated cells, reproducing the FFA action on macrophages. lEV‐FFA also induced the expression of extracellular matrix components. sEV‐FFA reduced the expression of genes involved in lipid oxidation and mitochondrial respiration and induced TAG accumulation without affecting muscle insulin resistance. By increasing myotube sphingomyelin concentrations, sEV‐FFA reduced muscle ceramides content. Thus, in diet‐induced obesity, macrophages release EVs able to participate in maintaining muscle integrity. The macrophage FFA/G15 condition reduced CD163+ and IL‐10 and increased IFNα, IL‐1beta, IL‐8. Therefore, macrophage‐FFA/G15‐released EVs lost the beneficial action of EVs from macrophage‐FFA and induced insulin‐resistance in C2C12.

Summary/Conclusion : The biological effects of EVs mirror the phenotype plasticity of the releasing macrophages recapitulating the different events leading to SkM insulin‐resistance in obesity‐induced diabetes.

Funding : This work is supported by the FRENCH AGENCY OF RESEARCH (# ANR‐21‐CE14‐0081).

Keywords : macrophages, extracellular vesicles, inflammation, diabetes, skeletal muscle, lipids

Platelet‐derived extracellular vesicles alter metastatic melanoma cell functionality

Alessandra de sousa Faria; Zeynep Tavukcuoglu; Mari Palviainen; Pia Siljander

University of Helsinki, Helsinki, Finland

Introduction : Skin melanoma is one of the most aggressive skin cancers and responsible for most of the skin cancer deaths. Experimental evidence shows that in addition to hemostasis, platelets play a role in cancer progression. Upon activation, platelets generate heterogeneous platelet‐derived extracellular vesicles (PEVs) that may educate cancer cells to favor cell proliferation and enhance metastasis in the tumor microenvironment. Here, we studied how the treatment of melanoma cells with different PEVs affected the 3D‐melanoma spheroid growth and cancer EV production.

Methods : Human platelets were activated by different platelet activators and PEVs were isolated by size exclusion chromatography and analyzed according to MISEV2018 guidelines. (ii) Melanoma: A2058 and MV3 cells were cultured as 3D spheroids by using GrowDex hydrogel. (iii) PEV‐melanoma co‐culture: EVs from PEV‐educated spheroids were isolated by ultracentrifugation and characterized by NTA and SP‐IRIS. (iv) Cells were isolated from MV3 and A2058 spheroids and intracellular signaling was determined by analysis of p‐p38, p‐FAK and p‐Src with western blotting.

Results : The normal growth of the MV3 and A2058 spheroids was altered depending on the type of PEV used to educate the spheroids. Also, the EV generation from the spheroids was shown to depend on the PEV education. CRP‐XL‐PEVs increased both the size of the spheroids and the EV production in the A2058 cell line. Fucoidan‐PEVs increased the spheroid size of the MV3 cells, whilst the highest EV production was achieved with ionophore‐PEVs. EVs from the different PEV‐educated melanoma spheroids exhibited different tetraspanin profiles. PEVs also altered signal transduction of the melanoma cells, showing modulation of proliferation pathways.

Summary/Conclusion : In conclusion, PEVs produced by different agonists modified the functions of 3D melanoma spheroids, e.g. proliferation, survival and altered EV production. Platelets and their EVs are novel immunomodulatory agents in the tumor microenvironment.

Funding : This work was supported by Academy of Finland PLEASER grant (330486), Turkish Ministry of National Education, Oskar Öflunds Stiftelse, Ida Montinin Foundation and Orion Research Foundation.

Keywords : platelets, melanoma, platelet educated tumor

Comparison of extracellular vesicles derived from porcine oviduct epithelial cells in different culture models

Jianchao Huo 1 ; Marcus Frank 2 ; Alireza Fazeli 3 ; Jennifer Schoen 1 ; Shuai Chen 1

1 Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research (IZW), Berlin, Germany, Berlin, Germany; 2 Medical Biology and Electron Microscopy Centre, Rostock University Medical Center, Rostock, Germany, Rostock, Germany; 3 Unit of Reproductive and Developmental Medicine, Department of Oncology and Metabolism, The Medical School, University of Sheffield, Sheffield, UK, Sheffield, United Kingdom, United Kingdom

Introduction : Oviductal extracellular vesicles (EVs) are key mediators of the maternal‐gamete/embryo crosstalk. Several culture approaches are available for cultivating oviduct epithelial cells in vitro, which may differ in terms of EV production and cargoes of EVs. We aimed to characterize and compare EVs derived from three culture models of porcine oviduct epithelial cells (POECs).

Methods : The following cell culture models were analyzed in a serum‐free medium: a) Submerged 2D culture of POEC cell line (CCLV‐RIE270) for 3 passages (3P, 3pools/P). b) Submerged 2D culture of primary POEC (N=3, 3P). c) Air‐liquid interface (ALI) culture of primary POEC (3w, N=5). The medium was refreshed 48h before collection, and cells were evaluated for morphology/histomorphometry. EVs were isolated by polyethylene glycol/ultracentrifugation and assessed by transmission electron microscopy (TEM), micro bicinchoninic acid (BCA) assay, and nanoparticle tracking analysis (NTA).

Results : The cell line exhibited a typical cobblestone shape. The 2D primary culture showed a similar phenotype, however, cell shape elongated over passaging. In contrast, ALI culture highly resembled oviduct epithelium, with both ciliated (55.9%) and secretory cell types (mean height: 19.93μm). NTA (normalized by 10⁶ cells) revealed that ALI culture mainly secreted EVs into the apical side (7.32x10⁷), with very low amount released into the basal medium (7.55x10⁵, P < 0.01). ALI culture produced more EVs than 2D primary culture (6.32x10⁶) and 2D cell line (1.4x10⁷), conforming to protein assessment. The size of EVs in the cell line was smaller than in other cultures (P < 0.05). The number of EVs secreted by the cell line slightly declined over passages, while an opposite tendency was noticed in 2D primary culture. TEM visualized the spherical shape of EVs and further confirmed the NTA findings.

Summary/Conclusion : Our preliminary data highlight the ALI culture as a model for EV production, and the directional release of EVs primarily to the apical pole of the oviduct epithelium.

Funding : The study was funded by the DFG (CH 2321/1‐1) and CSC (201908350115).

Staphylococcus aureus‐derived extracellular vesicles impair the osteogenesis‐angiogenesis coupling therefore causing bone loss

Yi Li 1 ; Haikuan Yu 1 ; Mingming Zhang 2 ; Ruijing Chen 1 ; Pengbin Yin 1

1 Department of Orthopedics, Chinese PLA General Hospital, Beijing 100853, China;, Beijing, China (People's Republic); 2 Department of Orthopedics, General Hospital of Chinese PLA, Beijing, China, Beijing, China (People's Republic)

Introduction : Bone infection is a catastrophic complication in fracture patients. This study aimed to investigate the effect of extracellular vesicles (EVs) derived from Staphylococcus aureus (S. aureus) destruct the osteogenesis‐angiogenesis coupling in both in vitro and in vivo bone infection models.

Methods : EVs isolated from S. aureus were examined the morphology and contents by transmission electron microscopy, nanoparticle tracking analysis and proteomic analysis. Preosteoclasts were cultured with EVs during osteoclast differentiation. The PDGF‐BB levels from preosteoclasts were assessed by ELISA and qPCR, respectively. A mouse model of bone infection was established by Intramedullary injection of S. aureus. At 8 weeks after the model was established, the femur samples were collected for analysis the destruction of the osteogenesis‐angiogenesis coupling.

Results : After endocytosed with the preosteoclasts in vitro, EVs from S. aureus could affect the osteoclast differentiation and inhibit the secretion of PDGF‐BB. In vivo models indicated the femur bone mineral density levels were significantly reduced after infection with S. aureus. Meanwhile, the EVs from S. aureus decreased CD31hiEmcnhi endothelial cell formation.

Summary/Conclusion : In summary, we mapped the detailed proteomic landscapes of EVs derived from S. aureus and showed that their potential biological roles are important in coupling osteogenesis‐angiogenesis during bone infection.

Saturday 28 May 2022

PS10: Late‐Breaking Poster Session 6: Physiology/Pathology II

Chair: Massimo Bottini – University of Rome Tor Vergata

Chair: Krizia Sagini, n/a – Oslo University Hospital Oslo, Department of Molecular Cell Biology, Institute for Cancer Research

The MHC Class I Peptidome of Extracellular Vesicles: Detection of Tumour Associated Antigenic Epitopes In Melanoma

Caitlin Boyne 1 ; Sydney Brown 2 ; Pankaj Kumar 2 ; Sally Shirran 2 ; Simon Powis 2

1 University of St Andrews ‐ School of Medicine, Cupar, United Kingdom; 2 University of St Andrews ‐ School of Medicine, St Andrews, United Kingdom

Introduction : Melanoma is the form of skin cancer associated with the highest rate of mortality and the incidence of the disease is increasing more rapidly than any other form of cancer (1). Extracellular vesicles (EVs) are small membrane vesicles that have been indicated as key players in the process of cell communication, in particular the communication that exists between cancer cells and the host microenvironment, both locally and distally (2). The MHC class I immunopeptidome present on EVs released by cancer cells may be the defining feature of EVs that allows them to have a significant impact on the anti‐tumour immune response. Thus, in this study we isolated EVs from a melanoma cell line in order to characterise their peptide ligandome.

Methods : The melanoma cell line ESTDAB‐056 was utilised. Immunoblot analysis of cell and EV lysates were carried out along with Nanoparticle Tracking Analysis of cell culture supernatant and flow cytometry of ESTDAB‐056 cells grown in culture to confirm the characterization of the EVs isolated and their associated MHC class I expression. Peptides were successfully eluted from triplicate cell and EV lysates using the immunoprecipitation technique and were analyzed by mass spectrometry.

Results : EVs were successfully isolated and characterized. A total of 804 peptides were detected from the cell surface and 472 peptides from the extracellular vesicles of the cell line studied. Within the extracellular vesicle ligandome we identified 24 peptides derived from tumour‐associated antigenic proteins, of which 3 have known T cell reactivity.

Summary/Conclusion : The data achieved supports the possibility of identifying peptides that could be recognized by T‐cells and mediate a reaction. The discovery of these key antigens would be a major step toward the development of a novel immunotherapy for melanoma, as well as possibly providing a novel diagnostic or prognostic tool for the disease.

Funding : Funded by Melville Trust Scotland

Keywords : melanoma; cancer; HLA‐1; extracellular vesicles; immunopeptidome; antigen presentation; mass spectrometry; cancer immunology

Adipocyte‐derived small extracellular vesicles from patients with metabolic dysfunction may induce insulin resistance in neural progenitor cells

Wade O'Brien 1 ; Rachael Batabyal 2 ; Laura Reck Cechinel 1 ; Evan Nadler 1 ; Kazue Hashimoto‐Torii 3 ; Robert J. Freishtat 1

1 Children's National Hospital, Washington, USA; 2 Children's National Hospital, Columbia, USA; 3 Children's National Hospital and The George Washington University, Washington, USA

Introduction : Insulin signaling in the brain is critical to neurodevelopment and cognitive function. It has been implicated in several neurological disorders. Specifically, the PI3K/AKT signaling cascade is critical for learning and memory. Adipocyte‐derived small extracellular vesicles (ad‐sEVs) are a potential mechanistic link to obesity‐related disease. In patients with adiposopathy, ad‐SEVs contain miRNAs predicted to induce systemic insulin resistance. We hypothesize that ad‐sEVs from patients with obesity and diabetes will downregulate insulin signaling in neural progenitor cells (NPCs), leading to insulin resistance.

Methods : Induced pluripotent stem cell derived NPCs were cultured for 72 hours, then exposed to 6μg/mL of ad‐sEVs isolated form visceral adipose tissue from adolescents with obesity and diabetes (n=3, OD NPCs) or obesity and insulin resistance (n=3, OI NPCs) for 24 hours while controls received no ad‐sEVs (n=3). At 96 hours, RNA was extracted and sequenced on Illumina NovaSeq. RNA sequencing data were analyzed on PartekFlow and differentially expressed genes identified using GSA.

Results : NPCs exposed to ad‐sEVs from patients with obesity and diabetes (OD NPCs) showed 2482 differentially expressed genes (FC ≥|1.9|, p < 0.05) compared to control NPCs. NPCs exposed to ad‐sEVs from patients with obesity and insulin resistance (OI NPCs) showed 114 differentially expressed genes compared to control NPCs. Between OD NPCs and OI NPCs there were 1576 differentially expressed genes. Among these differentially expressed genes, we identified downregulation of mRNAs related to insulin signaling pathways between metabolic conditions in neural progenitor cells. INSR and PI3K were downregulated in the OD NPCs compared to OI NPCs. Compared to control NPCs, OD NPCs showed downregulation of INSR, PI3K, and AKT. No change in mRNA expression was observed in OI NPCs relative to control NPCs.

Summary/Conclusion : These data suggest that ad‐sEVs from patients with diabetes may induce insulin resistance in NPCs which could affect neurocognitive function.

Funding : Children's Research Institute

Pregnancy exosome release and hsa‐miR‐126‐3p expression vary according to pancreatic β‐cell functionality

Melissa Razo‐Azamar 1 ; Rafael Nambo‐Venegas 2 ; Martha Guevara‐Cruz 3 ; Berenice Palacios‐González 4

1 National Institute of Genomic Medicine, MEXICO CITY, Mexico; 2 Laboratorio de Bioquímica de Enfermedades Crónicas Instituto Nacional de Medicina Genómica, MEXICO CITY, Mexico; 3 Departamento de Fisiología de la Nutrición, Instituto Nacional de Nutrición y Ciencias Médicas Salvador Zubirán, CDMX, MEXICO CITY, Mexico; 4 Unidad de Vinculación Científica Facultad de Medicina UNAM en Instituto Nacional de Medicina Genómica (INMEGEN), CDMX, MEXICO CITY, Mexico

Introduction : Gestational diabetes mellitus (GD) affects around 15% of all pregnancies worldwide. Recently, it has been suggested that maternal and placental exosomes play a key role throughout gestation. However, the mechanisms underlying maternal metabolic adaptations in GD remain poorly understood. Thus, we assessed serum exosome profiles and the EV‐associated hsa‐miRNA‐126‐3p in early pregnancy stages in normal and pregnancies with GD according to low or high pancreatic β‐cell functionality (HOMA‐%β).

Methods : We collected serum from 81 pregnant women before 13 weeks of gestation; 19 women with GD (preDG+), and 62 women with a healthy pregnancy (preDG‐). Later, women were subdivided according to low HOMA‐%β or high HOMA‐%β. Exosomes were isolated from maternal serum obtained at early gestation using the exoRNeasy Midi Kit. Immunoblotting was used to assess the presence of exosome‐associated proteins (CD63, PLAP & Alix). RT‐PCR evaluated the EV‐associated miRNA hsa‐miRNA‐126‐3p.

Results : According to the diagnosis of GD vs NGT, there was no difference in CD63‐positive and PLAP‐positive exosomes. Interestingly, when evaluating exosome abundance according to diagnosis and β‐cell functionality, we found that the preDG+ low HOMA‐%β group had the highest abundance of CD63‐positive and PLAP‐positive exosomes (∼2‐fold and ∼3‐fold greater, respectively) vs preDG‐ high HOMA‐%β. Findings on exosome‐associated hsa‐miR‐126‐3p expression showed that the preDG+ high HOMA‐%β group had the highest expression of this miRNA (∼8‐fold greater) vs preDG‐ high HOMA‐%β) while still maintaining an adequate insulin‐sensitivity HOMA‐IR index among all the study groups assessed.

Summary/Conclusion : These findings suggest that either in normal or pregnancies that develop GD, exosome abundance variations and a miRNA targeted to regulate IRS on insulin‐sensitive tissues depend on the pancreatic β‐cell functionality status.

Keywords : gestational diabetes, exosome, early pregnancy, miRNA

Mesenchymal stem cell‐derived extracellular vesicles for corneal epithelial repair

Vasudeva Tati 1 ; Sreya Mitra 1 ; Prabhudatta Das 1 ; Sayan Basu 2 ; Sachin Shukla 1

1 Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Centre for Ocular Regeneration, Prof. Brien Holden Eye Research Centre, L V Prasad Eye Institute, Hyderabad‐500034, India, Hyderabad, India; 2 Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Centre for Ocular Regeneration, Prof. Brien Holden Eye Research Centre, The Cornea Institute, L V Prasad Eye Institute, Hyderabad‐500034, India, Hyderabad, India

Introduction : Corneal injuries are the leading cause of blindness. While mesenchymal stem cells (MSCs) have been shown to promote tissue repair at the ocular surface, the role of MSC‐derived extracellular vesicles (EVs) in corneal epithelial repair is largely unknown.

Methods : This study was approved by the institutional ethical committee (LEC‐BHR‐P12‐20‐559 and LVPEI‐IC‐SCR‐05‐21‐007). Human bone marrow‐derived MSCs (BM‐MSCs, Lonza Bioscience, #PT‐2501) and corneal epithelial cells (HCECs, ATCC, #HCE‐2 [50.B1] CRL‐11135) were cultured, characterized, and maintained in vitro as per the manufacturer's instructions. EVs were isolated from cultured BM‐MSCs using Total Exosome Isolation Reagent (#4478359, Invitrogen) and differential ultracentrifugation. Briefly, at 70% confluency, MSCs were starved in serum free media for 24 hours. Thereafter, culture medium was harvested and centrifuged in following steps: 300xg for 10 minutes, 2000xg for 20 minutes, 10000xg for 40 minutes, and 100000xg for 120 minutes using SORVALL MX 150+ Micro‐Ultracentrifuge (Thermo Scientific). All centrifugation steps were carried out at 40C, and after each step supernatant was transferred in a fresh tube and centrifuged further. Finally, the pellet was resuspended in PBS (filter‐sterilized). The EVs, thus isolated, were quantified (using EXOCET, #EXOCET‐96A, System Biosciences), characterized (as per MISEV 2018 guidelines using western blotting: CD9, #10626D; CD63, #10628D; CD81, #MA5‐13548, Calnexin, #MA3‐027; electron microscopy, etc.), and used in in vitro wound healing assay with HCECs. In EV‐based experiments, cells were cultured in basal medium supplemented with 10% exosome‐depleted FBS. Non‐EV‐treated HCECs were used as control.

Results : The BM‐MSCs and HCECs were characterized as CD90+CD105+CD45 CD34‐ and CK3/12+, respectively. The EVs were CD63+ and Calnexin‐. The scratched HCECs treated with BM‐MSC‐EVs (1x107) showed faster wound closure, relative to control.

Summary/Conclusion : BM‐MSC‐EVs may facilitate repair of the injured corneal epithelium in vitro.

Funding : Science and Engineering Research Board (SRG/2020/000842) and Department of Biotechnology (BT/PR32404/MED/30/2136/2019), Govt. of India, Hyderabad Eye Research Foundation

Keywords : Mesenchymal Stem Cells, Bone Marrow, Corneal Injury, Tissue Repair, Extracellular Vesicles, Secretome

DASH diet mechanism dissected by urine exosomes

Dana Bielopolski 1 ; Luca Musante 2 ; Henrik Molina 3 ; Douglas Barrows 3 ; Tom Carrol 3 ; Jonathan N. Tobin 4 ; Rhonda Kost 3 ; Uta Erdbruegger 2

1 Rockefeller University, Tel Aviv, Israel; 2 University of Virginia, Charlotessvile, USA; 3 Rockefeller University, New York City, USA; 4 Clinical Director Network, New York City, USA

Introduction : The DASH (Dietary Approach to Stop Hypertension) diet is a proven intervention in lowering blood pressure, yet it's mechanism has never been fully elucidated. We sought to characterize changes in urine exosome protein abundance pattern during nutritional changes, shifting from Western style diet to DASH diet.

Methods : The study was approved by the Rockefeller University IRB and informed consent was obtained prior to any research procedure. 9 hypertensive volunteers were admitted for a 2‐week period and transitioned from American style diet to DASH diet. Spot urine samples were collected on days 0, 5 and 11, spun down after addition of protease inhibitor, and frozen in (‐80). Urinary EVs were enriched by differential centrifugation to generate a low centrifugation pellet (20,000G, P20) and from the supernatant, a high (100,000G, P100) centrifugation pellet. Uromodulin was removed from both pellets by TCEP as validated by western blotting of TSG101. The P100 pellet was resolubilized in 6M guanidine in acidic condition and applied to a size exclusion chromatography (70nm). Both pellets were combined for mass spectrometry. Proteins were precipitated and re‐dissolved in 50uL 8M Urea/50mM ABC/20mM DTT followed by alkylation. Proteins were digested with LysC at less than 4M Urea and then diluted to less than 2M Urea for trypsinization. Digestions were halted by adding neat TFA and peptides were subjected to Solid Phase Extraction. Data were acquired on mass spectrometer coupled to a 12cm packed‐in‐emitter column and searched against a Human database. Statistical analyses were performed using R. An ANOVA test was used to identify all proteins changing across the time course (p < 0.05) and their levels were visualized in a heatmap. Hierarchical clustering of the genes from the ANOVA analysis, combined with pairwise comparisons of individual time points, were used to identify proteins throughout the time course.

Results : 1800 proteins were detected in the combined P20 and P100 urinary EV pellet. 292 of them were clustered into 5 different expression patterns: 38 in cluster 1, 24 in cluster 2, 87 in cluster 3, 61 proteins in cluster 4, and 82 proteins in cluster 5. Two proteins’ expression patterns coincided with our model: AQP2 (aquaporine 2) expression decreased from day 0 to day 5 and day 11. SLC12A3 (sodium chloride symporter; NCC) expression increased from day 0 to day 5 and day 11.

Summary/Conclusion : An increase in urinary EV's content of NCC and decrease in AQP2 might provide a mechanistic explanation to clinical changes of increased urine volume and blood pressure reduction following DASH diet implmentation.

Funding : These projects were supported in part by the National Center for Advancing Translational Sciences, National Institutes of Health, through Rockefeller University, Grant # KL2TR001865.

This work was supported by the Young Investigator Grant of the National Kidney Foundation

Keywords : DASH; uEV's, hypertension, NCC, AQP2

Effects of Montmorency tart cherry (L. Prunus cerasus) juice on inflammatory responses in human cells subjected to hyperglycaemia and hyperlipidaemia

Charlotte Lawson 1 ; Soumya Sharma 1 ; Natasha Ostrowski 1 ; Terun Desai 2 ; Lindsay Bottoms 2

1 Royal Veterinary College, London, United Kingdom; 2 University of Hertfordshire, Hatfield, United Kingdom

Introduction : There is a global obesity epidemic, leading to increases in insulin resistance, metabolic syndrome and type two diabetes (T2D), which are associated with more than 50% greater risk of cardiovascular diseases (CVD), due to chronic systemic inflammation within the vasculature and other tissue. We have previously cultured human monocytes and endothelial cells in the presence of high glucose (10‐25mM) and palmitic acid (500nM) and shown increase in pro‐inflammatory CCL‐2 chemokine production, reactive oxygen species (ROS) production and extracellular vesicle (EV) release.

Montmorency tart cherry juice (MTCJ) provides high levels of beneficial polyphenols and supplementation has been shown to improve exercise parameters in healthy participants and in patients with metabolic syndrome.

Methods : Healthy volunteers were given MTCJ prior to exercise as published previously. Serum was collected pre and post exercise. EV were phenotyped by flow cytometry using fluorescently labelled antibodies to CD31, CD61, CD45, CD235 and Annexin V together with sizing beads and enumeration beads and confirmed by NTA. THP‐1 monocytes were treated with 18mM glucose, glucose oxidase with or without MTCJ (Cherry Active) for 24h. For ROS analysis, cells were pre‐loaded with dihydrorhodamine‐1,2,3. CCL‐2 was measured by ELISA, EV were analysed by flow cytometry.

Results : MTCJ reduced post‐exercise release of EV from platelets in vivo, reduced THP‐1 EV release, pro‐inflammatory chemokine production and reactive oxygen species production in vitro. Furthermore, the phenolic compounds vanillic acid, protocatechuic acid and chlorogenic acid, which have been identified in plasma after supplementation with MTCJ, modulated EV release, ROS production and CCL‐2 release in THP‐1.

Summary/Conclusion : MTCJ supplementation may be a useful dietary supplement for non‐pharmacologic intervention for patients at risk of obesity and associated chronic inflammatory syndromes.

Funding : Society for Endocrinology

Keywords : cardiovascular; inflammation

Investigating the role of extracellular vesicles in fibrosis with an in vitro model of Peyronie's disease

Marcus M. Ilg 1 ; David J. Ralph 2 ; Selim Cellek 1

1 Anglia Ruskin University, Chelmsford, United Kingdom; 2 University College London Hospital, London, United Kingdom

Introduction : Peyronie's disease (PD) is a fibrotic condition characterized by plaque formation in the penile tunica albuginea (TA), driven by persistent myofibroblast presence. The role of extracellular vesicles (EVs) has not been studied in PD.

Methods : Human primary fibroblasts were isolated from TA of PD patients. Conditioned media was generated from cells with and without Transforming Growth Factor‐ß1 (TGF‐ß1) to induce myofibroblast transformation. EVs were isolated using size‐exclusion chromatography columns after concentrating media via centrifugal filters. EVs were analyzed via Tunable Resistive Pulse Sensing (TRPS). Fibroblasts were treated with various concentrations of EVs in presence/absence of TGF‐ß1 and myofibroblast transformation was quantified measuring myofibroblast marker α‐SMA using In‐Cell ELISA (ICE).

Results : TRPS analysis revealed that the TGF‐ß1‐treated cells produced more EVs (5.4x109 particles/mL) than untreated cells (9.2x108 particles/mL). Mean diameter was 92nm and 71nm for vesicles of untreated and TGF‐ß1‐treated cells, respectively. TGF‐ß1‐treated cells produced particles with increased blockage duration at various particle diameters, indicating different shape. Interestingly, EVs from TGF‐ß1‐treated cells exerted a concentration‐dependent anti‐fibrotic effect, preventing TGF‐ß1‐induced myofibroblast transformation, whilst fibroblast EVs showed no effect. No anti‐myofibroblast effect was observed for co‐isolated soluble protein fractions. The effect of EVs could be abrogated when cells were treated with 10μM of dynasore to inhibit vesicle uptake.

Summary/Conclusion : This is the first study to characterize the EVs derived from fibroblasts and myofibroblasts in PD and to demonstrate an anti‐myofibroblast effect of myofibroblast‐derived EVs. Further work is needed to identify the contents of the vesicles to see what drives the effect on fibroblast/myofibroblast biology.

Funding : European Society for Sexual Medicine (RG19‐04)

Keywords : Fibrosis, Peyronie's disease, Myofibroblast, Fibroblast

Unraveling the role of extracellular vesicles in Alzheimer's disease: from disease progression to neuroprotection

Andreu Matamoros‐Angles 1 ; Mohsin Shafiq 1 ; Emina Karadjuzovic 1 ; Behnam Mohammadi 2 ; Feizhi Song 1 ; Santra Brenna 3 ; Berta Puig 4 ; Hermann Altmeppen 1 ; Michaela Schweizer 5 ; Friederike Zunke 6 ; Markus Glatzel 1

1 Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany, Hamburg, Germany; 2 Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 3 Department of Neurology, Experimental Research in Stroke and Inflammation (ERSI), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany., HAMBURG, Germany; 4 Department of Neurology, Experimental Research in Stroke and Inflammation (ERSI), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany., Hamburg, Germany; 5 Core Facility of Electron Microscopy, Center for Molecular Neurobiology ZMNH, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany, Hamburg, Germany; 6 Department of Molecular Neurology, University Hospital Erlangen, Friedrich‐Alexander University Erlangen‐Nürnberg (FAU), 91054 Erlangen, Germany., Erlangen, Germany

Introduction : Alzheimer's disease (AD) is the leading cause of dementia worldwide. The trigger is still unknown, and available symptomatic treatments show modest efficacy at best. AD is characterized by a slow yet progressive cognitive decline affecting brain regions related to memory, personality, and daily activity performances. In AD brains, there is a progression of amyloid‐β (Aβ) aggregation in extracellular plaques and the formation of intracellular neurofibrillary tangles composed of phosphorylated Tau protein, suggesting that this propagation participates in the pathology. Extracellular vesicles (EVs) have been related to crucial brain functions, such as myelin maintenance and neurotransmission. However, the functionality and regulation of brain‐EVs are not fully understood. Recently, EVs emerged as relevant in AD and were described as biomarkers in AD patients' serum. On the one hand, EVs trap Aβ, which reduces Aβ free oligomers load and Aβ‐mediated toxicity, and participate in prosurvival signaling, thereby exerting neuroprotective functions. Conversely, EVs enhance the propagation of Aβ and Tau aggregates, thus promoting the disease progression, highlighting a seemingly dual role of these entities in AD.

Here we will describe in detail the AD brain‐derived EVs’ cargo to characterize the main protein actors in the aforementioned dual role of EVs.

Methods : EVs are isolated from AD patients’ brains by differential ultracentrifugation and iodixanol‐based density gradient. Nanoparticle tracking analysis, western blotting, and transmission electron microscopy are used to characterize them. Their RNA and protein profiles will be analyzed by transcriptomic and proteomic work‐ups. Lastly, using hypothesis‐driven experiments in human iPS cell models, we will study the role of differentially expressed genes/proteins in brain‐derived EVs biology in AD's pathophysiology.

Results : Our optimized EVs’ isolation protocol shows two subpopulations of EVs with a similar average mean size (∼170‐200 nm). The samples express the EV markers CD81, CD9, and Flotilin‐1 and AD‐relevant proteins, such as ADAM10 and the prion protein. The transmission electron microscopy reveals the expected cup shape of the purified particles. Omics analyses are currently being conducted, and the human iPSC‐derived neuronal model is established.

Summary/Conclusion : Our optimized EVs’ isolation protocol drastically reduces the generation of proteolytic artifacts. These EVs will be useful to accomplish our primary goal: to achieve better mechanistic insight into EVs’ function in AD, especially into their neuroprotective role.

Funding : This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska‐Curie grant agreement N°101030402 and from the PIER initiative Hamburg (Project PIF 2020‐10).

Angiostatic effects of autologous and heterologous extracellular vesicles on coronary endothelium

Anca D. Dobrian 1 ; Nigeste Carter 2 ; Allison M. Mathiesen 1 ; Noel Miller 1 ; MIchael Brown 1 ; Ruben Colunga Biancatelli 3 ; John Catravas 4

1 Eastern Virginia Medical School, Norfolk, USA; 2 Virginia Commonwealth University, Richmond, USA; 3 Old Dominion University, Norfolk, USA; 4 Old Dominion UNiversity, Norfolk, USA

Introduction : We previously demonstrated that EVs released by endothelial cells (EC) of adipose tissue are angiostatic. However, it is not clear whether this is specific to adipose cells or is a general feature of EC‐EVs. Here, we compared the effects of EVs produced by human coronary artery endothelial cells (HCAEC, autologous) and adipose microvasculature (heterologous) on the endothelium, under basal and inflammatory conditions.

Methods : HCAEC and adipose ECs were treated for 6 days with a combination of pro‐inflammatory cytokines (PIC) TNFa, IFNg, and TGFb. EVs were isolated by ultracentrifugation, characterized by NTA and EM, and tetraspanin expression determined by ExoView R100. EV internalization was measured by imaging flow cytometry. HCAEC were then treated with EVs for 24 hours and in vitro tube formation and proliferation quantified. Barrier function and wounding were assessed using ECIS 1600R. Nanostring miRNA panel was used to identify miRNA cargo.

Results : All EV subtypes had similar morphology and size distribution. CD63 expression increased in autologous and heterologous EVs from PIC‐stimulated cells. EC showed reduced relative mesh formation in response to each EV subtype, compared to control cells. ECs displayed a significant reduction in proliferation, in response to each EV subtype. Autologous EVs isolated from PIC treated cells showed to damage HCAEC barrier function, while the same EVs isolated in basal conditions participated in wound recovery. We identified 8 miRNAs amongst all EV subtypes. IPA analysis identified target genes involved in VEGF signaling, cell cycle and extracellular matrix formation.

Summary/Conclusion : EVs from control‐ and PIC‐treated HCAEC reduced angiogenesis and proliferation possibly by targeting of genes via their miRNA cargo. EVs may exert an angiostatic effect during post‐ischemic myocardial events by competing with the pro‐angiogenic cues and by disrupting repair mechanisms.

Funding : American hear Association, NIH/NHLBI

PS11: Late‐Breaking Poster Session 7: Biomarkers I

Chair: Michael W. Pfaffl, n/a – Chair of Animal Physiology & Immunology

Chair: Zach A. Troyer, PhD – Johns Hopkins University

Proteomic analysis of extracellular vesicles derived from high‐grade serous ovarian cancer cells

Laura Lehtinen 1 ; Milla Hollmén 2 ; Katja Kaipio 2 ; Johanna Hynninen 3 ; Seija Grenman 3 ; Olli Carpén 4

1 Cancer Research Unit, Institute of Biomedicine and FICAN West Cancer Centre, University of Turku, Turku, Finland, Paimio, Finland; 2 Cancer Research Unit, Institute of Biomedicine and FICAN West Cancer Centre, University of Turku, Turku, Finland, Turku, Finland; 3 Department of Obstetrics and Gynecology, University of Turku and Turku University Hospital, Turku, Finland, Turku, Finland; 4 Department of Pathology, University of Helsinki and HUSLAB, Helsinki University Hospital, Helsinki, Finland, Helsinki, Finland

Introduction : High‐grade serous ovarian cancer (HGSOC) is a prime example of a disease, in which increased molecular knowledge has not yet translated to outcome improvement. The discussion between tumor cells and surrounding stroma is a critically important feature in disease progression in HGSOC, as most patients are diagnosed with extensively disseminated disease. Extracellular vesicles (EV) provide intercellular communication between tumor and stromal cells via several types of bioactive molecules, and may provide additional information on HGSOC biology.

Methods : Conditioned cell culture medium was collected from three commonly available HGSOC cell lines and three patient derived primary cell cultures (PD‐HGSOC). EVs were isolated from conditioned culture media with size‐exclusion chromatography and characterized for particle size and concentration with TRPS measurements. The EVs were visualized with transmission electron microscopy. The presence of known EV markers was studied with ELISA assays and western blotting. The protein composition of the individual EV samples was explored using LC‐ESI‐MS/MS. FunRich analysis tool was used to further explore the differences of EV protein compositions between commonly available and PD‐HGSOC cells.

Results : The characterization was conducted following MISEV2018 guidelines and confirmed successful isolation of highly pure EV samples. The identified proteins showed sample profiles typical for EVs. Preliminary analysis of proteomics data showed that HGSOC derived EVs contain various types of functionally important proteins. The protein composition differed between individual samples as well as between commonly available and PD HGSOC cells.

Summary/Conclusion : This study indicates interesting differences in EV protein composition between HGSOC cell lines and primary cell cultures. Comprehensive analysis of the proteomics data as well as in vitro validation assays continue.

Funding : The study was funded by The Academy of Finland.

Keywords : High‐grade serous ovarian cancer, patient derived cells, proteomics

Circulating small EV‐related alterations in early stage of atherosclerosis

Krisztina Pálóczi 1 ; Bernadett György 2 ; András Försönits 3 ; Júlia Opra 4 ; Boldizsár Csernai 3 ; Hargita Hegyesi 5 ; András Gézsi 6 ; Ádám Domonkos Tárnoki 7 ; Dávid László Tárnoki 7 ; Helga Szabó 7 ; Dóra Melicher 8 ; Edit I. Buzás 3

1 Semmelweis University Department of Genetics, Cell‐ and Immunobiology, Budapest, XVIII. kerület, Hungary; 2 University of Physical Education‐Research Center of Molecular Exercise Science, Budapest, Hungary; 3 Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, Budapest, Hungary; 4 Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, Budapest, Hungary; 5 Semmelweis University Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary; 6 Department of Measurement and Information Systems, Budapest University of Technology and Economics, Budapest, Hungary; 7 Medical Imaging Center, Semmelweis University, Budapest, Hungary; 8 Department of Emergency Medicine, Semmelweis University, Budapest, Hungary

Introduction : Atherosclerosis is a disease with complex genetic background. Multiple environmental and genetic factors are responsible for its development. Here we focused on circulating extracellular vesicles (EVs) and studied their changes in atherosclerosis.

Methods : Atherosclerosis was assessed by carotid and femoral artery ultrasonography. Blood samples were collected from healthy controls (n=21), from patients with early‐stage atherosclerosis (=15) and from individuals with more advanced atherosclerosis (n=18). ACD anticoagulated blood samples were used to prepare platelet free plasma. The PFP samples were subjected to 18,000g centrifugation to remove the medium sized EVs. Then the supernatant was filtered by gravity through a 0.22 micron filter, and was concentrated by ultrafiltration (100kDa cut‐off). The filtrate was further purified by SEC (qEV‐70), and the EVs were analyzed by Western blotting, NTA, ELISA and by flow cytometry (MACSPlex Exosome Kit). We also measured the blood plasma Lp(a), MMP12 and MCP1 levels, and analyzed the ApoE polymorphism.

Results : We found that MMP12 concentrations were significantly elevated both in the PFP samples and in the small EV preparations in the advanced atherosclerosis group as compared to healthy subjects. Importantly, we also found a significant elevation of MMP12 in small EVs of early‐stage atherosclerotic patients, while there was no elevation in the PFP samples of the same patients at this early stage of the disease. We also detected early‐stage atherosclerosis‐related increase in the number of CD14+ and CD142+ sEVs, while the CD146+ sEVs were significantly elevated only in the advanced atherosclerotic group.

Summary/Conclusion : Our data suggest that by analyzing circulating small EVs, certain atherosclerosis‐related alterations can be detected at an earlier stage of the disease than by the analysis of blood plasma samples.

Funding : This work was supported by National Research, Development and Innovation Office NKFIH, Hungary, NVKP_16‐1‐2016‐0017, the Hungarian Scientific Research Fund (OTKA K120237), VEKOP‐2.3.2‐16‐2016‐000002, VEKOP‐2.3.3‐15‐2017‐00016, H2020‐MSCA‐ITN‐2017‐722148 TRAIN EV and Higher Education Excellence Program (FIKP) Therapeutic Thematic Programme, Hungarian Thematic Excellence Programme No TKP2020‐NKA‐26, EU H2020 grant agreement No. 739593.

We would like to thank the ÚNKP‐20‐5 New National Excellence Program of the Ministry for Innovation and Technology, from the source of the National Research, Development and Innovation Fund as well as the Bólyai János research scholarship supported by the Hungarian Academy of Sciences."

Keywords : Extracellular vesicles, atherosclerosis, MACSPlex Exosome

Extracellular RNA (exRNA) as a biomarker for neurodegenerative diseases

Elizabeth Hutchins 1 ; Cherae Bilagody 2 ; Rebecca Reiman 2 ; Jerry Antone 2 ; Amber Logemann 2 ; Laura Cox 2 ; Bessie Meechoovet 2 ; Dorothy Marie Palomares 2 ; Farah Bitza‐Ray 2 ; Kendall Jensen 1

1 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA; 2 Translational Genomics Research Institute, Phoenix, USA

Introduction : The study and treatment of neurodegenerative diseases has been hindered by the lack of biomarkers to monitor disease development and progression. In this study we focus on using extracellular RNA (exRNA) as a biomarker for Alzheimer's disease (AD) and Parkinson's disease (PD). ExRNAs are carried by extracellular vesicles and can be found in all biofluids. Biofluids such as blood and urine are non‐invasive, cost effective, and can easily be collected for multiple time points. To ascertain exRNA as a viable biomarker we plan to establish a profile of exRNAs associated with disease and to confirm that it is reliable over time.

Methods : Plasma and urine samples were collected from individuals with Alzheimer's disease (AD) or Parkinson's disease (PD), and healthy individuals (control) at two different time points. Collection was performed with Informed patient consent through a contracted vendor under an approved IRB protocol and in accordance with Declaration of Helsinki. Libraries were prepped from vesicular RNA and sequenced using Illumina NovaSeq6000 platform. Post hoc analysis included correlation studies between visits in addition to differential expression.

Results : For plasma and urine we detected >15,000 genes and > 60,000 transcripts. Kidney specific genes have a higher correlation in urine, and brain specific genes have a higher correlation in plasma. Urine shows a higher percentage of protein coding and long non‐coding RNA, while plasma has a higher percentage of non‐coding RNA. Plasma exhibits more uniquely mapped genomes in comparison to urine.

Summary/Conclusion : Urine and plasma are viable sources of biofluids for biomarkers of CNS diseases based on the abundance of CNS specific genes. Plasma samples were found to be more reproducible and consistent than urine, but urine has the potential to be reproducible by removing variable genes and had greater diversity. Additionally, we identified candidate genes for disease based on differential expression.

Identification of cardiomyocyte‐derived circulating extracellular vesicle in the bloodstream

Hargita Hegyesi 1 ; Eva Pallinger 1 ; Annamária Huszár 2 ; Balazs Hornyak 1 ; Csenger Kovácsházi 3 ; Gabor B Brenner 4 ; Zoltán Giricz 5 ; Krisztina Paloczi 1 ; Delaram Khamari 1 ; Péter Ferdinandy 5 ; Edit I. Buzás 1

1 Semmelweis University Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary; 2 Department of Anatomy, Cell and Developmental Biology, Eotvos Lorand University, Budapest, Hungary; 3 Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary, Budapest, Hungary; 4 Semmelweis University Department of Pharmacology, Budapest, Hungary; 5 Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary; Pharmahungary ltd., Szeged, Hungary, Budapest, Hungary

Introduction : Extracellular vesicles (EVs) play a role in regulating the physiological and pathological functions of cardiomyocytes and it gives the potential to monitor cardiovascular diseases. The release of EVs is increased under cellular stress and cardiomyocyte damaging conditions. However, whether cardiomyocyte‐derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Conditional transgenic mice (MerCreMer/mTmG) expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)‐induced systemic inflammatory response syndrome (SIRS). We aimed to detect cardiomyocyte‐derived EVs in the blood.

Methods : EVs were separated both from platelet‐free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte‐derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography.

Results : In LPS‐treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF‐15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small‐ and medium‐sized EVs in the LPS‐injected mice. Importantly, we detected GFP+ cardiomyocyte‐derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration. The cardiomyocyte‐derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle‐associated glycogen phosphorylase (PYGM).

Summary/Conclusion : These data provide direct evidence that cardiomyocyte‐derived EVs are present in the circulation and that the increased number of cardiac‐derived EVs in the blood reflects cardiac injury in LPS‐induced systemic inflammation.

Funding : NVKP_16‐1‐2016‐0017,National Laboratories Excellence No NLP‐17

Keywords : Extracellular vesicles, Cardiomyocyte, Cardiomyopathy, Inducible transgenic mice, SIRS

Fast Isolation and Analysis of Functional Tumor‐related Exosomes (TREXs) from Head and Neck Squamous Cell Carcinoma (HNSCC) Plasma by Galectin‐based Glycan Recognition Particles

Dapi Menglin Chiang 1 ; Laura Benecke 2 ; Laurent Muller 2 ; Michael W. Pfaffl 1

1 Chair of Animal Physiology & Immunology, Freising, Germany; 2 Department of Otorhinolaryngology, Head and Neck Surgery, Basel, Switzerland

Introduction : Head and Neck Squamous Cell Carcinoma (HNSCC) is the eighth common cancer worldwide in 2021. Treatment failures are the main reason for the moderate overall survival, even though novel diagnostic methods and therapies have entered the clinics. Thus, better diagnostic methods are urgently needed for HNSCC follow‐up. Extracellular vesicles (EVs) have been recently studied as potential new targets in liquid biopsy for a successful and reliable cancer diagnosis. But, traditional high‐purity exosomes isolation method is a time‐consuming process, often with high degree of contaminating protein aggregates.

Methods : In this work, we present an EV isolation method for ‘pure’ and functional tumor‐related exosomes (TREXs) by galectin‐based glycan recognition particles, EXÖBead®. Bead‐based flow cytometry (BFC) results showed high abundant EV markers such as CD9, CD63, CD81 and TSG101. Intact eluted exosomes were observed by transmission electron microscopy (TEM) and Cryo‐EM after lactose buffer elution.

Results : A low lipoprotein contamination was demonstrated by measuring Apolipoprotein A1 which compared to size exclusion chromatography isolation. Classical PanEV markers (premixed CD9, CD63, and CD81 antibodies), common leukocyte antigen (CD45) and HNSCC biomarkers (PD‐L1, EpCAM and PanCK) were measured as TREXs biomarkers by BFC. CD45Neg PanEV+, EpCAM+ PanEV+, PD‐L1+ PanEV+, CD45Neg PanEV+ with triple HNSCC markers positive and CD45+ PanEV+ with double HNSCC markers positive were significantly higher in tumor patients (n=9) compared with healthy controls (n=9) and present an EV related circulating biomarker in HNSCC patients. Furthermore, HNSCC plasma eluted EVs by EXÖBead® isolation showed significantly higher immune suppression ability compared to Polyethylene glycol isolation (PEG; ExoQuick®). In peripheral blood mononuclear cells functional assay, PD1+ CD69neg and PD‐L1+ CD69neg T cells were also significantly increased from treatment with different patients’ plasma EVs (n=13) compared to elution buffer only controls (n=3).

Summary/Conclusion : EXÖBead® isolation revealed a fast and reliable method with low lipoprotein contamination and remained TREXs function from patient's plasma. Therefore, EXÖBead® showed great potential as a new EVs isolation tool with great diagnostic potentials in cancer diagnostics and further clinical settings.

Funding : This work was supported by the “pro patient” grant (pp18‐08) and Krebsliga 462 beider Basel (# 18‐463 2016).

Keywords : HNSCC, tumor‐derived EVs, low contamination, novel isolation technique, biomarker, galectin‐based glycan recognition particles, beads‐based flow cytometry

Computational deconvolution of plasma EV RNA by cell of origin improves detection of RNAs of interest

Robert Fullem 1 ; Emily Laplante 2 ; Aleksandar Milosavljevic 1

1 Baylor College of Medicine, Houston, USA; 2 BCM, Houston, USA

Introduction : Extracellular RNA (exRNA) that is contained in extracellular vesicles (EVs) is a mixture of RNA originating from a number of cell types of origin. This creates challenges where informative EV RNA biomarkers of disease can be masked by more numerous EVs from non‐disease related cell types. The deconvolution of plasma EV RNA by cell type of origin can address this challenge by identifying RNA contributed by EVs originating from proximal cell types, and thus improving detectability of RNAs originating from a distal tissue of interest.

Methods : We performed RNA‐Seq of EVs isolated from Red Blood Cells, Platelets, and Neutrophils. Utilizing the XDEC tool, these reference samples were used to deconvolute a study of pre and post exercise plasma EV exRNA by cell type of origin. Additionally, these reference samples were used with publicly available eCLIP data to predict the presence of RNA‐Binding Protein (RBP) EVs through permutation testing of RBP associated exRNAs. Downstream analysis predicted target genes of miRNAs of interest that were not identified through non‐deconvolution differentiation analysis.

Results : 22 RNAs were found to be significantly upregulated in a post exercise associated deconvoluted EV profile. This included novel RNAs not identified when analyzing the bulk plasma exRNA samples. Downstream analysis of the gene targets of the 16 differentially expressed miRNAs identified exercise related processes as the top scoring gene ontology results. Additionally, permutation testing for RBP presence in cell types of origin EVs allowed prediction of which RBPs occupied cell type specific EVs under pre and post exercise conditions.

Summary/Conclusion : Using limited reference data, our analysis revealed novel characteristics of EV biology in pre and post exercise conditions through deconvolution and imputation testing. With further reference data of additional proximal cell types of origin, we can leverage these methods to further interrogate exRNA studies of interest.

Exploring the role of Rab3 in exosome release from prostate cancer cells

Silvana Romero 1 ; Krizia Sagini 2 ; Kirsten Sandvig 2 ; Alicia Llorente 3

1 Dept. of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 2 Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 3 Oslo University Hospital Oslo, Department of Molecular Cell Biology, Institute for Cancer Research, oslo, Norway

Introduction : Exosomes are extracellular vesicles released by cells upon fusion of multivesicular bodies with the plasma membrane. However, the mechanisms underlying the secretion of exosomes are still unclear. Rab proteins play important roles in vesicular membrane trafficking along the endocytic and the secretory pathway. In particular, Rab3 has been involved in cellular secretion, and specifically Rab3D and Rab3B have been found to be enriched in urinary exosomes from prostate cancer patients compared to healthy males. In this study we investigated the role of Rab3 isoforms in exosome release in prostate cancer cells.

Methods : The role of Rab3A, Rab3B and Rab3D, in exosome release was investigated by siRNA depletion from the highly metastatic cancer cell line PC‐3. Knockdown efficiency was verified by western blot (WB) and specificity by qPCR. Small extracellular vesicles (sEVs) were then isolated by differential centrifugation from cell media and analyzed by nanoparticle tracking analysis (NTA) and WB. Moreover, confocal microscopy was used to investigate potential alteration of the endo‐lysosomal system in depleted cells. Alterations in other cellular pathways was investigated by following the trafficking of the toxin ricin.

Results : Our results indicate that depletion of Rab3D or Rab3B, but not Rab3A, in PC‐3 cells increase the release of sEVs enriched in exosomal markers such as Alix and Syntenin. Moreover, Rab3 depletion did not affect sEV size distribution, as shown by NTA. No significant changes in the transport of externally added ricin to the Golgi apparatus and cytosol were shown, suggesting that Rab3 is mainly associated with secretory processes.

Summary/Conclusion : This study reveals that Rab3D and RabB depletion in prostate cancer cells may play a role in exosome secretion, but further experiments are required to verify the endosomal origin of these sEVs.

Keywords : Exosomes, prostate cancer, Rab proteins.

Plasma extracellular vesicles: a promising tool for predicting ischemic stroke severity

Paola Monti 1 ; Angelica Carandina 2 ; Chiara Favero 3 ; Mirjam Hoxha 3 ; Nicola Montano 2 ; Luca Ferrari 4 ; Eleonora Tobaldini 2 ; Valentina Bollati 5

1 1) Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy, Saronno, Italy; 2 1) Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy 2) Department of Internal Medicine, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy, Milan, Italy; 3 1) Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy, Milan, Italy; 4 1) Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy 2) Occupational Health Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy, Milan, Italy; 5 1) Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy 2) Occupational Health Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy, Milano, Italy

Introduction : Ischemic stroke (IS) is a leading cause of death globally, with a huge burden in terms of mortality‐to‐incidence and disability. Non‐invasive tools for IS prognosis are currently lacking. Here, we focused on circulating EVs as potential predictive markers for acute and mid‐term IS outcomes.

Methods : After approval by local Ethics Committee, we enrolled 47 patients with acute IS, whose severity was evaluated at admission (T0) and after one week (Tw) using NIH stroke scale to assess neurological impairment, and after 3 months (Tm) using the mRs scale to assess functional disability. After giving informed consent, blood samples were collected at T0, at T1 i.e. soon after recanalization treatment or after 2 hours in non‐treated patients, and at Tw. Plasma EVs were enriched by ultracentrifugation. Total EV count and characterization of EV subpopulations (CD14+: monocytes/macrophages, CD61+: platelets, CD105+: total endothelial cells, CD25+: T‐cells, and CD62e+: activated endothelial cells) were obtained by NTA and flow cytometry, respectively. Multivariable regression models were applied to evaluate the relationship between IS outcomes and EVs.

Results : No association was found between EV count and early/long‐term outcomes. However, CD61+ EVs at T0 was positively associated with IS severity at T0 (∆%=0.18, p=0.002), and with severity of mid‐term outcome (∆%=0.30, p=0.016). CD25+ EVs at T0 were positively related to both early (∆%=3.93, p=0.009) and mid‐term IS outcomes (∆%=3.94, p=0.009). Finally, CD25+ EVs at T1 were positively related to mid‐term IS outcome (∆%=0.91, p=0.047).

Summary/Conclusion : The present study suggests that specific EV subtypes could be predictive of IS severity and both short‐ and long‐term outcomes. Our results provide new insights into the pathophysiological process of IS and propose EVs as a promising tool for better stratification of IS patients, for the choice of medical therapy, and for post‐recanalization treatment monitoring.

Extracellular Vesicles Expressing Tissue Factor, a Novel Biomarkers for Thrombotic Risk Assessment in Pancreatic Ductal Adenocarcinoma

Amineh Dadkhah 1 ; Dimitar Tasev 2 ; Moniek De Maat 3 ; Kees Kluft 4

1 Good Biomarker Sciences (GBS) Leiden enrolled at the Department of Haematology, Erasmus University Medical Center, Leiden, Netherlands; 2 Good Biomarker Sciences (GBS) Leiden, Leiden, Netherlands; 3 Department of Haematology, Erasmus University Medical Center, Rotterdam, Netherlands; 4 Good Biomarker Sciences (GBS Leiden), Leiden, Netherlands

Introduction : Cancer patients are at high risk for blood coagulation and venous thromboembolism (VTE) with a poor prognosis. The risk depends on different factors, including the type and stage of cancer, patient age and health background, and therapeutic methods used for cancer treatment. Pancreatic adenocarcinoma (PDAC) is a type of cancer with a high frequency of VTE incidents, but what makes PDAC such a thrombogenic disease remains unclear. Studies suggest that extracellular vesicles (EV) expressing tissue factor (TF) may play a crucial role in initiating thrombosis and causing VTE. Tissue factor (TF) is the primary initiator of blood coagulation and plays an essential role in haemostasis. Higher expression of this molecule in the pancreatic tissue can contribute to the local VTE in PDAC patients. Extracellular vesicles are a heterogeneous group of membranous lipid‐containing structures, including microvesicles and exosomes released from almost all types of cells under physiological conditions; however, pathological conditions, including cancer stimulate releasing a higher amount of EV in the tumour environment. These cancer‐derived particles contain procoagulant phospholipids, including phosphatidylserine. Circulating EV containing TF might be the key of VTE in far distant sites from primary tumours. This study set out to identify a novel biomarker for better risk assessment of thrombotic events in PDAC patients.

Methods : The pancreatic cancer cell line, BXPC‐3 was used as a cell model for a generation of EVs. Isolated EVs were characterized by nanoparticle tracking analysis (NTA) and different size‐fractions (1000, 450, 200 and 100nm) were isolated and assessed for TF by quantitative ELISA. A TF activity assay was used to determine the procoagulant activity of TF+ EVs.

Results : Our findings suggest that the EVs released from cancer cell lines under our experimental conditions express TF. The EVs are mostly in a range of 100‐400nm and smaller EVs are containing a higher concentration of TF.

Summary/Conclusion : Our study provides evidence that TF+‐EVs of a certain size are highly pro‐coagulant and might be used as biomarkers for thrombotic risk assessment in PDAC patients.

Can extracellular vesicles derived from different temozolomide‐treated glioblastoma multiforme cell lines be exploited as liquid biopsy?

Diana Vardanyan 1 ; Stefano Tacconi 2 ; Simone Dinarelli 3 ; Maria Concetta Cufaro 4 ; Elisa Panzarini 5 ; Piero Del Boccio 6 ; Luciana Dini 2

1 Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento,, Lecce, Italy; 2 Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy; 3 Institute for the Structure of Matter (ISM), National Research Council (CNR), Rome, Italy; 4 Department of Innovative Technologies in Medicine and Dentistry, "G. d'Annunzio", University of Chieti‐Pescara, Chieti, Italy; 5 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy; 6 Department of Pharmacy ,"G. d'Annunzio", University of Chieti‐Pescara, Chieti, Italy

Introduction : Extracellular vesicles (EVs) have been recently suggested as possible biomarkers of glioblastoma (GBM) multiforme, and thus used as "liquid biopsy" for diagnostic information, monitoring disease progression and response to clinical treatment. The overall purpose of the study is to define a specific molecular fingerprinting of EVs from four different GBM cell lines in presence or absence of temozolomide (TMZ).

Methods : EVs from four GBM cell lines (U87MG, T98G, U373MG, U251MG), in presence or in absence of 200μM temozolomide (TMZ) were isolated by ultrafiltration combined with size exclusion chromatography (SEC). The morpho‐quantitative analysis and size distribution of EV were conducted using high‐resolution microscopy (i.e., Atomic force microscopy, AFM, and scanning transmission electron microscopy, STEM). AFM was also used to determine the topographical distribution and size of budding vesicles directly on GBM cell surface. Further, the WB identification of EV‐associated markers and the proteomic analysis of isolated EVs will allow to determine the molecular profiles associated with each cell line in presence or absence of TMZ.

Results : The amount and the size of EVs produced under normal conditions varies between the four GBM cell lines and is further affected following treatment with TMZ. The cell surface topographical distribution of budding vesicles and their number was significantly different among the four GBM cells (U87MG>U251MG>U373MG>T98G) and, influenced by TMZ treatment. Specific proteins that can serve as biomarkers of the disease were identified by using proteomic analysis and discriminated the different cell lines in untreated and TMZ‐ treated conditions.

Summary/Conclusion : The combined ultrastructural and proteomic approaches are useful to categorize the four GBM cells‐derived EVs in the attempt to use them as non‐invasive diagnostic tools.

Funding : No external funding

Keywords : Temozolomide, Glioblastoma Multiforme, Atomic force microscopy, Scanning transmission electron microscopy, Ultrafiltration, Size‐exclusion chromatography, Proteomic analysis, molecular fingerprinting, budding vesicles

Effect of X‐ray irradiation on quantity and tetraspanin markers expression of extracellular vesicles (EVs) derived from peripheral blood mononuclear cells (PBMCs)

Zi Huai Chew; Christelle Chua

National University of Singapore, Singapore Nuclear Research and Safety Initiative, Singapore, Singapore

Introduction : EVs are involved in cell‐to‐cell communication after exposure to ionizing radiation (IR), although their exact functions remain to be fully elucidated. PBMCs are known to be particularly sensitive to IR, but it is only more recently that researchers have begun to characterize PBMC‐derived EVs 1. Previous studies have focused mainly on the impact on EV cargo after high doses of radiation typically applied for radiotherapy, and frequently at later time points of more than 24h2 .This study aims to fill in a gap by investigating if X‐ray irradiation at lower doses between 0 to 4 Gy would alter the basic characteristics of PBMC‐derived EVs after 24 hours in culture.

Methods : Whole blood or PBMCs collected from healthy donors were exposed to X‐ray irradiation and cultured in plasma for 24 hours. Plasma was collected and size exclusion chromatography was used to purify and isolate the EVs. Presence of EVs were confirmed by flow cytometry using a newly‐developed water‐soluble membrane dye (developed by the National University of Singapore, GRIP program). Once the presence of EVs were identified, interferometric imaging was used to quantify the difference in particle count and tetraspanin markers (CD9, CD63 and CD81) between irradiated and non‐irradiated samples.

Results : Interferometric imaging analysis revealed that the EVs averaged between 50‐70nm in size. Interestingly, the overall quantity of EVs does not appear to increase significantly, 24 hours after exposure. This is in contrast to expectations that the EV population always increases in response to IR, and suggests that at earlier time points this may not be the case. We also observed that CD9 is generally highly expressed and that the percentage of CD63 expression appears to increase with radiation exposure.

Summary/Conclusion : Our results suggest that exposure of PBMCs to ionizing radiation have differing extent of effects on the quantity of EVs and expression of various tetraspanin markers.

Funding : National Research Foundation (Singapore)

Identification of surface protein markers of human adult astrocyte extracellular vesicles

Keerthanaa B. Balasubramanian Shanthi 1 ; Ravindra Daddali 1 ; Antti Kiviniemi 2 ; Abhishek Sharma 1 ; Ulrich Bergmann 1 ; Seppo Vainio 1 ; Genevieve Bart 1

1 University of Oulu, Oulu, Finland; 2 University of Oulu, oulu, Finland

Introduction : In the frame of MindGap FET‐open project, our aim is to identify markers of successful mindfulness practice in cancer survivors, carried by brain‐derived EVs. To identify suitable surface protein for brain‐derived EV capture from blood we characterized EVs from adult human primary astrocytes in culture. Astrocytes are central nervous system restricted glial cells with a wide range of functions, evolving with brain activity. In the healthy brain they are involved in maintenance of blood brain barrier (BBB), regulation of synaptic function, and regulation of blood flow in the CNS. New discoveries also highlight their potential role in mood and other brain disorders. From our proteomics data, Calsyntenin1 (CLSTN1), a single pass transmembrane protein involved in synapse formation and synaptic plasticity was selected for further analysis.

Methods : Primary human adult astrocytes were grown to 85‐90% confluency, then switched to FBS free collection medium. EVs were prepared using serial low speed centrifugations, 0,8μm filtration and 2 hours 100k ultracentrifugation (EV‐TRACK ID EV220009 ). EV were characterized by NTA, Exoview, transmission electron microscopy and western blot, associated proteins were identified by mass spectrometry.

U87 glioma cell line was transduced with lentivirus encoding human CLSTN1 isoform 2 with C‐terminus EGFP tag, overlayed with rat tail collagen I and imaged by confocal microscopy.

Results : Mass spectrometry identified 194 proteins with high combined FDR, and GO term enrichment for: nervous system development, synapse and extracellular vesicles. CLSTN1 association with astrocyte EVs was confirmed by western blot.

3D confocal imaging of live CLSTN1‐EGFP U87 cells show they secrete fluorescent EV‐like particles.

Summary/Conclusion : Analysis of Astrocyte EVs and fluorescent EVs produced by CLSTN1‐EGFP U87 glioma cells show that CLSTN1 is a potential marker of brain‐derived EVs.

Funding : FET Open MindGap (ID: 829040) Horizon2020‐EU 1.2.

PS12: Late‐Breaking Poster Session 8: Biomarkers II

Chair: Saara Laitinen, PhD – Finnish Red Cross Blood Service

Chair: Antonia Reale, MD – Monash University

Urinary EVs and SEC‐enrichment: a 150‐experience summary

Marta Clos Sansalvador 1 ; Sergio G. Garcia 2 ; Miriam Moron‐Font 3 ; Marcella Franquesa 3 ; Francesc E. Borràs 3

1 1. IVECAT, REMAR‐IGTP Group & Nephrology Service, “Germans Trias i Pujol” Health Science Research Institute & University Hospital, Can Ruti Campus, Badalona, Spain 2. Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain, Badalona, Spain; 2 1 IVECAT, REMAR‐IGTP Group & Nephrology Service, “Germans Trias i Pujol” Health Science Research Institute & University Hospital, Can Ruti Campus, Badalona, Spain, Badalona, Spain; 3 1. IVECAT, REMAR‐IGTP Group & Nephrology Service, “Germans Trias i Pujol” Health Science Research Institute & University Hospital, Can Ruti Campus, Badalona, Spain, Badalona, Spain

Introduction : Urinary extracellular vesicles (uEV) show great potential as a source of non‐invasive biomarkers for chronic kidney disease (CKD). Our lab was pioneer in using size exclusion chromatography (SEC) for uEV enrichment. Here we summarize the experience and main results of more than 150 SEC‐enriched uEV samples.

Methods : Urine samples from healthy controls (n=17), normo‐functional kidney transplanted patients (n=17) or chronic kidney disease (CKD) patients (n=98) had been frozen for several months/years at ‐80°C before enrichment. Thawed samples (20 mL each) were processed for uEV enrichment by SEC and screened for the EV markers CD9 and CD63 by bead‐based flow cytometry. Protein content was measured using either BCA or Nanodrop.

Results : While no differences were observed between the mean fluorescence intensity (MFI) of both CD9 and CD63 in all samples, analyzing the CD9/CD63 ratio in each individual sample clearly segregated two groups. Specifically, the CD9 MFI was increased in one group (G1, n= 69) and decreased in the other (G2, n=85), while CD63 MFI remained more stable among all samples. The reason for this difference is not clear, but it can be attributed neither to the protein content of the sample nor their pathological state. In fact, when comparing healthy controls and CKD patients, a significant reduction on the expression of both EV markers was observed in the pathological samples, maybe reflecting the pathology itself or the chronic treatment.

Technical reproducibility of the enrichment method has been tested in up to 34 urine samples. Each of these were isolated twice at different time points and using two independent home‐made SEC columns. The ratio MFI‐1/MFI‐2 (MFI from first isolation / MFI from second isolation) was 1.059 ± 0.29 and 1.037 ± 0.31 for CD9 and CD63 respectively, highlighting a great level of reproducibility.

Summary/Conclusion : Overall, our data clearly show that SEC is a robust and reproducible uEV isolation technic. In our experience, CD63 MFI seems to be more reliable for uEV labelling in normalization intended studies. Finally, we found the expression of both CD9 and CD63 is decreased in chronic kidney patients compared to healthy controls. An unexplained observation is the detection of two groups of samples based on the CD9/CD63 ratio.

Exovesicle concentration and cargo in patient derived ALS cellular model are modulated upon treatment with TDP‐43 modulators

Carmen Pérez de la Lastra 1 ; Carlota Tosat‐Bitrián 2 ; Alejandro Bueso 3 ; Ana Martinez 2 ; Angeles Martin‐Requero 4 ; Manuel Izquierdo 5 ; Valle Palomo 6

1 Centro de Investigaciones Biológicas Margarita Salas CSIC, Madrid, Spain; 2 Centro de Investigaciones Biológicas Margarita Salas CSIC, Calle Ramiro de Maeztu, 9, 28040 Madrid; CIBERNED Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Calle de Valderrebollo 5, 28031 Madrid, Spain, Madrid, Spain; 3 IMDEA Nanociencia, Calle Faraday 9, Madrid 28049, Spain, Madrid, Spain; 4 Centro de Investigaciones Biológicas Margarita Salas CSIC, Calle Ramiro de Maeztu, 9, 28040 Madrid; CIBERNED Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Calle de Valderrebollo 5, 28031 Madrid, Spain, MADRID, Spain; 5 Departamento de Metabolismo y Señalización Celular, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC‐UAM, 28029 Madrid, Spain, Madrid, Spain; 6 IMDEA Nanociencia, Calle Faraday 9, Madrid 28049, Spain; 3CIBERNED Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Calle de Valderrebollo 5, 28031 Madrid, Spain, MADRID, Spain

Introduction : Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by the degeneration of the upper motor neurons in the brain and lower motor neurons in the spinal cord, which are responsible for voluntary muscle movement. Due to the heterogeneity of the disease and the lack of biomarkers the pharmacological evaluation of new drugs is a challenging task. Current animal models do not reflect the human pathology and diversity. Therefore we utilize a model of lymphoblasts derived from patients to characterize the pathology and its response to treatment. Extracellular vesicles (EVs) have shown to be dysregulated in the disease and here we explore whether pharmacological treatment would alter EVs biogenesis and cargo load in our model. We focused in TDP‐43 modulators since this protein is found aggregated in 97% of sporadic ALS patients and a severe dysregulation has been shown to be present in the disease.

Methods : Lymphoblastic cell lines derived from ALS patients and healthy controls were incubated in RPMI‐1640 media supplemented with 10% exosome depleted FBS and 1% penicillin streptomycin. They were treated with 2 μM CK‐1δ inhibitor IGS‐2.7 for 72 h. EVs were isolated by ultracentrifugation and utilizing Thermofisher Total Exosome Isolation Reagent. EVs were characterized by TEM, WB, nanoparticle tracking analysis, and TDP‐43 content was also analyzed by TR‐FRET. Finally EVs were incubated 2 h with biotinylated antibody anti‐human CD63, followed by a overnight incubation with streptavidin‐conjugated Quantum Dot (QDs) nanoparticles and were analyzed by nanoparticle tracking analysis.

Results : ALS lymphoblasts present a higher concentration of EVs than healthy control lymphoblasts, and it is reduced upon pharmacological treatment. In addition, the cargo loading of the isolated EVs was also modulated after μM CK‐1δ inhibitor IGS‐2.7 treatment considering an equal number of vesicles. TDP‐43 protein was able to be quantified using a TR‐FRET assay showing a correlation with both pathological events that was also modulated upon pharmacological recovery. Lastly, we present a method to characterize our EV samples using fluorescent QDs.

Summary/Conclusion : Patient derived models of sporadic ALS show EVs alteration when compared to healthy controls. This alteration was shown to be modulated upon pharmacological treatment against the most prominent pathological feature of ALS, which involves TDP‐43 phosphorylation.

Keywords : Amyotrophic lateral sclerosis

Are cytokines associated with extracellular vesicles? Investigation of EVs from biofluids and stem cell‐derived astrocytes and microglia

Tanina Arab 1 ; Zhaohao Liao 1 ; Rajini Nagaraj 2 ; Evan Gizzie 3 ; Vasiliki Mahairaki 4 ; David A. Routenberg 3 ; Kenneth W. Witwer 5

1 Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA, Baltimore, USA; 2 MesoScale Diagnostics, LLC, Rockville, MD, USA, Bethesda, USA; 3 MesoScale Diagnostics, LLC, Rockville, MD, USA, Rockville, USA; 4 Department of Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. The Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease, Johns Hopkins University School of Medicine, Baltimore, MD, US., Baltimore, USA; 5 Department of Molecular and Comparative Pathobiology, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA. The Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease, Johns Hopkins University School of Medicine, Baltimore, MD, US, Baltimore, USA

Introduction : Several reports indicate that EVs are associated with cytokines. However, it remains unclear if cytokines are true EV components or co‐isolates. Using an ultrasensitive multiplexed ELISA method, we measured nine cytokines in EV preparations from cerebrospinal fluid (CSF), blood plasma, and cell culture of stem cell‐derived astrocytes and microglia.

Methods : CSF, blood plasma, and saliva were fractionated by differential ultracentrifugation (dUC, including high‐speed pellets and supernatant) and by SEC with 70 nm cutoff and/or 35 nm (CSF only). Induced pluripotent stem cells (iPSCs) were differentiated into astrocytes and microglia and activated or not with LPS. Cell culture EVs were separated by dUC and SEC. EVs were characterized per MISEV2018. Nine cytokines were measured by ultrasensitive multiplexed ELISA (Meso Scale), sensitive to fg/mL levels.

Results : IL‐1beta and IL‐6 were the major detected cytokines in the samples used here and showed large differences in abundance between EV‐depleted and EV‐enriched fractions. Levels in UC pellets and SEC EV fractions were much lower; e.g., 70 nm SEC EV fractions had ∼1.6% on average, across samples, of the level in protein fractions, and 4.0% for 35 nm SEC. Across all sample types, these differences were smallest for plasma fractions. In saliva samples, IL‐1beta was the central detectable cytokine, with least abundance in the high‐speed UC pellet. IL‐6 was quantifiable in all astrocyte releasate fractions, including EVs, and LPS stimulation increased IL‐6 concentration in EV and protein fractions by 7.5 and 2.0‐fold, respectively. In contrast, released very little IL‐6 in EV fractions compared with protein fractions, with no differences under LPS stimulation.

Summary/Conclusion : Ultrasensitive multiplexed ELISA revealed that IL‐6 and IL‐1beta were the main measured cytokines in EV fractions from three human biofluids and several cell types. Some signal was associated with EVs, but at levels much lower than those in EV‐depleted fractions. Protease protection assays are currently underway to determine if these cytokines are located inside or outside EVs.

Funding : This work is supported by The Michael J. Fox Foundation

Keywords : cytokine, extracellular vesicles, IL‐6, IL‐1b, microglia, astrocytes, plasma, CSF,

Extracellular Vesicle‐Enriched miRNA‐Biomarkers Show Improved Utility For Detecting Alzheimer's Disease Dementia and Medial Temporal Atrophy

Gunnar H. Poplawski 1 ; Yuek Ling Chai 2 ; Lea Strohm 2 ; Yanan Zhu 2 ; Rachel Chia 2 ; Joyce Ruifen Chong 2 ; Li Han Zhou 3 ; Heng Phon Too 2 ; Saima Hilal 2 ; Edward Koo 2 ; Christopher Chen 2

1 Duke‐NUS, Singapore, Singapore; 2 National University of Singapore, Singapore, Singapore; 3 MiRXES, Singapore, Singapore

Introduction : It is hypothesized that extracellular vesicles (EVs) protect circulating miRNA‐biomarkers from degradation within the patient blood. To test this hypothesis we compared miRNAs isolated from EVs and from serum from the same AD and control patient cohorts. miRNA profiling revealed a significant diagnostic advantage of utilizing EV‐derived miRNAs.

Methods : A total of 25 patients with AD dementia and 34 controls with no cognitive impairment (NCI) from a Singapore‐based clinical cohort were included in this study. miRNA profiling was performed on both serum and blood‐derived EVs from the same patient cohort, followed by association analyses with AD dementia and brain medial temporal atrophy.

Results : We identified almost twice as many EV‐miRNAs with strong associations and diagnostic ability in detecting AD dementia and medial temporal atrophy, as compared to serum‐miRNAs. We further developed combination panels of serum‐ miRNAs and EV‐miRNAs with improved performance in identifying AD dementia from patient samples.

Summary/Conclusion : These data support the further utilization and development of EVs as carriers for disease predicting biomarkers for neurodegenerative diseases and provide potentially new biomarkers for the diagnosis of AD.

Funding : This work was supported by the National Medical Research Council (NMRC/CSA‐SI/007/2016 to CPC and MKPL; NMRC/CG/M006/2017,

NMRC/CG/013/2013 and NMRC/CG/NUHS/2010 to CPC), OFYIRG19MAY‐0024, The miRNA discovery work was conducted at the NUS ncRNA Core Facility and supported by the joint NUSMed‐MiRXES ncRNA Core Service Grant (2018).

Keywords : Alzheimer's disease, dementia, microRNA, extracellular vesicle, medial temporal atrophy, blood biomarker, diagnosis

The first ToF‐SIMS‐based studies on glycan composition of endothelial cell EVs under normoglycemic and hyperglycemic conditions

Magdalena E. Marzec 1 ; Carina Rząca 2 ; Małgorzata Przybyło 3 ; Ewa Ł Stępień 2

1 Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University in Krakow, Poland, Krakow, Poland; 2 Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland, Kraków, Poland; 3 Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University in Krakow, Poland, Krakow, Poland

Introduction : Secondary ion mass spectrometry with a time‐of‐flight analyzer (ToF‐SIMS) is the method of choice for the analysis of biomolecules in tissues, cells and membranes. It enables the qualitative semi‐native examination without extraction, fixation or marking of target elements with simultaneous high‐resolution 2D imaging. Due to the analyzed mass range, the research objects are mainly amino acids and lipids, e.g. sphingolipids. As glycosylation is not a specific template process, the final glycan formation is determined by the expression of genes and proteins involved in glycan biosynthesis pathways, as well as the availability of sugar donors, to name these major factors. It is scarcely proven whether external sugar concentrations can modify glycoaminoglycans (GAGs) and glycolipids. Here, we explore the GAGs changes by comparative ToF‐SIMS analysis in extracellular vesicles (EVs) derived from TIME cells culture in normoglycemic and hyperglycemic conditions.

Methods : Cells were exposed to normoglycemic and hyperglycemic (HG) conditions at 35 mM glucose. A Low‐Vacuum Filtration (L‐VF) and ultracentrifugation were used to separate different subpopulations of EVs: exosomes and ectosomes . For measurements, EVs were applied in PBS solution on cleaned silicon surfaces. TOF‐SIMS equipped with the Bi3+ gun was used.

Results : Differences in the composition of glycosphingolipids, as well as glycans coupled with ceramide, in EVs were observed in EVs. Culturing cells in HG caused changes in the glycosaminoglycans profile, such as heparin, chondroitin sulphates and heparan in EVs.

Summary/Conclusion : The modified composition of the specific pattern of sugar antigens on the surface of exosomes and exosomes may interfere with basic processes related to cell behaviour, recognition and cell adhesion, thereby disrupting the proper flow of information and physiological intercellular interactions. The examined glycans on the surface of EVs derived from HG cells distinguishes them from normoglycemic EVs and indicate that they could be used to detect, determine the advancement of, among others, diabetes as well as be the target of personalized therapy.

Funding : This work was supported by the National Science Center (NCN), grant OPUS 17 to prof. E. Stępień (No. 2019/33/B/NZ3/01004).

Keywords : ToF‐SIMS; glycan; hyperglycemia; endothelial cell

EV‐miRNA sequencing with IsoSeek for response prediction in patients with refractory Multiple Myeloma

Cristina Gómez‐Martín 1 ; E.E.E. Drees 2 ; M.A.J. van Eijndhoven 3 ; Nils Groenewegen 2 ; Ernesto Aparicio‐Puerta 4 ; E.J. Bradley 1 ; Leontien Bosch 3 ; C. Scheepbouwer 1 ; Karin Oudshoorn 5 ; Johan de Rooij 6 ; Michael Hackenberg 7 ; Niels van der Donk 8 ; Dirk M. Pegtel 3

1 Amsterdam University Medical Centers, Amsterdam, Netherlands; 2 amsterdam university medical centers, amsterdam, Netherlands; 3 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC location Vrije Universiteit, Amsterdam, The Netherlands, Amsterdam, Netherlands; 4 Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany; 5 Department of Health Technology and Services Research, University of Twente, Twente, Netherlands; 6 ExBiome B.V., Amsterdam, Netherlands; 7 Department of Genetics, University of Granada, Granada, Spain; 8 Department of Hematology, Amsterdam University Medical Centers, Amsterdam, Netherlands

Introduction : Patients diagnosed with Multiple Myeloma as incurable cancer of the bone marrow, have many treatment options but personalizing treatment is difficult. The only current method to assess response to therapy in clinical routine is measuring M‐protein levels in either the blood or urine, despite a suboptimal specificity and sensitivity. It is well known that miRNAs are shed by cancer cells and their microenvironment into the circulation. MiRNA profiling with sequencing is the preferred method but challenges for their faithful detection have hampered implementation in routine diagnostics. Especially when input is limited ligation/amplification bias and generation of artificial variants (isomiRs) may occur. We developed an unbiased, UMI‐informed and low‐input optimized IsoSeek protocol that measures miRNAs with single‐nucleotide accuracy in EV‐enriched plasma fractions and applied this method to predict response to therapy in MM patients.

Methods : We developed ‘IsoSeek’ a method that diverges from prior protocols combining randomized adapters and 10N unique molecular identifiers. Upon exhaustive rounds of optimization, we show extensive isomiR detection and strongly reduced PCR‐bias compared to commercial protocols. We sequenced 71 samples from MM patients undergoing 2 types of treatment with the IsoSeek method. Lasso regression models (glmnet R package) were built with the data to obtain prognostic miRNA signatures to distinguish responders from non‐responders.

Results : We found that a 9 miRNA model can discriminate MM patients with progressive disease from those responding to treatment (Based on M protein the current gold standard) with an AUC of 0.89 when tested in an independent dataset. More complex signatures with 179‐499 features using canonical miRNA sequences only or 3’end isomiRs (non‐templated additions), enhanced the AUC to 0.91 (0.8‐1.00) and 0.92 (0.82‐1.00) respectively. These data are currently being validated in plasma EVs from an independent MM cohort.

Summary/Conclusion : We present here a tentative model based on 9 miRNAs that can be used to predict the response to treatment in Multiple Myeloma patients with high sensitivity and specificity and that has been validated in an independent test dataset. IsoSeek advances our knowledge of miRNA diversity and supports the development of small RNA sequencing as a robust platform for liquid biopsy.

Funding : AQrate, KWF‐5510, CCA2021‐9‐77, Amgen

Keywords : miRNAs, biomarkers, Multiple Myeloma

Estrogen deficiency caused by menopause blunts acute exercise‐induced circulating microRNA response in extracellular vesicles

Sira M. Karvinen 1 ; Tia‐Marje Korhonen 1 ; Tero Sievänen 1 ; Jari Karppinen 1 ; Hanna‐Kaarina Juppi 1 ; Veera Jakoaho 1 ; Urho Kujala 2 ; Jari Laukkanen 3 ; Maarit Lehti 2 ; Eija Laakkonen 4

1 Gerontology Research Center and Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland; 2 Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland; 3 Institute of Clinical Medicine, University of Eastern Finland, Jyväskylä, Finland; 4 Gerontology Research Center and Faculty of Sport and Health Sciences, University of Jyväskylä,, Jyväskylä, Finland

Introduction : Menopausal decrease in circulating estrogen levels affects metabolic health. However, the detailed mechanisms underlying this process remain unclear. Both estrogens and exercise improve metabolic health, which may be partly mediated by small non‐coding RNAs, such as microRNAs (miRs). In recent years, extracellular vesicles (EVs) have attracted interest in the field of tissue crosstalk. However, in many studies on EVs, the co‐isolation of high‐density lipoprotein (HDL) particles has not been considered. This study investigated the role of EVs as estrogen and exercise‐responsive small RNA (sRNA) carriers.

Methods : We utilized plasma samples from a subset of the Estrogen, microRNAs and the risk of metabolic dysfunction (EsmiRs) study consisting of 14 postmenopausal women. Of them, seven used estrogen‐based hormonal therapy (HT) and seven were nonusers. Groups were matched for age, peak aerobic capacity (VO2peak), body mass index, and body fat percentage. Subjects performed a maximal incremental VO2peak test on a bicycle ergometer and had plasma samples harvested PRE, POST and 1 h POST exercise test. EVs from plasma were isolated using sequential ultracentrifugation followed by size‐exclusion chromatography to separate HDL particles from EVs. We carried out a Next‐Generation Sequencing (NGS) analysis for the EV samples. The resulting sequences were categorized by sRNA origin and differential expression analysis was conducted by DESeq2.

Results : When examining HT users and nonusers together, we observed an increase in the relative abundance of miRs (+3.1% points) and transfer RNA‐derived sRNAs (+2.1% points), while the relative amount of ribosomal RNA‐derived sRNAs decreased (‐5.0% points) immediately after exercise (POST‐PRE). When investigating the differentially expressed sRNAs, miRs 191‐5p, 223‐3p, and 146b‐5p were significantly upregulated in HT users immediately after exercise (POST vs. PRE, p< 0.05). In addition, miR‐486‐5p was upregulated and miR‐340‐5p downregulated in HT users 1h POST vs. POST (p< 0.05). There were no significant findings in the studied time points in nonusers.

Summary/Conclusion : Our results highlight that the estrogen deficiency caused by menopause diminishes the acute exercise‐induced miR‐response in EVs.

Funding : This study was funded by the Academy of Finland (grants 332946, 275323, 309504, 314181, and 335249).

Electrically evoked contractions in the C2C12 myotubes increase miR‐1‐3p packing into extracellular vesicles

Juulia H. Lautaoja 1 ; Sira M. Karvinen 2 ; Juha J. Hulmi 1 ; Satu Pekkala 1

1 Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland; 2 Gerontology Research Center and Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland

Introduction : Exercise‐induced health benefits are thought to be mediated partly via skeletal muscle‐derived signaling molecules, which are carried via extracellular vesicles (EVs). However, only in vitro approaches enable exclusive examination of the EVs and their cargo originating from the skeletal muscle. Myotube contractions evoked by exercise‐like electric pulse stimulation (EL‐EPS) in vitro induce many key physiological responses both intra‐ and extracellularly as in vivo exercise. This model can be used to verify whether the contracting skeletal muscle cells release specific micros‐RNAs (miRs) within the EVs as has been shown after in vivo exercise.

Methods : The 24‐hour low‐frequency EL‐EPS (1 Hz, 2 ms, 12 V) was applied on the murine C2C12 myotubes under serum‐free conditions via carbon electrodes attached to C‐Pace EM stimulator (Ionoptix Corporation). The electrodes were placed on all plates to exclude possible electrode‐mediated effects. The media were collected immediately after EL‐EPS and the EVs were isolated by using exoRNeasy serum/plasma kit (Qiagen). Nanoparticle tracking analysis (NTA) and electron microscopy (EM) were used to validate successful EV isolation. Real‐time qPCR was used to analyze the EV miR content.

Results : Based on the NTA and EM analyses, myotube‐derived EVs were successfully isolated and of expected size (∼100 nm). The content of miR‐1‐3p increased in the EVs after EL‐EPS (P < 0.05), while miR‐133a‐3p also increased by several fold, but due to high variation, this result was non‐significant (P = 0.242).

Summary/Conclusion : In agreement with the in vivo exercise studies, we showed that myotube contractions increased the packing of miR‐1‐3p, and possibly miR‐133a‐3p, into the released EVs. To the best of our knowledge, this is the first study to examine in vitro the contraction‐induced effects on the EV miR content released by the myotubes.

Funding : The Academy of Finland (grant no. 332946 to S.K. and 308042 to S.P.) and the Finnish Cultural Foundation (to J.H.L) funded this work.

Keywords : Exercise, skeletal muscle, myotube, contraction, microRNA

Proteomic profiling of small extracellular vesicles from head and neck squamous cell carcinoma cells uncovers signatures for tumor hypoxia and angiogenesis

Alicja Głuszko 1 ; Mirosław J. Szczepański 1 ; Theresa L. Whiteside 2 ; Torsten E. Reichert 3 ; Jacek Siewiera 4 ; Nils Ludwig 5

1 Chair and Department of Biochemistry, Medical University of Warsaw, 1 Banacha St., 02‐097 Warsaw, Poland, Warsaw, Poland; 2 UPMC Hillman Cancer Center, University of Pittsburgh, Cancer Institute Pittsburgh, Pennsylvania, USA, Pittsburgh, USA; 3 Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, 93053 Regensburg, Germany, Regensburg, Germany; 4 Department of Hyperbaric Medicine, Military Institute of Medicine, 04‐141 Warsaw, Poland, Warsaw, Poland; 5 Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, 93053 Regensburg, Germany, Regensburg, Poland

Introduction : Molecular and functional alterations in head and neck squamous cell carcinomas (HNSCCs) are a result of commonly observed tissue hypoxia and it is well known that exposure to hypoxic conditions triggers the formation of pathological vasculature, which ultimately compromises patient outcome.

Objectives : The aim of this study was to characterize the influence of hypoxic vs. normoxic conditions on the release and proteomic content of tumor‐derived small extracellular vesicles (sEVs).

Methods : Material & Methods: HNSCC cells (FaDu, PCI‐30, SCC‐25) and HaCaT keratinocytes were exposed to 21, 10, 5, 1% oxygen supply. sEVs were isolated from supernatants using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis, electron microscopy, immunoblotting, and high‐resolution mass spectrometry.

Results : Results: Isolated sEVs ranged in size from 125–135 nm and carried CD63 and CD9 but not Grp94. HNSCC cells adapted to hypoxic conditions with increased release of sEVs, which were enriched in unique protein profiles. The hypoxic profile of HNSCC parent cells was reflected in the proteome cargo of sEVs: about 15% of the total detected proteins in sEVs were unique for hypoxic cells. Seven hypoxia‐related proteins (KT33B, DYSF, STON2, MLX, LIPA3, NEK5, P12L1) represented a common signature of hypoxic sEVs, which were also enriched in pro‐angiogenic proteins.

Summary/Conclusion : Conclusion: The degree of tumor hypoxia was reflected in protein profiles of sEVs, therefore, sEVs could potentially serve as clinical biomarkers for hypoxic conditions.

Funding : This work was partially funded from the National Science Centre, Poland UMO‐2017/26/M/NZ5/00877# to M.J.S. and from Medical University of Warsaw MB/M/48(79)# to A.G., T.L.W. was supported in part by the NIH grant U0‐1 DE 029759. N.L. was supported by the Walter Schulz Foundation.