Medicine · 2024

Proceedings of the CIMAGO Congress - Environment, Genetics and Oncobiology in the 21st Century - Challenges and Opportunities.

Название и аннотация: Перевод в очереди

Обработка статьи · этапы, ошибки и управление

Загрузка состояния…

История · было → стало

Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.

Аннотация

Аннотация не предоставлена источником.

Перевод полного текста: Перевод в очереди

Читать найденный полный текст

https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC) , where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

ORAL COMMUNICATIONS

Coimbra, 25 th and 26th January 2024

CO-1-Can tumor educated platelets identify and predict survival of multiple myeloma patients?

Joana Jorge 1,2,3 , Ana Cristina Gonçalves 1,2,3 , Alberto Berral-Gonzalez 4 , Raquel Alves 1,2,3 , Javier De Las Rivas 4 , Ana Bela Sarmento Ribeiro 1,3,5

1 Laboratory of Oncobiology and Hematology (LOH) and University Clinics of Hematology and Oncology, Coimbra Institute for Clinical and Biomedical Research (iCBR) – Group of Environment, Genetics and Oncobiology (CIMAGO), FMUC, University of Coimbra, Coimbra, Portugal; 2 Center for Innovative Biomedicine and Biotecnology (CIBB), Coimbra, Portugal; 3 Clinical Academic Centre of Coimbra (CACC), Coimbra, Portugal; 4 Bioinformatics and Functional Genomics Group, Cancer Research Center (CiC-IBMCC, CSIC/USAL), Consejo Superior de Investigaciones Cientificas (CSIC) and University of Salamanca (USAL), Salamanca, Spain; 5 Hematology Service, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal.

Context/aims: Tumor-educated platelets (TEPs) have emerged as a promising biosource of peripheral specific biomarkers for the detection of various cancer types. However, its role in hematological neoplasias is still poorly understood. Multiple myeloma (MM) is a clonal plasma cell disorder genetically complex, with a high biological and clinical heterogeneity. MM diagnosis and monitoring is highly dependent on bone marrow aspirate/biopsy, an invasive and painful procedure, that does not capture the clinical and spatial heterogeneity of this disease. Our goal was to identify a MM TEP gene expression signature and correlate it with MM prognosis and survival.

Methods: Two different available databases were used. The GSE183635 that includes platelet RNA-seq data of 22 MM patients and 183 asymptomatic healthy donors (aHD); and the GSE24080 with gene expression microarray data of CD138+ cells and survival data of 559 MM patients. All the analyses were performed using R and R Studio (version 2023.06.1) and different R packages (GEOquery, limmaVoom, edgeR, ggplot2, etc).

Results: After a rigorous bioinformatic analysis we identified 223 differentially expressed genes between platelets from MM patients and aHD. Nineteen genes presented greater statistical significance with p-value <0.000001 and a fold change >2. Among these, 15 genes ( HBD, CA1, GZMH, IFI27, LY6E, ITGB7, ITGAL, NKG7, RAD23A, NADSYN1, DUSP2, PAXX, GZMB, TCIRG1 , and OSBPL5 ) were upregulated and 4 genes ( WDR11-DT, ZNF385D, GRHL1 , and HSD17B3 ) were downregulated. The prognostic value of these 223 gene MM TEP signature was further explored. Ninety-seven genes were significantly correlated with patient survival (p<0.05). Moreover, 11 of these genes ( EFHD2, CMTM6, ARPC5L, TNNC2, GSTP1, RASAL3, GAK, ANGPT1, DNAJC7, PLAAT4 and TLR5 ) presented a higher impact on MM prognosis with p<0.001 and hazard ratios over 1.5 (1.636±0.119).

Conclusions: These results demonstrated a significantly different transcriptomic profile between TEPs of MM patients and aHD platelets. This newly identified MM TEP signature can possibly be used as a new screening test and prognostic biomarker in MM patients.

CO-2-Therapeutic efficacy of PARP and CHK1 inhibition in combination with cytarabine in acute myeloblastic leukemia: preliminary study in vitro

Beatriz Santos Lapa 1,2 , Maria Inês Costa 1,2 , Joana Jorge 1,2 , Raquel Alves 1,2 , Ana Bela Sarmento-Ribeiro 1,2,3 , Ana Cristina Gonçalves 1,2

1 Laboratory of Oncobiology and Hematology (LOH) and University Clinics of Hematology and Oncology, Coimbra Institute for Clinical and Biomedical Research (iCBR) – Group of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra (FMUC), Coimbra, Portugal; 2 Center for Innovative Biomedicine and Biotecnology (CIBB) and Clinical Academic Centre of Coimbra (CACC), Coimbra, Portugal; 3 Service of Hematology, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal.

Context/aims: Genome integrity is crucial for cell survival, but it faces threats from various sources, triggering the DNA damage response (DDR). Essential elements of the DDR comprise sensor proteins (e.g., PARP1/2), mediators and signaling transducers, and effector molecules (e.g., CHK1/2). Increased DNA damage and altered DDR are linked to genetic instability, which presumably, may be involved in the development of acute myeloblastic leukemia (AML). This work aimed to assess the therapeutic potential of CCT245737 (CCT; CHK1 inhibitor) and niraparib (Nir; PARP1/2 inhibitor) in monotherapy and combination in AML, using in vitro model.

Methods: The THP-1 AML cell line was incubated with increasing concentrations of CCT and Nir, both alone and in combination with each other or with cytarabine. Cell density and viability were assessed using the trypan blue assay for 72h. Flow cytometry was employed to analyze the cell cycle, through propidium iodide/RNAse staining, and the cell death, using Annexin V/7-AAD double staining. The results were statistically analyzed, considering a significance level of 95%.

Results: CCT and Nir, in monotherapy, induced a dose and time-dependent reduction in cell proliferation and viability. In THP-1 cells the estimated IC 50 for CCT and Nir at 72h was 45µM and 24µM, respectively. The drugs in combination also exhibited a dose and time-dependent decrease in cell proliferation and viability and showed an enhanced cytostatic and cytotoxic effect at lower doses. After 72 hours of treatment with 2.5µM of CCT and Nir or 2.5µM of CCT and 0.1µM of cytarabine, cell viability decreased by 90% and 95%, respectively. Moreover, 2.5µM of Nir and 0.5µM of cytarabine resulted in a viability decrease of 50%. Treatment with the inhibitors not only increased the percentage of cells in apoptosis but also induced a cytostatic effect by causing cell cycle arrest in the G 2 /M phase.

Conclusions: CCT and Nir, in monotherapy or combination, reduced cell proliferation and viability in THP-1 cells. The synergistic effects, even at lower doses, and the amplifying impact of cytarabine suggest promising paths for developing new effective strategies in AML therapeutics

CO-3-Preliminary results of SNP Array in molecular stratification of pediatric Acute Lymphoblastic Leukemias

Luís Pires 1 , Pedro Veiga 1 , Mariana Val 1 , Susana Ferreira 1 , Paula Gameiro 2 , Maria João Martins 2 , Margarida Coucelo 3 , Sónia Silva 4 , Manuel Brito 4 , Ximo Duarte 5 , Joana Melo 1,6,7 , Isabel Carreira 1,6,7

1 Laboratório de Citogenética e Genómica – Faculdade de Medicina da Universidade de Coimbra, Portugal; 2 Laboratório de Hemato-Oncologia – Instituto Português de Oncologia de Lisboa, Portugal; 3 Unidade Funcional de Hematologia Molecular, Centro Hospitalar e Universitário de Coimbra EPE, Hospital Pediátrico, Coimbra, Portugal; 4 Oncologia Pediátrica, Centro Hospitalar e Universitário de Coimbra EPE, Hospital Pediátrico, Coimbra, Portugal; 5 Serviço de Pediatria e do Adolescente – Instituto Português de Oncologia de Lisboa, Portugal; 6 CIBB- Centro de Inovação em Biomedicina e Biotecnologia, Universidade de Coimbra, Portugal; 7 iCBR CIMAGO – Centro Investigação em Meio Ambiente Genética e Oncobiologia, Faculdade de Medicina da Universidade de Coimbra, Portugal

Context/aims: Acute Lymphoblastic Leukemia (ALL) is a malignancy characterized by uncontrolled proliferation of abnormal lymphocytes. It stands as the most prevalent malignancy among children, characterized by various genetic alterations with an impact on survival rate, prognosis, and therapy response.

A therapeutic algorithm called ALLTogether was developed to improve survival and therapeutic response in ALL. Young people have excellent results, with survival rates reaching 90% in children and 75% in young adults. Array Comparative Genomic Hybridization combined with Single Nucleotide Polymorphism array (SNPa) plays a key role in ALLTogether, identifying ploidy, whole-genome alterations, and isodisomy. SNPa also allows the detection of specific genetic alterations, such as deletions involving key genes BTG1, CDKN2A/B, EBF1, ETV6, IKZF1, PAX5, RB1, CRLF2 , and iAMP21.

In this study, we present the preliminary results of genetic alterations detected in a group of ALL patients characterized by SNPa, which is used for risk stratification.

Methods: A cohort of 103 ALL samples were analyzed by SNPa. The analysis included the evaluation of ploidy, whole-genome alterations, and loss of heterozygosity (LOH) regions affecting ALLTogether critical genes.

Results: In our study, 67% of the samples analyzed were diploid, 26% were hyperdiploid, and 6% were hypodiploid (poor prognosis). It is worth highlighting a greater frequency of ploidy changes in males. The most frequent genetic alteration was the homozygous deletion of the CDKN2A/B gene, followed by deletions in ETV6 and PAX5 . Relevant copy number variations and relevant alterations were found in every chromossome. We detected only one case with iAMP21. LOH was more significant in chromosomes 2, 5, 6, 9, 11 and 16.

Conclusions: Risk stratification is essential to improve the prognosis of pediatric ALL. SNPa is critical in identifying specific genetic alterations in ALL, influencing survival rates and therapeutic response. Integrating diverse techniques and areas of expertise in healthcare benefits the patient by allowing healthcare professionals to make accurate decisions that optimize ALL treatment approaches.

CO-4-Pre-metastatic lung fibroblasts boost the metastatic potential of osteosarcoma cells

Gabriela Sampaio-Ribeiro 1,2 , Sara Almeida 1,3 , Célia Gomes 1,2

1 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 2 Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; 3 Institute for Nuclear Sciences Applied to Health (ICNAS) and Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, Portugal

Context/aims: Lung metastasis is the leading cause of osteosarcoma (OS)-related death. It has been shown that primary tumors evoke phenotypic changes in lung fibroblasts to form a permissive pre-metastatic niche (PMN), but remains unclear the reciprocal effect on tumor cells. Herein, we aimed to explore how activated resident lung fibroblasts impact OS cell behavior.

Methods: Fibroblasts were isolated from fresh lungs of mice bearing a primary OS or pretreated with the secretome of 143B OS cells and assayed for activation markers and inflammatory cytokines. The 143B OS cells were then incubated with the fibroblast-derived secretome at different time points, to assess how it imparts to OS cells the ability to resist chemotherapy, activation of stemness and dormancy states, and adhesion properties.

Results: Lung fibroblasts from mice with a primary OS or treated with 143B secretome displayed an activation phenotype with expression of α-SMA, fibronectin, FAP, and PDGFR and of pro-inflammatory cytokines. After exposure to the fibroblast secretome, OS 143B cells became more resistant to doxorubicin and enriched in stemness properties namely increased SOX2 and KLF4 expression. At 24h, 143B cells entered into a dormancy-like state, as evidenced by increased NR2F1 expression and p38 high /pERK low ratio that progressively switched to a p38 low /pERK high proliferative phenotype. Moreover, 143B cells upregulate the expression of the adhesion receptor integrin-β1, which favored the adhesion of OS cells to the extracellular matrix fibronectin.

Conclusions: These data demonstrated the reciprocal paracrine interaction between OS cells and fibroblasts at the pulmonary PMN, and how it cooperates to enhance OS cell fitness for lung metastasis formation.

CO-5-Dedifferentiated gingival fibroblasts present stem-like properties and multilineage-differentiation capacity

Carlos Miguel Marto 1,2,3 , Mafalda Laranjo 2,3,4 , Ana Cristina Gonçalves 2,4,5 , Anabela Paula 2,4,6 , Joana Jorge 2,4,5 , Ana Bela Sarmento-Ribeiro 2,4,5 , Manuel Marques-Ferreira 2,4,7 , Bárbara Oliveiros 2,4,8 , António Cabrita 1 , Maria Filomena Botelho 2,3,4 , Eunice Carrilho 2,4,6

1 Univ Coimbra, Institute of Experimental Pathology, Faculty of Medicine, Coimbra, Portugal; 2 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 3 Univ Coimbra, Institute of Biophysics, Faculty of Medicine, Coimbra, Portugal; 4 Clinical Academic Centre of Coimbra (CACC), Coimbra, Portugal; 5 Univ Coimbra, Laboratory of Oncobiology and Hematology (LOH) and University Clinic of Hematology, Faculty of Medicine, Coimbra, Portugal; 6 Univ Coimbra, Institute of Integrated Clinical Practice and Laboratory of Evidence-Based and Precision Dentistry, Faculty of Medicine, Coimbra, Portugal; 7 Univ Coimbra, Institute of Endodontics, Faculty of Medicine, Coimbra, Portugal; 8 Univ Coimbra, Laboratory of Biostatistics and Medical Informatics, Faculty of Medicine, Coimbra, Portugal.

Context/aims: Using synthetic materials to replace lost or damaged oral tissues is the main approach in modern dentistry. However, this does not allow proper tissue regeneration or the recovery of all lost functions. To surpass such limitations, regenerative dentistry (RD) uses stem cells to regenerate lost or damaged tissues. Although several sources of oral stem cells exist, all present relevant limitations, so new sources of stem cells are fundamental. Gingival fibroblasts (GFs) are easy to collect and expand from patients. This work explores the potential of GFs as a novel source of stem-like cells to be used in RD.

Methods: Mouse GFs were dedifferentiated using reversine. The expression of key markers CD11b, CD45, CD90, CD105, CD106, and telomerase reverse transcriptase (TERT), was evaluated by flow cytometry. The alkaline phosphatase (ALP) gene expression was determined by RT-PCR, and the protein expression by live immunofluorescence. Oct-3/4 and Nanog protein expression was evaluated by western blot and the DNA methylation using an ELISA kit. Following dedifferentiation, the cells were cultured in a specific differentiation medium, and osteogenic, adipogenic, and chondrogenic differentiation were evaluated through staining and subsequent quantification.

Results: The dedifferentiation protocol significantly increased mesenchymal stem cell markers CD90 and CD145, while CD11b and CD45 expression remained undetectable. Moreover, CD106-positive cells increased, corresponding to a sub-population with stem properties. Although no changes were detected in ALP gene and protein expression, Nanog protein expression, and DNA methylation, a significant increase in TERT expression was seen. Dedifferentiated cells successfully underwent osteogenic, adipogenic, and chondrogenic differentiation, verified from the 7th day of culture. The osteogenic differentiation increased from the 7th to the 14th day of culture.

Conclusions: This study demonstrates the successful dedifferentiation of GFs into stem-like cells with trilineage differentiation potential. The dedifferentiation process induced genetic and functional changes, enhancing the stem cell properties of the cells.

CO-6-a-C:H(:N) coatings for orthodontic applications: corrosion resistance and in vitro biocompatibility

António Fróis 1,2,3 , Cristina Santos Louro 1 , Ana Cristina Santos 1,2,3

1 CEMMPRE, ARISE, Department of Mechanical Engineering, Faculty of Sciences and Technology, University of Coimbra, Portugal; 2 Coimbra Institute for Clinical and Biomedical Research/Centre for Innovative Biomedicine and Biotechnology (iCBR/CIBB), Biophysics Institute, Faculty of Medicine, University of Coimbra, Portugal; 3 Area of Environment Genetics and Oncobiology (CIMAGO), University of Coimbra, Portugal.

Context/aims: Biometallic alloys thrive in fixed orthodontics for their unmatched balance of mechanical, corrosion resistance and biocompatibility properties. Nitinol, Ti-based alloys, and several stainless steel (SS) grades are widely used for manufacturing fixed appliances that will remain inside the oral cavity for approximately 2 years. However, intraoral environment is highly dynamic and aggressive for any biomaterial, inevitably leading to the in vivo degradation of metals and alloys, and consequently to the release of metallic ions. Some ions are toxic and can elicit hypersensitive reactions (allergies), such as Nickel (Ni).

This study aims to develop carbon-based protective coatings to improve the overall corrosion resistance and biocompatibility of metallic alloys used in Orthodontics, namely the medical-grade SS 316L (AISI).

Methods: Hydrogenated amorphous carbon (a-C:H) coatings were deposited on SS 316L substrates by reactive magnetron sputtering. Variable CH 4 /Ar flow ratios provided different H contents, while N 2 inflow allowed doping with N. Samples were immersed for 30 days in a modified Fusayama-Meyer artificial saliva (pH 2.3). Released Ni, Cr and Fe ions were quantified by Inductively Coupled Plasma Optical Emission Spectrometry. Fibroblasts and macrophages (mono- and co-cultures) were incubated with corrosion eluates to assess cell viability through the MTT colorimetric test. Statistical analysis was performed with the R Studio software.

Results: The results revealed that coatings were able to reduce metallic ions release by up to ~41% when compared to the uncoated samples. Moreover, statistically significant increases in cell viability for coated samples were obtained for both mono- and co-cultures.

Conclusions: This study indicates that a-C:H-based coatings have the potential to reduce metal release by protecting the substrates against corrosion. The overall in vitro cell viability of SS 316L substrates could be improved. The a-C:H-based coatings are, therefore, promising candidates for enhancing the biocompatibility of orthodontic alloys. Animal models are required to confirm this protective behaviour in vivo .

Acknowledgements: This work was funded by Foundation for Science and Technology (FCT), Portugal (SFRH/BD/143905/2019 grant, and UIDB/00285/2020 and LA/P/0112/2020 projects); COMPETE-FEDER-POCI programs CENTRO-01-0247-FEDER-039880 and PTDC/EMD-EMD/21402020. A special thank you to Centro Hospitalar da Universidade de Coimbra (CHUC), to iCBR-FMUC Vivarium , to the Biophysics Institute of FMUC, to University of Aveiro, and to RI.TE.

CO-7-Characterization of a human amniotic membrane extract and its fractions: a proteomic study

Ricardo Jorge Teixo 1,2,3 , Susana Aveiro 4,5 , Beatriz Serambeque 1,2,3 , Mafalda Laranjo 1,2,3 , Maria João Carvalho 1,2,6 , Ana Margarida Abrantes 1,2,3 , Maria Filomena Botelho 1,2,3 , Pedro Domingues 4

3 Clinical Academic Center of Coimbra, Coimbra, Portugal; 4 Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, Aveiro, Portugal; 5 GreenCoLab - Green Ocean Association, University of Algarve, Faro, Portugal; 6 Gynecology Service, Coimbra Hospital and University Centre, Coimbra, Portugal.

Context/aims: Human amniotic membrane (hAM) presents anti-tumor properties, such as anti-angiogenic and pro-apoptotic activity. We previously showed that total hAM extract (hAME) and its fractions obtained by solubility fractionation leads to decreased viability on hepatocellular carcinoma (HCC) cells. Thus, we aimed to characterize the hAME and fractions proteomic profile.

Methods: Fractionation of hAME was performed through ammonium sulphate (AS) precipitation by sequentially adding 10, 25 and 50% AS to hAME, on ice, 15min, centrifuged at 14000 G, 15min. Soluble fractions (10S/25S/50S) were submitted to salting out with PBS by centrifugation (4000G, 60 min, VivaSpin® tubes, 30kDa cutoff).

SDS-PAGE electrophoresis was performed with 30μg of protein of hAME, 10S, 25S and 50S in a 12.5% acrylamide gel, resolved at 150V. Gel was stained with 0.12% commassie blue G-250 solution. In gel digestion was performed and identification of proteomic profile was determined by mass spectrometry (MS) and subsequent bioinformatics analyses.

Results: We identified 1295 proteins with at least 2 peptides by analysis of whole gel lanes by MS. PCA analysis showed a clustering of proteomic data from hAME and 50S fraction, and no differences on proteomic data from 10S and 25S fractions.

The heatmap represents the 50 most abundant proteins and relative abundance among hAME, 10S, 25S and 50S samples. These proteins cover a wide range of cellular functions.

Comparing relative abundance between hAME and fractions, we identified 16 proteins at 10S, 5 proteins at 25S and 355 proteins at 50S with lower relative abundance. There was 1 protein with higher relative abundance at fractions 10S and 50S comparing to hAME.

Conclusions: We successfully determine the proteomic profile of hAME and fractions. Lower relative abundance of proteins in fractions are due to lower protein solubility in ammonium sulphate.

CO-8-Genomic Landscape of Glioblastoma Multiforme: Molecular characterization of tumor samples by aCGH

Pedro Veiga 1 , Domingos Roda 2 , Alexandra Mascarenhas 1 , Francisco Caramelo 3,4 , Clara Romero 5 , Joaquim Correia 5 , Pedro Abreu 5 , Pedro Teles 5 , José Luís Alves 6 , Joana Barbosa Melo 1,4,7 , Isabel Marques Carreira 1,4,7 , Ilda Patrícia Ribeiro 1,4,7

1 University of Coimbra, Cytogenetics and Genomics Laboratory, Institute of Cellular and Molecular Biology, Faculty of Medicine, Coimbra, Portugal.

2 Algarve Radiation Oncology Unit – Joaquim Chaves Saúde (JCS), Faro, Portugal.

3 Laboratory of Biostatistics and Medical Informatics, iCBR - Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

4 University of Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) and Center of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, Coimbra, Portugal.

5 Centro Hospitalar e Universitário do Algarve, CHUA, Serviço de Neurocirurgia, Faro, Portugal.

6 Centro Hospitalar e Universitário de Coimbra, CHUC, EPE, Serviço de Neurocirurgia, Coimbra, Portugal.

7 University of Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal and Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal.

Context/aims: Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor, originating from glial cells, with a survival rate of only 15 months after diagnosis. The molecular pathogenesis of glioblastoma multiforme is complex and involves multiple alterations in various genes, cell signalling pathways, and genome regulatory elements. Therefore, analysis of the molecular profile of the tumor could allow a better stratification of patients, improving treatment outcomes and contributing to a better management of the disease. The main objective of this work is to characterize the genomic profile of glioblastoma by Array Comparative Genomic Hybridization (aCGH), using tumor biopsy samples.

Methods: Tissue samples collected from the biopsy procedure of 45 patients were subjected to DNA extraction, followed by analysis using aCGH.

Results: Following aCGH analysis of the molecular profile of 35 of the 45 glioblastoma samples, the landscape of alterations consisted of trisomies and monosomies affecting chromosomes 1, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17,20, 21, 22, and Y. Trisomy of chromosome 7 and monosomy of chromosome 10 were identified as the most prevalent alterations. The genomic profile was further characterized by deletions and duplications in specific regions, notably involving tumor suppressor genes and oncogenes. Most of the patients from this cohort exhibited duplications involving the EGFR and MET genes and deletions of CDKN2A, PTEN, RB1 and NF1 were also relevant. These genes are related to several cell signaling pathways whose dysregulation is associated with the development and progression of the disease. Our findings are consistent with those reported in the literature.

Conclusions: Our findings showed the heterogeneous landscape of chromosomal alterations in glioblastoma. The identification of recurrent genomic alterations provides a foundation for further research where we aim to establish genotype-phenotype correlations. Integration of these results with clinical data and findings from other omics methodologies will also contribute to enriching our comprehensive understanding of glioblastoma pathogenesis.

CO-9-Ectonucleotidases CD39 and CD73 in T cells: a study in the peripheral blood of bladder cancer patients

Frederico Furriel 1,2 , Margarida Pereira 3 , Ana Lúcia Santos 4 , Sandra Silva 4 , Isabel Silva 4 , Paula Laranjeira 4 , Hugo Ferreira 3 , Gabriela Sampaio Ribeiro 3 , Vítor Sousa 5 , Célia Gomes 3 , Artur Paiva 4 , Belmiro Parada 1,6

1 Faculty of Medicine of University of Coimbra, Portugal; 2 Leiria Hospital Center (CHL), Leiria, Portugal; 3 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 4 Unit of Operational Management of Cytometry, Coimbra Hospital and University Centre (CHUC), Coimbra, Portugal; 5 Department of Pathology, Centro Hospitalar e Universitário de Coimbra, Portugal; 6 Urology and Renal Transplantation Department, Centro Hospitalar e Universitário de Coimbra, Portugal.

Context/aims: Emerging evidence demonstrates the role of the adenosinergic pathway (AP) as an immunosuppressive mechanism playing a pivotal role in cancer.

In this study, we aim to quantify the expression of two components of the AP, the cell surface ectonucleotidases CD39 and CD73, on different subpopulations of T cells (CD4+, CD8+, regulatory T cells [Treg] and Gamma-Delta [GD]) in peripheral blood of bladder cancer (BC) patients. This is part of study aiming to trace an immunologically based signature of the AP in BC.

Methods: We conducted a study with 39 sequential patients with urothelial carcinoma of the bladder who were programmed for surgery. Peripheral blood was collected before surgery, and the CD39 and CD73 frequency and expression on T cells were evaluated by flow cytometry with a FACSCanto™ II cytometer. Results were compared with a control group of 14 gender- and age-matched individuals. Institutional Ethical Committee approval was obtained.

Results: Compared to control, BC patients showed a statistically significant increase in the frequency of CD8+ T cells (p=0.047), regulatory T cells CD4+ (CD25+bright, CD127+dim), (p=0.007) and Gamma-Delta (GD) T cells (p=0.002).

When assessing the ectonucleotidases in T cells, patients had a significantly higher frequency of CD73+GD (p=0.03) and CD39+CD73+CD4+ T cells (p=0.04). Moreover, patients with lower GD T cells frequency (<2%) had a lower relapse-free survival at 18 months (HR 3.47), as compared with patients with higher GD counts. No survival differences were found based on CD73+ frequency or expression in GD. Additionally, we could not observe significant differences between high-risk (p≥2 and/or high-grade) and low-risk (pTa-1, low-grade) BC patients.

Conclusions: The peripheral blood of bladder cancer patients shows a combination of pro-immunosuppressive and pro-inflammatory changes in T cell populations, among which the gamma delta subset may have prognostic implications. This is associated with increased activity of CD73, which suggests the role of the AP in the pathophysiology of BC. This encourages further research on this pathway and exploration of targeted therapies.

CO-10-Procurement Graft Biopsy versus the KDRI in predicting kidney transplant outcomes - A cross-sectional study

Ana Marta Ferreira 1 , João Lorigo 1 , Edgar Tavares da Silva 1 , Luís Rodrigues 2 , Carolina Pimenta 2 , Vasco Quaresma 1 , Miguel Eliseu 1 , Pedro Nunes 1 , Arnaldo Figueiredo 1

1 Serviço de Urologia e Transplantação Renal, Centro Hospitalar e Universitário de Coimbra; 2 Serviço de Nefrologia, Centro Hospitalar e Universitário de Coimbra

Context/aims: The histological evaluation of renal grafts prior to a transplant, and donor related factors (last seen creatinine value, sex, age and comorbidities) are some of the elements that are proved to influence kidney transplantation outcomes. In our center the Remuzzi score is used to select grafts for transplantation (simple transplant for scores of 1 and 2, double for 3 and 4, and above that the grafts are excluded). The Kidney Donor Risk Index (KDRI) is a clinical score, applicable to deceased kidney donors, that reflects relative graft failure risk associated with deceased donor characteristics. It has been proposed as a substitute of, or complementary to preimplantation renal biopsy.

The aim of this study is to compare the predictive value of the KDRI score of kidney donors and graft’s Remuzzi score with clinical outcomes at 12 months.

Methods: Cross-sectional study that included all patients receiving a biopsied kidney graft from 2011 to 2020 at the University Hospital of Coimbra (n=109). Clinical data was collected from the hospital and the kidney transplant unit database. Statistical analysis was performed using SPSS software ver. 22.0 (IBM, Armonk, NY, USA). The serum creatinine (sCr) during the first year of follow up was correlated with the observed Remuzzi and the KDRI scores. The samples were then divided in two groups, according to an optimal cut-off value (KDPI ≤ or > 1,75) identified in a ROC curve.

Results: Median age of the population was 63,7±8,4 years old. The median sCr at 1, 3, 6 and 12 months were 1,76 mg/dL, 1,77 mg/dL, 1,81 mg/dL and 1,83 mg/dL, respectively. Patients receiving a kidney from a donor with higher KDRI showed a greater sCr at 12M (r=+0,04, p<0.05), however, higher Remuzzi’s scores of the graft’s biopsy were not correlated with greater sCr at 1, 3, 6 or 12 months (p=n.s.). Patients with a KDRIs ≤ 1,75 (45%) showed lower sCr at 12 months vs. the ones with a KDRIs > 1,75 (1,59 vs 2 mg/dL, p<0.05).

Conclusions: KDRI showed better correlation with clinical outcomes (sCr at 12 months) than the graft’s biopsy Remuzzi score.

Poster Highlight

PH-1-Telaglenastat, a glutaminase inhibitor, as a potential therapeutic target in chronic myeloid leukemia: an in vitro study

Raquel Alves 1,2 , Rafael Gomes 1 , Simão Furtado 1 , Joana Jorge 1,2 , Beatriz Lapa 1,2 , Maria Inês Costa 1,2 , Ana Cristina Gonçalves 1,2 , Ana Bela Sarmento Ribeiro 1,2,3

1 Univ Coimbra, Faculty of Medicine, Coimbra Institute for Clinical and Biomedical Research (iCBR), Area of Environmental Genetics of Oncobiology (CIMAGO), Laboratory of Oncobiology and Hematology (LOH) and University Clinics of Hematology and Oncology, and Center for Innovative Biomedicine and Biotechnology (CIBB), Portugal; 2 Clinical Academic Center of Coimbra (CACC), Portugal; 3 Hematology Service, Centro Hospitalar e Universitário de Coimbra, Portugal.

Context/aims: Metabolic reprogramming is a fundamental characteristic of tumor cells to sustain their high biosynthetic and proliferative needs. This characteristic is relevant in several neoplasms, including chronic myeloid leukemia (CML). Glutaminolysis provides intermediates for Krebs cycle and is involved in de novo synthesis of amino acids, nucleotides, fatty acids, and reduced glutathione (GSH). Currently, different inhibitors of glutamine metabolism have been evaluated in various neoplasms. The potential of these inhibitors is little explored in CML, especially in cases of resistance to imatinib. Therefore, the therapeutic potential of telaglenastat, a glutaminase inhibitor, was evaluated in in vitro models of CML sensitive and resistant to imatinib. Methods: Three CML cell lines were used: K562 cells (imatinib sensitive), K562-RC and K562-RD cells (imatinib resistant). Cells were incubated with increasing concentrations of telaglenastat for 72h. Dose-response curves were determined by resazurin metabolic assay. Cell death was analyzed by optical microscopy (May-Grunwald Giemsa stain) and flow cytometry (FC; annexin V/7-AAD). Cell cycle was assessed by FC with propidium iodide/RNase stain. Mitochondrial membrane potential (ψmit), ROS, and GSH levels were analyzed by FC. Statistical analysis was carried out with a significance level of 95%.

Results: Telaglenastat reduced metabolic activity in a time-, dose- and cell-line-dependent manner. Cell lines resistant to imatinib were the most sensitive to telaglenastat, with an IC50 at 48h of 0.75μM in K562-RD and 4.9μM in K562-RC, compared to K562 cells (85.8μM). This inhibitor induces apoptosis, as demonstrated by FC and analysis of cell morphology by microscopy. A decrease in ψmit was observed in K562 and K562-RC cells, proving the induction of apoptosis via mitochondrial pathway. Telaglenastat induced S-phase cell cycle arrest in K562-RD cells. Exposure to this drug led to an increase in ROS and GSH levels in all models, and a state of oxidative stress was observed in the K562 cells.

Conclusions: Our results suggest the therapeutic potential of telaglenastat in CML, particularly in cases of imatinib resistance.

PH-2-CD20+ T cells in monoclonal B cell lymphocytosis and chronic lymphocytic leukemia

Cristiana Rodrigues 1 , Paula Laranjeira 1 , Aryane Pinho 2 , Isabel Silva 1 , Sandra Silva 1 , Margarida Coucelo 3 , Catarina Geraldes 4 , Artur Paiva 1

1 Flow Cytometry Unit, Department of Clinical Pathology, Centro Hospitalar e Universitário de Coimbra, Portugal; 2 Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Portugal; 3 Unidade Funcional de Hematologia Molecular, Serviço de Hematologia Clínica, Centro Hospitalar e Universitário de Coimbra, Portugal; 4 Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal

Context/aims: Monoclonal B Lymphocytosis (MBL) and Chronic Lymphocytic Leukemia (CLL) are characterized by the accumulation of clonal B lymphocytes, with a mature phenotype and abnormal function, in peripheral blood. CLL-like MBL shares immunophenotypic, biological, and molecular features with CLL. MBL is a pre-malignant state that, in some cases, precedes CLL. In CLL and CLL-like MBL there is evidence of disruption in the interaction between pathological B cells and T cells. CD20+ T cells are a subpopulation of T cells that appear to be involved in autoimmune diseases and cancer. The aim of this study was to quantify and phenotypically characterize CD20+ T cells from subjects with MBL type CLL, with CLL and from a control group, in order to understand if these cells could be involved in the pathophysiology of these entities.

Methods: For this purpose, 13 subjects of low-count MBL type CLL, 12 subjects of high-count MBL type CLL and 27 patients of CLL were studied. The phenotypic study of CD20+ cells was performed by flow cytometry, while the search for mutations in the TP53 gene was performed using Next-Generation Sequencing (NGS) and the determination of the mutational status of the immunoglobulin heavy chain variable region (IGHV) gene was performed using PCR/fragment analysis and Sanger sequencing.

Results: Our results show that CD20+ T cells are less represented in patients with MBL and CLL, specifically in those with unmutated IGVH gene. CD20+ T cells were predominantly present in the CD8+ T cell compartment, exhibiting a predominantly Tc1 memory phenotype. An increase in follicular CD4+ CD20+ T cells and expansion of CD8+ CD20+ Tc1 cells was observed in the MBL and CLL groups. Our results also showed an expansion of Vβ 5.1 family in CD4+ CD20+ T cells from CLL patients.

Conclusions: These changes observed in CD20+ T cells suggest their involvement in the pathophysiology of MBL and CLL, and also suggest that studies studies in larger cohorts of patients with a long follow-up are needed to validate these results and evaluate the possible role of these cells in the prognosis and stratification of patients based on the risk of progression from MBL to CLL.

PH-3-Enhancing Kidney Transplant outcomes: evaluating prognostic significance of preservation solutes and mesenteric arteries for optimal recovery

Rafaela B. Clamote 1 , Edgar T. Silva 1,2,3 , Ana S. Pires 1,3,4 , Vasco Quaresma 2,3 , Ana J. Guerra 2 , Inês A. Marques 1,3,4 , Ana M. Abrantes 1,3,4 , Arnaldo Figueiredo 1,2,3 , Maria F. Botelho 1,3,4

1 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Institute of Biophysics, Faculty of Medicine, Coimbra, Portugal; 2 Centro Hospitalar e Universitário de Coimbra (CHUC), Urology and renal Transplantation Service, Coimbra, Portugal; 3 Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; 4 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal.

Context/aims: Despite donor shortage, renal transplantation is the best treatment for end-stage renal disease. This study explores the connection between donor endothelial dysfunction, reactive oxygen species (ROS) production, and transplant outcomes (TO) to develop new selection criteria and techniques to improve patient outcomes. By understanding the role of ROS and endothelial dysfunction, we can optimize preservation solutions (e.g. Celsior) and adopt approaches to minimize graft damage, enhance function, and expand donor availability. This research aims to improve recipient prognosis, quality of life, and long-term transplant success for end-stage renal disease patients.

Methods: We examined 39 mesenteric artery fragments, assessing endothelial damage using the American Heart Association (AHA) classification. Graft and donor characteristics were also examined. Renal TO were evaluated based on graft function and serum creatinine levels. Preservation solute samples were collected from deceased kidney donor recipients between January 2023 and June 2023, and ROS levels were analyzed using fluorimetry.

Results: The deceased kidney donor recipients consisted largely of males (67%), averaging 55years. Most donors met extended criteria for kidney donation. ROS levels decreased by half, after preservation, but there was no significant difference between immediate and delayed graft function. The AHA classification revealed mild endothelial damage in 18/33 analyzed arteries. Overall, 34 recipients experienced immediate graft function, 8 had delayed function, and four kidneys experienced primary dysfunction. Rejected kidneys are of poor quality with atherosclerosis and abnormal ostium, leading to early failure. Successful transplants have better quality, less atherosclerosis, and healthier donors.

Conclusions: The study indicates that Celsior solution reduces ROS levels during static cold storage, although without correlation between ROS levels and short-term TO. Larger studies are needed to comprehend a possible correlation with long-term kidney transplant success. Despite sample size and statistical limitations, the study suggests a link between atherosclerosis and TO.

Funding: Foundation for Science and Technology (FCT), Portugal (Strategic Projects UIDB/04539/2020, UIDP/04539/2020); COMPETE-FEDER (POCI-01-0145-FEDER-007440); Bolsa APU2023.

PH-4-Myeloid lineage Next Generation Sequencing fusion panel –validation and experience in a diagnostics laboratory

Ana Teresa Simões 1 , Ana Catarina Oliveira 1 , Joana Azevedo 1 , Teresa Fidalgo 1 , Catarina Geraldes 1,2,3 , Emília Cortesão 2,3 , Margarida Coucelo 1

1 Unidade Funcional de Hematologia Molecular, Serviço de Hematologia Clínica, Centro Hospitalar e Universitário de Coimbra, Portugal; 2 Serviço de Hematologia Clínica, Centro Hospitalar e Universitário de Coimbra, Portugal; 3 Coimbra Institute for Clinical and Biomedical Research (iCBR) – Grupo de Investigação em Ambiente, Genética e Oncobiogia (CIMAGO), Faculdade de Medicina, Universidade de Coimbra.

Context/aims: Next Generation Sequencing (NGS) technology has completely shifted the genetic studies routine in the haematology laboratory, allowing the definition of the molecular spectrum of haematologic neoplasms in a single study. More recently, the use of RNA panels (RNAseq) in the identification of fusion genes has granted the recognition of rare translocations, many of them cryptic and non detectable using conventional cytogentics.

The main aim of this study was to evaluate and implement a NGS panel for myeloid lineage fusion genes in our molecular diagnostics laboratory.

Methods: Thirty seven samples were studied with a RNAseq NGS panel for 29 fusion driver genes (Oncomine Myeloid Research Assay, ThermoFisher): 16 Acute Myeloid Leukaemia (AML) with complete genetic studies but no recurrent translocations defining AML; 1 AML with KMT2A rearrangement (@ KMT2A ) without known fusion partner and 15 eosinophilias. In order to validate the panel, 5 samples with previously known translocations were also included.

Results: In all five samples with known translocations these were properly identified, as well as their breakpoints, using the RNAseq panel. In one AML without translocations previously identified, the fusion gene ETV6::MECOM was detected. In the AML @KMT2A the partner CREBBP was identified and in one of the eosinophilias the panel detected the fusion gene FIP1L1::PDGFRA. In the remaining 28 samples the study was negative.

Conclusions: The NGS panel detected the recurrent alterations in all known samples. The use of this panel allowed the identification of ETV6::MECOM fusion gene in one AML, reclassifying it as “AML with MECOM rearrangement”, accordingly to WHO2022. In the AML @KMT2A the fusion partner, previously unrecognized, was identified.

The implementation of this NGS panel in the molecular study routine allows the identification of fusion genes, some of them rare – this capacity, as well as the correct identification of breakpoints, has great utility in the development of monitorization studies. This approach brings a better classification, risk stratification and follow up to haematologic neoplasms, namely AML.

PH-5-Unraveling the Mysteries of Oral Cancer through Epigenomics and Cell-Free DNA Analysis

Ilda Patrícia Ribeiro 1,2,3 , Leonor Barroso 4 , Francisco Marques 5 , Francisco Caramelo 2,6 , Luís Miguel Pires 1 , Joana Barbosa Melo 1,2,3 , Isabel Marques Carreira 1,2,3

1 University of Coimbra, Cytogenetics and Genomics Laboratory, Institute of Cellular and Molecular Biology, Faculty of Medicine, Coimbra, Portugal; 2 University of Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) and Center of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, Coimbra, Portugal; 3 University of Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal and Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; 4 Maxillofacial Surgery Department, Coimbra Hospital and University Centre, CHUC, EPE, 3000-075 Coimbra, Portugal; 5 Department of Dentistry, Faculty of Medicine, University of Coimbra, 3000-075 Coimbra, Portugal; 6 Laboratory of Biostatistics and Medical Informatics, iCBR - Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Context/aims: Oral cancer (OC) is posing a global concern with alarming rates worldwide, marked by high recurrence and mortality rates. Therefore, it is imperative to identify biomarkers for diagnosis and prognosis, along with the development of targeted therapies. This study aimed to perform a genome and epigenetic characterization of OC samples, identifying the most common altered chromosomes and genes. Additionally, we also explore the potential of liquid biopsies to follow-up these patients.

Methods: The epigenomic characterization of 32 oral cancer tissue samples was performed using arrayCGH and MS-MLPA techniques. Copy number variation and methylation data was analyzed using specific bioinformatic pipelines. Cell-free DNA (cfDNA) from plasma and urine was quantified at several timepoints during patient´s treatment.

Results: We observed that chromosomes 3, 4, 5, 14, 15 and X were the most frequently altered in our cohort. Several genes related to carcinogenesis are mapped in the chromosomes, such as ADAM6, ZIC4, RAD17 and CCNB1 .The most statistically significant pathways associated with the amplified and deleted genes were PI3K-Akt and metabolic signaling pathways, respectively. Several chromosomal alterations were observed to be concurrent, meaning they were present simultaneously in the same patient, potentially playing a vital role in carcinogenesis. Among the chromosomal arms with gains and losses, those showing the strongest correlation were 20p/11p and 1q/16p, respectively. Additionally, the levels of cfDNA seem to increase right after initiation of treatment in most patients, before decreasing. Sequencing of ctDNA showed not only mutations in pivotal genes for carcinogenesis process such as TP53 and PIK3CA but also the identification of new alterations that arise during the treatment course. In certain patients who experienced metastasis/relapse during the clinical follow-up, a notable elevation in cfDNA levels was observed in biofluids collected during the follow-up period.

Conclusions: A comprehensive molecular characterization of OC samples, combined with non-invasive molecular monitoring of patients, appears to be useful for achieving precision medicine for this neoplasm.

PH-6-NIPT: A Game-changer in Prenatal screening

Isabel M. Carreira 1,2,3 , Luís M. Pires 1 , Susana Ferreira 1 , Mariana Val 1 , Pedro Veiga 1 , Ilda P. Ribeiro 1,2,3 , Joana B. Melo 1,2,3

Context/aims: The advent of circulating fetal DNA (cfDNA) in maternal blood, coupled with next-generation sequencing (NGS) technologies, has allowed the incorporation of non-invasive prenatal testing (NIPT) into Prenatal Diagnosis (PND) with high sensitivity and specificity for detecting aneuploidies. This study aims to present the outcomes of implementing a NIPT test by sequencing circulating cfDNA in maternal blood using NGS technologies in a public laboratory.

Methods: Since the introduction of NIPT at the Laboratory of Cytogenetics and Genomics – FMUC, 2178 pregnant women have undergone the test.

Results: The NIPT test revealed 16 trisomies of chromosome 21 (0.7%), 1 trisomy 18 (0.04%), 5 trisomies of chromosome 13 (0.23%), 197 inconclusive results (9%), and 1959 with absence/low risk of aneuploidy (89,9%). Most of the inconclusive results were due to different sampling parameters associated with low fetal fraction and high pregnant body mass index. In addition to trisomies 21, 18 and 13, we also detected trisomies of chromosomes 7, 8, 15 16 and 20, unexpected maternal deletions and duplications in the short arm of the X chromosome and mosaicism involving the sex chromosomes.

Conclusions: The introduction of NIPT has expanded the accessibility of a screening test for pregnant women at risk of aneuploidy, thereby diminishing the necessity for invasive procedures. In terms of NIPT performance, the verification of all positive results through amniocentesis underscores its high sensitivity, ensuring dependable outcomes even with a minimum fetal fraction of 3%. The knowledge obtained through NIPT has enabled the investigation of cell-free DNA in various biofluids for monitoring oncological diseases during and after treatment.

POSTERS

Evaluation of the effect of sublethal photodynamic therapy on endometrial cancer

Beatriz Serambeque 1,2 , Genniffer Oliveira 1,3 , Guilherme Maia 1 , Margarida Silva 1 , Ana Cristina Gonçalves 2,4,5 , Raquel Alves 2,4,5 , Ana Bela Sarmento-Ribeiro 2,4,6 , Nelson Pereira 7 , Marta Pineiro 7 , Teresa Pinho e Melo 7 , Maria Filomena Botelho 1,2,5 , Maria João Carvalho 2,8,9 , Mafalda Laranjo 1,2,5

1 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Institute of Biophysics, Faculty of Medicine, Portugal; 2 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Portugal; 3 Instituto Politécnico de Coimbra, Escola Superior Agrária de Coimbra, Portugal; 4 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) Area of Environment, Genetics and Oncobiology (CIMAGO), Laboratory of Oncobiology and Hematology (LOH) and University Clinics of Hematology and Oncology, Faculty of Medicine, Portugal; 5 Clinical Academic Centre of Coimbra (CACC), Portugal; 6 Hematology Service, Centro Hospitalar e Universitário de Coimbra, Portugal; 7 Univ Coimbra, Coimbra Chemistry Centre - Institute of Molecular Sciences and Department of Chemistry, Portugal; 8 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Institute of Biophysics and Universitary Clinic of Gynecology, Faculty of Medicine, Portugal; 9 Gynecology Service, Centro Hospitalar e Universitário de Coimbra, Portugal

Context/aims: Cancer stem cells (CSC) are a subpopulation implicated in tumorigenesis and resistance to chemotherapy. Therefore, to develop an effective therapy to eliminate CSC, it is crucial to understand the impact of an ineffective therapy. Due to the ease of uterus hysteroscopic access, photodynamic therapy (PDT) can be a promising minimally invasive approach. Thus, this study aimed to explore the effects of sublethal PDT on endometrial cancer.

Methods: Sublethal concentrations of a 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorin (PX1) and Temoporfin in endometrial cancer cell lines, ECC-1 and RL95-2, were determined from dose-response curves, and its effect on viability and cell cycle were assessed through flow cytometry. After conditioning cells with sublethal PDT, and to evaluate the response to therapy, cells were treated with therapeutic concentrations of PX1 and Temoporfin (7.5mW/cm 2 ,10J), and carboplatin. Metabolic activity was measured using MTT. Endometrial CSC, isolated through a sphere-forming protocol on conditioned cells, were characterized by sphere-forming capacity, self-renewal, and projection area. Conditioned CSC were treated with PX1 and carboplatin, and the viability was assessed through flow cytometry.

Results: We proved that the used concentrations were sublethal, with a slight decrease in viability and no influence on the cell cycle. In both cell lines, conditioned cells did not show higher susceptibility or resistance to therapy. Regarding CSC, conditioning with Temoporfin showed a significant decrease in the sphere-forming ( p =0.029) and self-renewal capacity ( p =0.041) compared with Naif CSC. Preliminary results of the CSC viability revealed that conditioning with PX1 seemed to induce resistance to PDT and carboplatin.

Conclusions: In conclusion, sublethal PDT seems to influence the CSC phenotype and suggest resistance to PDT and chemotherapy, encouraging future research.

Funding: CIBB (UIDB/04539/2020, UIDP/04539/2020) and CQC (UIDB/00313/2020, UIDP/00313/2020) funded by Fundação para a Ciência e Tecnologia (FCT), co-funded by COMPETE. PTDC/QUI-QOR/0103/2021 (FCT, I.P./MCTES, PIDDAC). PhD Scholarship (2020.07672.BD) to BS - FCT/ESF.

Zinc increases cytotoxic, cytostatic and genotoxic effects of therapeutic compounds in acute myeloblastic leukemia

Maria Inês Costa 1,2 , Beatriz Lapa 1,2 , Joana Jorge 1,2 , Raquel Alves 1,2 , Ana Bela Sarmento Ribeiro 1,2,3 , Ana Cristina Gonçalves 1,2

1 Laboratory of Oncobiology and Hematology (LOH) and University Clinics of Hematology and Oncology, Coimbra Institute for Clinical and Biomedical Research (Icbr) Group of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra (FMUC), Coimbra, Portugal; 2 Center for Innovative Biomedicine and Biotechnology (CIBB) and Clinical Academic Centre of Coimbra (CACC), Coimbra, Portugal; 3 Service of Hematology, Centro Hospitalar e Universitário de Coimbra, Portugal;

Context/aims: DNA damage response (DDR) is key for genomic stability and zinc (Zn) is a DDR cofactor. In acute myelobastic leukemia (AML), Zn decreases and DDR defects are often observed, but the role of Zn supplementation as adjuvant to AML therapy are not well understood. We evaluated the effects of Zn in combination with cytarabine (AraC, conventional drug) and Olaparib (Ola, DDR inhibitor) in AML models.

Methods: HEL, K-562, NB-4, and THP-1 cell lines were incubated (72h) with increasing AraC or Ola concentrations in monotherapy and combination with ZnSO 4 (IC 25 ). Cell density and viability were evaluated by trypan blue assay. We analysed the type of cell death by flow cytometry (FC), using annexin V/7-AAD staining, the expression of the apoptotic markers cleaved PARP and active caspase-3 (FC), and cell morphology by optical microscopy (May Grunwald-Giemsa staining). Cell cycle distribution and proliferation was assessed by PI/RNAse staining and BrdU incorporation (FC). Chromosomal damage (micronucleus assay), genomic damage (yH2AX expression by FC), and the expression of 18 DDR genes by qPCR were also assessed.

Results: Zn addition to Ola and AraC decreased cell density and viability in all cell lines compared to the observed with drugs in monotherapy (p<0.05). The IC 50 (48h) were considerably reduced by Zn (AraC: 5.9-57.6x; Ola: 2.7-8.7x). Zn combinations increased the late apoptosis/necrosis fraction (p<0.05) and the expression of cleaved PARP (p<0.05) and active caspase-3 (p<0.0001). Morphological analyses shown features of apoptosis. Zn altered the cytostatic profile of AraC (S) and Ola (G 2 /M) in HEL and K-562 cells, inducing G 0 /G 1 arrest. In general, BrdU incorporation decreased with Zn addition. Chromosomal and genomic damage increased by the combination strategies (p<0.05). Several DDR genes were differentially modulated by combination treatments, with PARP1 (p<0.05) and XRCC6 (p<0.01) genes consistently upregulated.

Conclusions: Zn potentiated the cytotoxic, cytostatic, and genotoxic effects of AraC and Ola in four AML models, while considerably reducing their IC 50 . These findings may translate into safer, less toxic, and more efficient therapeutic regimens in AML with Zn supplementation.

Effects of Cold Atmospheric Plasma in Breast Cancer Stem Cells

Catarina Almeida-Ferreira 1,2,3,# , Gonçalo Lopes 4,# , Rafael Silva-Teixeira 5 , Francisco Caramelo 1,3,6 , Maria Filomena Botelho 1,3,6,* , Mafalda Laranjo 1,3,6,*

1 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment, Genetics and Oncobiology Research (CIMAGO), Institute of Biophysics, Faculty of Medicine, Coimbra, Portugal; 2 Univ Coimbra, Faculty of Pharmacy, Coimbra, Portugal; 3 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal; 4 Polytechnic of Coimbra, Coimbra Agriculture School (ESAC), Coimbra, Portugal; 5 Department of Cardiology, Hospital Center of Vila Nova de Gaia/Espinho, EPE, Vila Nova de Gaia, Portugal; 6 Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; #These authors contributed equally for this work; *Shared senior authorship.

Context/aims: Breast cancer has the highest number of new cases and the second-highest mortality rate in the world for both sexes and all ages. Current treatments for this disease include surgery, chemotherapy, radiotherapy, and hormonal therapies, which are effective but also have limitations and side effects. Cold Atmospheric Plasma (CAP) has emerged as a potential treatment, exhibiting selectivity to cancer cells while preserving healthy cells. Cancer stem cells (CSCs) play a critical role in tumour recurrence and metastasis due to their heightened self-renewal and invasion among the cancer population. The main goal of this study was to assess the effects of CAP on breast CSCs.

Methods: These studies were performed in two breast cell lines representing triple negative (HCC1806) and hormone-dependent (MCF-7). The evaluation included plasma cytotoxicity through metabolic activity, sphere formation capacity, self-renewal, and projection area regarding different exposure times.

Results: Metabolic activity significantly reduced after 60 seconds (71.02±5.56%, p=0.0137) of plasma treatment, most marked after 240 seconds (23.64±5.65%, p=0.0054) in the MCF-7 cell line. HCC1806 significantly decreased after 240 seconds (39.59±5.45%, p=0.0081). Sphere formation capacity and self-renewal decreased with increasing exposure to cold plasma in stem cells from both lines after 120 and 240 seconds. The projection area of the spheres decreased in HCC1806 after 240 seconds from 3686±319.3 pixels to 419.3±242.1 pixels, p=0.0179; however, it increased in MCF-7 after 120 seconds of exposure.

Conclusions: Plasma demonstrated therapeutic potential in targeting breast CSCs, encouraging future studies.

Funding: PhD studentship grant by the Portuguese National Funding Agency for Science and Technology (FCT), 2022.12228.BD. Institutional funding from FCT strategic projects (UIDB/04539/2020, UIDP/04539/2020) and by COMPETE-FEDER (POCI-01-0145-FEDER-007440).

In vitro & in vivo adjuvant impact of green tea on breast cancer treatment – imaging methodologies

Vladyslava Trachenko 1,2,3 , Catarina Pereira 1,2,3 , Bruna Santos 1,2,3 , Filipa Machado 1,2,3 , Nuno Braz 1,2,3 , Glória Figueiredo 1,2,4 , Sofia Brás 1,2,3 , Benedetta Marin 2,3,5 , Mariana Marques 1,2,3 , Fabiana Ribeiro 4 , Bárbara Oliveiros 6 , Pedro Encarnação 4 , Pedro Correia 4,7 , Ana Luísa Silva 4,7 , Filipe Castro 7 , João Veloso 4,7 , Ana Cristina Santos 1,2

1 Institute of Biophysics, Faculty of Medicine of the University of Coimbra, Coimbra, Portugal; 2 Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine of the University of Coimbra (FMUC), Center for Innovative Biomedicine and Biotechnology (CIBB) - Area of Environment Genetics and Oncobiology (CIMAGO), Coimbra, Portugal; 3 Faculty of Sciences and Technology of the University of Coimbra, Coimbra, Portugal; 4 Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, University of Aveiro, Aveiro, Portugal; 5 Dipartimento di Ingegneria dell’Informazione DEI, Università Degli Studi di Padova, Italy; 6 Laboratory of Biostatistics and Medical Informatics, Institute of Biophysics, Faculty of Medicine of the University of Coimbra (FMUC), Coimbra, Portugal; 7 Radiation Imaging Technologies Lda (RI-TE), Ílhavo, Portugal.

Context/aims: Breast cancer is one of the most common cancer types worldwide in females. Chemotherapy is a common cancer treatment, although potentially causing drug resistance and several side effects. Green tea (GT) is a natural product rich in bioactive compounds (catechins, flavonoids, phenolic acids, & methylxanthines). Molecular mechanisms including antioxidant activity, inhibition of cancer stem cell expansion, downregulation of oncogenes, and regulation of telomerase activity are some domains of its action. Its combination with conventional chemotherapy can increase treatment effectiveness, while reducing side effects. This project aims to evaluate a potential adjuvant effect of a green tea extract (GTEpo), in combination with clinical chemoagents for breast cancer treatment using MCF-7 and HCC-1806 cell lines, both in vitro & in vivo .

Methods: MTT (colorimetric cell viability assay) and flow cytometry (cell death type & phase of the cycle) are used to analyse in vitro results. EasyPET.3D in vivo imaging (tumour induction with either breast cancer cell lines in a non-orthotopic animal model - BALB/c female nude mice), using [ 18 F]-FDG radiopharmaceutical, enables to assess tumour evolution and treatment protocol outcomes. Collected samples of tumour/induction site are prepared for histopathological routine studies.

Results: On-going study results show that in HCC-1806, the conjugation of 5 μg/mL GTEpo with the clinical combination of Paclitaxel (Pacli) & Carboplatin is significantly active (𝑝< 0.05). For MCF-7, the joint action of conjugation with GTEpo (5 μg/mL) and the clinical combination Pacli & Gemcitabine is significantly active (𝑝< 0.01). GTEpo can enhance cytotoxic effects of some chemotherapeutic agents against MCF-7 cells, leading to a better quality of life.

Conclusions: GTEpo enhances chemoagents effectiveness, crucial for triple negative tumours and even for ER+ types and may allow the decrease of dosages. Statistically stronger in vitro data and a larger number of in vivo studies will enable to draw valuable conclusions. This project is just beginning with great expectations.

Acknowledgements: This work was funded by projects CENTRO-01-0247-FEDER-039880 and PTDC/EMD-EMD/2140/2020. A special thank you to Centro Hospitalar da Universidade de Coimbra (CHUC), to iCBR-FMUC Vivarium , to the Biophysics Institute of FMUC, to University of Aveiro, and to RI.TE.

Green tea extract conjugation with chemoagents for breast cancer bone metastasis: in vitro & in vivo imaging evaluation

Catarina Pereira 1,2,3 , Vladyslava Trachenko 1,2,3 , Bruna Santos 1,2,3 , Filipa Machado 1,2,3 , Nuno Braz 1,2,3 , Glória Figueiredo 1,2,4 , Sofia Brás 1,2,3 , Benedetta Marin 2,3,5 , Mariana Marques 1,2,3 , Fabiana Ribeiro 4 , Bárbara Oliveiros 6 , Pedro Encarnação 4 , Ana L. Silva 4,7 , Pedro Correia 4,7 , Filipe Castro 7 , João Veloso 4,7 , Ana C. Santos 1,2

Context/aims: Breast cancer is the most prevalent oncological disease within the female population worldwide. Its preferential predisposition for bone metastasis encompasses significant mortality rate. Green tea is rich in several catechins, with antioxidant, anti-inflammatory and antimutagenic properties. Evaluation of the effect of a green tea extract (GTEpo), combined with chemoagents used in the clinic to treat breast cancer bone metastasis, is the aim of the project.

Methods: In vitro studies are being carried out with the MCF-7 cell line, testing the effect of different GTEpo concentrations, in combination with treatment protocols, such as Vincristine (Vin) (2 mg/m 2 ) + Epirubicin (Epi) (75 mg/m 2 ) + Cyclophosphamide (CFA) (1,200 mg/m 2 ). To access in vitro cell viability the MTT colorimetric assay is used. An in vivo model of bone metastasis induction (skull of BALB/c female nude mice) is being studied using imaging techniques (best in vitro protocols with GTEpo). [ 18 F]-FDG (i.p. administration) is used for microPET (easyPET.3D) imaging. Histopathological studies of collected samples will be done.

Results: I n vitro combination of GTEpo with the chemodrugs highlighted its coadjuvant effect. GTEpo 5 μg/mL proved to be more effective. A more relevant reduction of MCF-7 cell’s viability was observed for Vin (2 mg/m 2 ) + GTEpo 0.5 μg/mL, although all tested concentrations of GTEpo + Vin are statistically (p < 0.05) and clinically (median < IC 50 ) significant. In contrast, the association GTEpo+Epi (75 mg/m 2 ), although being clinically significant, did not induce comparingly higher cell death. PET functional scans are important to control tumours’ development and/or the need to reinject the animals with MCF-7 cells.

Conclusions: An in vitro adjuvant effect of GTEpo was observed for the tested protocol. In vivo ongoing experiments proved the potential use of the easyPET.3D technology in this field and will continue to run. Other clinical protocols shall be tested, using both MCF-7 and HCC-1806 cell lines; reduction of clinical dosages will also be considered.

easyPET technology in an experimental animal model of bone metastasis induction: study of human colon and breast cancer

Glória Figueiredo 1,2 , Fabiana Ribeiro 2 , Rute Santos 1 , Mariana Marques 1 , Bárbara Oliveiros 4 , Pedro Encarnação 2 , Pedro Correia 2 , Ana Luísa Silva 2,3 , Filipe Castro 3 , João Veloso 2,3 , Ana Cristina Santos 1

1 Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine of the University of Coimbra (FMUC) - Area of Environment Genetics and Oncobiology (CIMAGO), Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, PORTUGAL; 2 Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, University of Aveiro, Portugal; 3 Radiation Imaging Technologies Lda (RI-TE), Ílhavo, Portugal; 4 Laboratory of Biostatistics and Medical Informatics, Institute of Biophysics, Faculty of Medicine of the University of Coimbra (FMUC), Coimbra, Portugal.

Context/Aims: Cancer is one of the main mortality causes, not enabling an increase in life expectancy. According to WHO, there is a substantial diversity of malignant neoplasms. The prevailing diagnosis of breast cancer in women is very relevant. Its evolution has a preferential predisposition for bone metastasis, which has high clinical relevance, given the significant associated morbidity and mortality. Colon cancer accounts for ~10% of diagnosed cancer cases worldwide, being the 3 rd most frequent malignant one in men, preferentially metastazing to bone, skin, and spleen. The project aims to early detect bone metastasis, based on an animal model to simulate tumour invasion, doing monitoring with microPET.

Methods: Tumours were induced, using WiDr and MCF-7 cell lines, in Balb/c(-) male or female mice, respectively (slight scarification of the skull, followed by injection of the cell suspension). Tumour development was monitored (7 weeks) using the easyPET.3D to acquire images using [ 18 F]-FDG or [ 18 F]-NaF, to assess glycolytic and osteoblastic activity.

Results: Animals inoculated with WiDr cells showed a generalised increase of glycolytic activity over the head (%ID/g 3.3 vs 9.1), due to tumoural cell invasion of cranial bone. It was not possible to externally perceive the presence of a tumour mass in most of the subjects, within the 1 st 2 weeks of study. Nevertheless, Standard Uptake Values (SUV), in the defined volume of interest, already showed a growth pattern (5.7%ID/g). MCF-7 cells are sensitive to oestradiol, hence female animals received a 17ßoestradiol injection (1-week post-cell’s injection) leading, in early stages, to a higher uptake in the thyroid and a decreasing uptake in the head. Histopathology of bone invasion confirmation was done.

Conclusion/Future perspectives: EasyPET.3D showed great potential to early detect tumours. The animal model of bone metastasis induction using WiDr cell line is elective. Breast tumour (MCF-7 cell line) requires a longer period of study. In vitro and in vivo studies are under way to assess the potential efficacy of coupling natural therapies & clinical chemotherapeutics at early stages of tumour development.

Naturally occurring abietane diterpenoids as potential antitumor agents againts urothelial carcinoma cells

Ana Isabel Sousa 1,2,# , Mariana Magalhães 1,2,3,# , Gabriela Espírito-Santo 1,2 , Raquel Alves 4,5 , Patrícia Rijo 6,7 , Anabela Mota Pinto 2,5 , Ricardo Leão 2,5 , Célia Cabra l,2,8

1 University of Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR), Clinic Academic Center of Coimbra (CACC), Faculty of Medicine, Coimbra, Portugal; 2 University of Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal; 3 University of Coimbra, Institute for Interdisciplinary Research, Doctoral Programme in Experimental Biology and Biomedicine (PDBEB), Portugal & CNC—Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 4 Laboratory of Oncobiology and Hematology, University Clinic of Hematology and Applied Molecular Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 5 iCBR, Group of Environment Genetics and Oncobiology (CIMAGO)—Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 6 CBIOS—Universidade Lusófona’s Research Center for Biosciences & Health Technologies, Lisbon, Portugal; 7 Faculty of Pharmacy, Instituto de Investigação do Medicamento (iMed.ULisboa), University of Lisbon, Lisbon, Portugal; 8 University of Coimbra, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal.

#These authors contributed equally to this work.

Context/aims: Upper urinary tract urothelial carcinoma (UTUC) is a highly aggressive form of urological malignancy affecting the renal pelvis and ureter, constituting 2-10% of urothelial carcinomas. Despite early diagnosis, a significative number of patients reach an invasive stage of the disease, impacting prognosis. Cisplatinum-based combined chemotherapy is the preferred treatment for metastatic cases unless existing contraindications. In response to the growing need for alternatives to chemotherapy, there is interest in exploring phytochemical antitumor agents like diterpenes, a biologically active group found in the genus Plectranthus L’Hér. This study focuses on evaluating the cytotoxic potential of natural occurring abietane diterpenoids, namely 7α-Acetoxy-6β-hydroxyroyleanone (Roy), 7β,6β-dihydroxyroyleanone6 (Diroy), and Parvifloron D (ParvD), in the 639-V cell line derived from ureteral transitional cell carcinoma.

Methods: Firstly, these diterpenes were isolated from South African plants belonging to the Lamiaceae family: Roy and Diroy were isolated from the acetonic extract of leaves and stems of Plectranthus hadiensis Schweinf., respectively, and ParvD was isolated from the acetonic extract of Plectranthus ecklonii Benth. Following, we investigated the cytotoxic/antiproliferative profile of these diterpenes in urothelial carcinoma cells. Briefly, cell viability was evaluated by Alamar blue® assay, while cell death and the impact on cell cycle was assessed by flow cytometry. The half-inhibitory concentration (IC50) was determined using GraphPad Prism.

Results: Roy and ParvD exhibited significant cytotoxic/antiproliferative effect, whereas DiRoy showed no therapeutic impact. Roy demonstrated the most promising results, supported by an increase in cells undergoing early apoptosis. Moreover, Roy induced cell cycle arrest in the S/G2/M phase, hampering cell proliferation.

Conclusions: This study highlights Roy as a potential drug lead for further exploration, regarding its antitumor mechanism, against UTUC, offering a promising profile for future investigations.

Revolutionizing bladder cancer treatment: exploring plasma-activated liquids as cutting-edge intravesical agents

Edgar Tavares-Da-Silva 1,2,3 , Eurico Pereira 1,3,4 , João Craveiro 1 , Ana S. Pires 1,3,4 , Lúcia Fernandes 1 , Francisco Caramelo 1,4 , Ana M. Abrantes 1,3,4 , Figueiredo Arnaldo, Maria F. Botelho 1,3,4

1 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Institute of Biophysics, Faculty of Medicine, Coimbra, Portugal; 2 Centro Hospitalar e Universitário de Coimbra (CHUC), Department of Urology and Renal Transplantation Coimbra, Portugal; 3 Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; 4 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal.

Context/aims: The Cold Atmospheric Plasma (CAP) research for cancer treatment has led to the investigation of its potential application in treating bladder cancer (BC). Specifically, CAP-activated liquids (CAPAL) emerge as a promising alternative to traditional intravesical agents for BC therapy. The thermal plasma (TP) generated by a surgical bipolar resectoscope, a common tool in medical procedures, influences the adjacent NaCl 0.9% solution used for irrigation. This interaction transforms the solution into TP-activated liquid (TPAL) directly in contact with the bladder wall. To expedite the potential integration of PAL in BC treatment, we compared the characteristics and biological impacts of CAPAL and TPAL.

Methods: Our group developed an electronic device to generate CAP, while TP was produced using a resectoscope. Short-term exposures (30, 60, 120, 180 seconds) of NaCl 0.9% to CAP or TP yielded CAPAL or TPAL, respectively. Reactive oxygen and nitrogen species (RONS) in each activated liquid were assessed using the OxiSelect™ RONS Assay Kit. BC cell lines, TCCSUP (grade IV) and HT-1376 (grade III), were incubated with CAPAL or TPAL. Metabolic activity and protein content were evaluated 24 hours post-incubation using the MTT and SRB assay, respectively.

Results: Both CAPAL and TPAL exhibited a significant increase in RONS compared to non-irradiated NaCl 0.9%. In terms of biological impact on BC cell lines, both CAPAL and TPAL led to an exposure-time-dependent reduction in cell metabolic activity and protein content.

CAPAL demonstrated a more potent antitumor effect on the high-grade cell line (TCCSUP), while TPAL exhibited a similar effect on both cell lines. Notably, CAPAL required longer exposure times to achieve effects comparable to TPAL at shorter exposure times.

Conclusions: This study underscores the potential therapeutic role of PAL in BC treatment. Both TPAL and CAPAL demonstrated similar biological effects on BC cells. Although TPAL is already in contact with the bladder wall during surgery, this study aims to provide evidence that CAPAL can be a safe and effective treatment for BC, bringing us a step closer to its clinical application.

Colorectal cancer assessment of cytotoxic effects - in vitro & in vivo - of Seaweed Extracts

Filipa Machado 1,2,3 , Carolina Bastos 2,3,4 , Bruna Santos 1,2,3 , Ana Catarina Pereira 1,2,3 , Vladyslava Trachenko 1,2,3 , Nuno Braz 1,2,3 , Sofia Brás 1,2,3 , Benedetta Marin 1,5 , Ana Valado 6,7 , Leonel Pereira 8 , João Cotas 8 , Fabiana Ribeiro 9 , Bárbara Oliveiros 10 , Pedro Encarnação 9 , Ana Luísa Silva 9,11 , Pedro Correia 9,11 , João Veloso 9,11 , Ana Cristina Santos 2,3

1 Faculty of Sciences and Technology of the University of Coimbra (FCTUC), Portugal; 2 Institute of Biophysics, Faculty of Medicine of the University of Coimbra, Coimbra, Portugal; 3 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 4 Coimbra Health School (ESTeSC), Portugal; 5 Dipartimento di Ingegneria dell’Informazione DEI, Università Degli Studi di Padova, Italy; 6 Polytechnic Institute of Coimbra, ESTeSC-Coimbra Health School, Biomedical Laboratory Sciences, Coimbra, Portugal; 7 CFE - Centre for Functional Ecology: Science for People & Planet Marine Resources, Conservation and Technology - Marine Algae Lab, Department of Life Sciences, University of Coimbra, Coimbra, Portugal; 8 Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, University of Aveiro, Aveiro, Portugal; 9 Laboratory of Biostatistics and Medical Informatics, Institute of Biophysics, Faculty of Medicine of the University of Coimbra (FMUC), Portugal; 10 Radiation Imaging TechnologiesLda (RI-TE), Ilhavo, Portugal

Context/aims: Colorectal cancer (CRC) is the third most common type of cancer worldwide and the second most fatal. The primary treatments for this type of cancer are surgery, radiotherapy, chemotherapy, and immunotherapy. However, they have many side effects, hence the great demand for alternative natural compounds. Seaweeds contain fundamental components for human nutrition (e.g., proteins, minerals, fibres & fatty acids). The main aim of the project is to study the cytotoxic effects of seaweed extracts (SE) (e.g., alginate from Phaeophyceae and carrageenans from Calliblepharis jubata, Rhodophyta), on CRC in vitro and in vivo .

Methods: The WiDr colon cancer cell line was used, and its viability was assessed with the MTT colorimetric assay. Studies were also carried out on fibroblasts to evaluate antioxidant and anti-inflammatory effects of SEs. Pilot in vivo studies were carried out on BALB/c (+) mice, with some SEs (daily gavage/6 days). Histopathological analysis (optical microscopy) of collected samples was done.

Results: In vitro Iota and CF carrageenans showed the best results (medians values for 3 and & 4.5 mg/mL under the IC50 line). In viv o CS did not induce remarkable alterations. In vitro sodium alginate (1.5 & 4.5 mg/mL) presents better results than alginate. In vivo alginate caused kidney haemorrhage, dilated sinusoids and hydropic degeneration of hepatocytes. In vitro fucoidan showed median values are > IC50, hence no antitumoral effects; in vivo some kidney haemorrhage and slight lung congestion were noted.

Conclusions: Carrageenans, sulphated polysaccharides fulfilling essential functions in cells walls interstitial spaces and intercellular adhesion, show the best results either in vitro & in vivo . Iota carrageenan has sulphate groups in C-2 position of D-galactose and 3,6-anhydro-galactose units sensitive to Ca2+, leading to distinct interactions with biological molecules. Kappa carrageenan sulphate groups are in C-4 & C-2 positions of D-galactose interacting with K + . CF has more sulphate groups, hence the best in vitro results.

Colon cancer and a green tea extract: evaluation of efficacy using imaging methods in vitro and in vivo

Benedetta Marin 2,3,5 , Bruna Santos 1,2,3 , Nuno Braz 1,2,3 , Glória Figueiredo 1,2,4 , Sofia Brás 1,2,3 , Mariana Marques 1,2,3 , Fabiana Ribeiro 4 , Bárbara Oliveiros 6 , Pedro Encarnação 4 , Pedro Correia 4,7 , Ana L. Silva 4,7 , Filipe Castro 7 , João Veloso 4,7 , Ana Cristina Santos 1,2

Context/aims: Green tea (GT) is a natural compound encompassing several benefits for human health. Recent studies reported its role in cancer prevention and also during chemotherapy. If coupled with chemoagents, it can amplify their action. The chemoagents dose could be reduced, therefore decreasing side effects, which represent a clinical drawback in cancer management. The aim of this project was to test a GT extract (GTE) in combination with a chemotherapy protocol against Colorectal Cancer, the 3rd most spread cancer, and the 2 nd cause of mortality worldwide.

Methods: The WiDr cell line was used both for in vitro and in vivo studies (non-orthotopic animal model in BALB/c nude male mice). The tested clinical-cocktail was 5-Fluorouracil (400 mg/m 2 ) + Cyclophosphamide (1,000 mg/m 2 ). In vitro studies were done using different concentrations of GTE (0.5-5-10 mg/mL). The effects of the coupled therapy (natural compound+chemoagents) were studied with the MTT assay, an indicator of cell viability and proliferation. This protocol was then tested in vivo (daily oral administration of GTE). PET imaging with the easyPET.3D system was done to monitor tumours’ behaviour, with [ 18 F]-FDG as radiotracer.

Results: Although statistical analysis of the results for the cocktail + GTE showed no significancy, the cell survival was clearly lower than IC 50 . None of the 3 tested GTE concentrations showed adjuvant action neither alone nor in combination with the drugs’ cocktail, with no statistical differences between them. However, the results in vivo were different: animals under conventional treatment had a shorter survival than those also taking GTE. The Standard Uptake Value of tumours of PET images showed the benefices of GTEpo: initial increase with a subsequential reduction along treatment.

Conclusions: In vitro results did not meet the expectations with these cocktail/concentrations. New chemodrugs could be coupled with GTE and tested in the future. In vivo experiments showed the adjuvant power of GTE, more cases should be done. Histopathological studies are on-going, and could help to understand the tumour behaviour, being useful to explain the SUV decrease.

easyPET technology: the adjuvant role of ECGC in chemotherapy in a rodent model of human colon cancer

Mariana Marques 1 , Fabiana Ribeiro 2 , Glória Figueiredo 1,2 , Rute Santos 1 , Bárbara Oliveiros 4 , Pedro Encarnação 2 , Pedro Correia 2 , Ana Luísa Silva 2,3 , Filipe Castro 3 , João Veloso 2,3 , Ana Cristina Santos 1

1 Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine of the University of Coimbra (FMUC) - Area of Environment Genetics and Oncobiology (CIMAGO), Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, PORTUGAL; 2 Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, University of Aveiro, PORTUGAL; 3 Radiation Imaging Technologies Lda (RI-TE), Ílhavo, PORTUGAL; 4 Laboratory of Biostatistics and Medical Informatics, Institute of Biophysics, Faculty of Medicine of the University of Coimbra (FMUC), Coimbra, PORTUGAL

Context/Aims: Currently, cancer is responsible for more than ¼ of deaths in EU (1.2 million). In 2020 colorectal cancer (CRC) was the 3 rd type of cancer with the highest incidence (1.94 million diagnoses), and the 2 nd one with the highest mortality rate (936,000 deaths) worldwide. Due to the high incidence of cancer and drug resistance, research for new alternative and more effective agents, such as green tea (GT), is rising. Natural compounds can be a good alternative to prevent the onset and proliferation of tumours. This study aims to evaluate the progression of CRC, in a non-orthotopic animal model, applying different therapeutic protocols in combination with GT, by using the easyPET.3D system.

Methods: CRC was induced inoculating WiDr cells in the tight of Balb/c(-) mice (28 males, 4 animals/group). According to the selected treatment protocols, tumour development was monitored for 5 weeks by [ 18 F]-FDG PET imaging, with the easyPET.3D. Mice were kept warm and under anaesthesia during PET scans. Cisplatin (Cis) & pemetrexed (Al) were selected as chemotherapeutics for CRC, isolated and in cocktail (Cis+Al), according to approved clinical protocols. A GT extract enriched in EGCG (epigallocatechin-3-gallate), an anticancer flavonoid present in GT, has been used as adjuvant: Cis+GTE Al+GTE, Cis+Al+GTE.

Results: Induced tumours were not externally observable within the 1 st 2 weeks of study, but microPET scans already showed increasing glycolytic activity in the right tight region. Standard uptake values (SUV) were calculated for all animals, expressing increasing values until each treatment begun to act. Cis+Al showed the best results (SUV mean 12.3 vs 5.0) (survival 15d), nonetheless adding EGCG (Cis+Al+EGCG) led to an even more favourable effect (SUV mean 5.5 vs 2.0) (survival 45d). Imaging results were confirmed by histopathology.

Conclusion/Future perspectives: EasyPET (in upgrading) showed great potential to detect early tumour development, helping to assess its progression and effectiveness of the chosen treatments. In vitro and in vivo studies are being done to assess the efficacy of other clinical chemotherapeutic protocols + GTE as potential CRC therapies.

Design, synthesis and evaluation as theranostics agents of novel metalloconjugates targeted at the mitochondria of prostate cells

Margarida Sobral 1,2,3 , Joana Santos 4 , Célia Fernandes 4,5 , Paula Raposinho 4,5 , Filipa Mendes 4,5 , Ana M. Urbano 6,7 , Sandra Mota 2,3 , Paulo J. Oliveira 2,3 , António Paulo 4,5

1 PDBEB Doctoral Programme in Experimental Biology and Biomedicine, Institute of Interdisciplinary Research, University of Coimbra, Portugal; 2 CNC UC Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 3 CIBB Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 4 C2TN -Center for Nuclear Sciences and Technologies, Instituto Superior Técnico, University of Lisboa, Portugal; 5 Department of Engineering and Nuclear Sciences, Instituto Superior Técnico, University of Lisboa, Portugal; 6 Molecular Physical Chemistry R&D Unit, Department of Life Sciences, University of Coimbra, Portugal; 7 Center of Investigation in Environment, Genetics and Oncobiology, University of Coimbra, Portugal

Context/aims: The prostate-specific membrane antigen (PSMA) is a transmembrane protein markedly overexpressed in over 80% of prostate cancers. 177Lu-PSMA-617 (Pluvicto™), a radiopharmaceutical targeted at PSMA, has recently received FDA approval for the treatment of metastatic castration-resistant prostate cancer. In this complex, 177Lu is chelated by PSMA-617, a derivative of dodecane tetraacetic acid (DOTA) containing a PSMA inhibitor for selective uptake by prostate cancer cells. 177Lu shares limitations of other beta minus particle (β–) emitters, such as nephrotoxicity and resistance to β– radiation, and its replacement by Auger electron-emitting radionuclides, such as 111In, might be a safer alternative, while higher efficacy might be achieved by specifically targeting the mitochondria, regarded as highly sensitive to ionizing radiation. To this end, we synthesized two chelators, TPP-PSMA-617 and TPP-G3-PSMA-617, containing a triphenylphosphonium (TPP) group in the PSMA-617 structure. One chelator also contains a cathepsin B cleavable (triglycine; G3) linker between the PSMA inhibitor and the DOTA moiety, for the intracellular generation of smaller radioconjugates.

Methods: The biological activity of the indium (111In and natIn) complexes of the novel chelators and of their single-targeted equivalents, directed at either PSMA (PSMA-617) or mitochondria (TPP-DOTA), was tested in three human prostate cell lines—two sublines of the PC-3 cancer cell line, one not expressing (PC-3 FLU) PSMA, the other overexpressing it (PC-3 PIP)—and in the PSMA-negative PNT2 cell line, derived from normal prostate tissue.

Results: All complexes exhibited low cytotoxicity against all cell lines and all those containing the PSMA-binding moiety strongly inhibited PSMA. Cell binding and internalization were substantial in PC-3 PIP cells and negligible in PC-3 FLU cells.

Conclusions: The preliminary assessment of our novel complexes showed that they possess characteristics suitable for highly targeted therapy against prostate cancer.

Funded by Foundation for Science and Technology, Portugal (UID/Multi/04349/2019, PTDC/MED-QUI/1554/2020, 2021.06783.BD, UIDB/00070/2020, UIDP/00070/2020).

Retroperitoneal lymphadenectomy in metastized testicular tumors: A retrospective analysis of patients treated at Centro Hospitalar Universitário de Coimbra

Mariana Vaz Pinto 1 , Edgar Tavares da Silva 1,2 , Ana Marta Ferreira 2 , Vasco Quaresma 2 , Belmiro Parada 1,2 , Arnaldo Figueiredo 1,2

1 Faculty of Medicine of University of Coimbra, Portugal; 2 Urology Department, Centro Hospitalar e Universitário de Coimbra, Portugal

Context/aims: Testicular cancer is the most common solid neoplasm in men between the ages of 20 and 40, during their peak of fertility. The incidence of this neoplasm has increased over the last decades, but its survival rate is high.

When metastasis occur, the retroperitoneal abdominal lymph nodes are most affected, leading to a 5-year-survival-rate of less than 40%. Therefore, retroperitoneal lymph node dissection (RPLND) plays a crucial role in the multimodal treatment of metastatic testicular tumors. It is a highly useful but complex surgical, associated with a relevant rate of complications and additional procedures.

Thus, the objective of this study is to analyse the experience of a Portuguese referral centre and compare it with national and international series.

Methods: We conducted a retrospective, single center, clinical study including all patients over 18 years old diagnosed with histologically confirmed germ cell neoplasia undergoing RPLND as part of the treatment for this tumor, between January 2007 and September 2023.

Statistical analysis was performed to study the type of surgery, neoplastic lesion, the patients’ age, hospitalization times, perioperative and long-term complications, as well as the need for accompanying procedures and consolidation chemotherapy.

Results: Out of 202 individuals, we obtained a total of 19 patients for whom RPLND was performed, 21% were seminomas and 74% were non-seminomas. Hospitalizations ranged from 3 to 58 days and the mean age was 35.2 years. Operative and perioperative complication rates were 21% and 26%; 30% of the patients required consolidation chemotherapy. Reconstruction of major vessels occurred in 26%, nephrectomy in 11%, and segmental enterectomies in 11%. Residual masses identified necrosis in 42%, teratoma in 32%, and viable germ cell tumor in 26%. At the data collection date, 84% of the patients were alive, with an average survival of 52 months. All deaths occurred within the first year.

Conclusions: The results obtained regarding complications and additional procedures show no significant differences from the reported in the literature. The histology of the primary tumor in most patients was non-seminoma.

Metabolomic Signature of Bladder Cancer

João Lorigo 1,2 , Edgar Tavares Silva 1,3,4 , Ana Salomé Pires 3,4,5 , Marco Alves 6 , Ana Margarida Abrantes 3,4,5 , Maria Filomena Botelho 3,4,5 , Arnaldo Figueiredo 1,3,4

1 Department of Urology, Centro Hospitalar e Universitário de Coimbra, Portugal; 2 Faculty of Medicine of University of Coimbra, Portugal; 3 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Institute of Biophysics, Faculty of Medicine, Coimbra, Portugal; 4 Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; 5 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal; 6 Centro Hospitalar Universitário de São João, Portugal

Context/aims: Bladder cancer is the ninth common tumor worldwide, and its incidence is slowly rising. Despite the high frequency and mortality associated with this disease, little has evolved recently regarding the diagnosis and follow-up methods of this type of tumor. In fact, cystoscopy and cytology are still standards for bladder cancer detection. It is critical for the development of less invasive and reliable diagnostic techniques for bladder cancer, other than cystoscopy and cytology. In this sense, metabolomics has recently emerged as a promising and powerful tool in the study of oncological diseases. It allows not only the discovery of new diagnostic biomarkers, but also markers of response to certain treatments and prognostic value. The objective of this study was to identify the metabolomic profile of bladder tumor specimens after transurethral resection (TURB).

Methods: It included 35 patients submitted to TURB in 2017. Bladder tumor fragments were analyzed using nuclear magnetic resonance spectrometry in order to characterize its metabolites. The differential metabolites were extracted and analyzed by univariate and multivariate analysis methods. Five years follow-up data were collected and compared with the corresponding metabolomic profile.

Results: From the total TURB specimens, 53,1% were low risk, 59,4% were classified as pTa, 31,3% as pT1, 9,4% as pT2 and 3,2% had associated CIS. At 5 years follow-up, 53,3% of the patients had recurrence of the disease with a median recurrence free survival of 20,1 months. In the recurrent group, we found a tendency to a different lactate (reduction of activity), phenylalanine and tryptophan metabolism (p<0.2). These findings meet previous observations.

Conclusions: Preliminary results showed changes in lactate, tryptophan and phenylalanine metabolic pathways. Studies of larger sample sizes are needed in order to identify useful biomarkers in clinical practice.

Unleashing the power: PARP inhibitor combinations for a breakthrough approach in pancreatic cancer

Eurico Pereira 1,2,3 , Joana Marques 1 , Paulo Simões 4 , Ana M. Abrantes 1,2,3 , Pedro Silva-Vaz 1,3,5 , Maria F. Botelho 1,2,3 , Ana S. Pires 1,2,3

Context/aims: Pancreatic cancer (PanC), with a 5-year survival rate of approximately 5%, is among the most lethal cancers globally needing novel and more effective treatment approaches. Poly(ADP-ribose) Polymerase inhibitors (PARPi) obstruct the ability of cancer cells to repair their damaged DNA. As a result, the combination of PARPi and DNA-damaging agents (DNA-DA) or external beam radiation therapy can escalate the quantity of DNA damage beyond the repair mechanisms, enhancing treatment effectiveness while minimizing adverse effects. This study aimed to investigate the potential of combining PARPi with DNA-DA or radiotherapy to improve PanC treatment outcomes.

Methods: Olaparib (OLA), a PARPi, was combined with DNA-DA (irinotecan or oxaliplatin) in the MIA PaCa-2 cell line. The drugs were combined at a constant ratio based on their IC 50 . Three combination regimens were tested: simultaneous administration, OLA administered 24 hours before or after each DNA-DA. The combinations were evaluated 24, 48, and 72 hours after treatment using the SRB assay. The Chou-Talalay approach was used to assess synergism. OLA was also combined with radiotherapy at a fixed non-cytotoxic concentration (1µM) to evaluate radiosensitization using the clonogenic assay. The sensitization enhancement ratio (SER) was used to quantify the radiosensitization effect.

Results: OLA combined with DNA-DA, especially irinotecan, showed a synergistic effect, that was more pronounced when OLA was administered 24 hours after the irinotecan. A slight synergistic effect was observed with OLA administered 24 hours after oxaliplatin. Additionally, OLA demonstrated a radiosensitizing effect in the MIA PaCa-2 cell line with a SER=1.39.

Conclusions: This study demonstrates the potential of combining OLA with irinotecan or oxaliplatin (in a sequential regimen) and highlights OLA’s radiosensitizing effect as a future therapeutic approach for PanC. The induction of single-strand breaks by the DNA-DA or radiotherapy with the consequent inhibition of their repair by OLA may help explain the synergistic and radiosensitizing effects observed. Further investigation is warranted to clarify such mechanisms.

Cellular response of human skin keratinocytes to external beam radiotherapy

Inês A. Marques 1,2,3* , Diana Figueiredo 1,4* , Ana Salomé Pires 1,2,5 , Raquel Alves 2,5,6 , Ana Cristina Gonçalves 2,5,6 , Paulo César Simões 7 , Maria Filomena Botelho 1,2,5 , Ana Margarida Abrantes 1,2,5 , Tiago A. Azevedo 1,2 , João Carvalho 1 , Ana Margarida Abrantes 2

1 Univ Coimbra, iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Institute of Biophysics, Faculty of Medicine, Coimbra, Portugal

2 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal; 3 Univ Coimbra, Faculty of Pharmacy, Coimbra, Portugal; 4 Univ Coimbra, Faculty of Science and Technology, Coimbra, Portugal; 5 Clinical Academic Centre of Coimbra (CACC), Coimbra, Portugal; 6 Univ Coimbra, iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Laboratory of Oncobiology and Hematology (LOH), University Clinics of Hematology and Oncology, Faculty of Medicine, Coimbra, Portugal; 7 Department of radiotherapy, Centro Hospitalar e Universitário de Coimbra, Portugal (*both authors contributed equally to the work) Contact: iafmarques@uc.pt

Context/aims: Despite innovations in external beam radiotherapy (ERT) techniques, skin complications and the possible secondary skin diseases associated with ionizing radiation (IR) continue to be a major concern for patients. Hence, knowledge of the biological effects of IR on the skin is essential and has not yet been clarified.

Thus, the main goal of this work is to evaluate the IR-induced biological effects of ERT in human skin keratinocytes.

Methods: A human skin keratinocytes cell line (HaCaT) were exposed to therapeutic doses of X-rays (0-10 Gy) within a proper phantom (0.5x10 6 cels/mL), using a TrueBeam linear acelarator of 6MV energy. Thus, clonogenic assay were done to access radiosensitibility and obtain mean lethal dose (DL50). Thus, flow cytometry was used for the evaluation of biological effects induced by ERT on cell viability (annexin V/prodium iodide) and cell cycle (propidium iodide/RNase) and genotoxicity was assessed by micronucleus assay, 7 days post-irradiation. Cell migration (scratch assay) was evaluated till 6 days post-irradiation.

Results: The exposition of HaCaT cells to 0-10 Gy of X-rays lead to a decrease in cell survival, adjusted to a linear quadratic model, with a LD50=3.64 Gy (CI95% 3.49-3.80 Gy) and a SF(2Gy) of 0.82 (CI95% 0.78-0.86). The irradiation with 2Gy or LD50 dose (4Gy) induced a decrease in cell viability to 78,25 ± 3,73% (p<0,05) and 66,00 ± 11,74%

(p<0,001), respectively. This is associated with an increase in cell death by initial apoptosis as well as with an arrest in S ir G2/M phases and a decrease in cell migration, after irradiation with LD50 or higher doses. The results showed an increase in genotoxicity, even after exposure to 2Gy.

Conclusions: Exposure of human keratinocytes to therapeutic doses of IR induces long-term radiobiological effects and genotoxicity, promoting cell death. Moreover, the decrease in cell migration, even at 2Gy, could be correlated with the severity of skin injuries, and this should be considered during ERT planning.

Funding: PhD Scholarship to I.A.M (SFRH/BD/136973/2018, FCT and FSE). FCT strategic projects (UIDB/04539/2020, UIDP/04539/2020); COMPETE-FEDER (POCI-01-0145-FEDER-007440).

Modeling the radiobiological effect of radiation using spheroids as an in silico model

Tiago A. Azevedo 1,2 , João Carvalho 1 , Ana Margarida Abrantes 2

1 CFisUC, Department of Physics, University of Coimbra; 2 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra

Context/aims: While our understanding of the hallmarks of cancer has advanced tremendously, current forms of therapy, such as radiotherapy, don’t yet make use of their full potential. Due to the enormous progress in computational power and tools, we can bridge the different domains of radiotherapy using computational simulations.

Methods: In this work we developed a computational model of cell growth based on a Cellular-Potts Model (CPM). This model is then imported into TOPAS-nBio software package. This software simulates the irradiation stage with the creation of radicals due to radiolysis of water and can be used to realistically describe the DNA structure. This allows us to evaluate the impact of direct and indirect effects, and the outcomes in cell survival due to double strand breaks (DSB), creating a coupled model of spheroid growth and radiation treatment.

Results: The change of cell type is accomplished by incorporating oxygen diffusion. As the spheroid grows, some regions start to become oxygen deprived, which corresponds to hypoxic and necrotic regions, while cells at the rim can access sufficient oxygen to support their proliferating activity. Irradiation stage can start at any chosen moment. With every irradiation, spheroid size decreases, with different treatment regimens leading to morphologically different spheroids. The exposure of a single cell to different X-ray irradiation doses generated a uniform distribution of DSB, with most damage being due to indirect effects, highlighting the need of simulation of the chemical stage. The number of clustered DSB can more accurately be fitted to the LQ equation than simply the total number of DSB.

Conclusions: The CPM produces a spheroid that grows following a Gompertz function and responds to the oxygen levels, leading to the formation a layered structure of hypoxic and necrotic zones. The implementation of the irradiation phase was successful as the volume of the spheroid decreases with the increase in dose given and in the number of irradiations. These preliminary results provide a roadmap to build a comprehensive toolkit for simulating multicellular growth and DNA damage.

Steroidal oximes as a potential new therapeutic for different types of cancer

Ana R. Gomes 1,2 , Elisiário J. Tavares-da-Silva 2 , Saúl C. Costa 3 , Ana M. Abrantes 1,4,5 , Ana C. Gonçalves 4,5,6 , Raquel Alves 4,5,6 , Ana Bela Sarmento 4,6,7 , Maria F. Botelho 1,4,5 , Fernanda M.F. Roleira 2 , Ana S. Pires 1,4,5

1 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Biophysics Institute of Faculty of Medicine, Azinhaga de Santa Comba, Pólo III - Pólo das Ciências da Saúde, Coimbra, Portugal

2 Univ Coimbra, CERES, Faculty of Pharmacy, Laboratory of Pharmaceutical Chemistry, Azinhaga de Santa Comba, Pólo III - Pólo das Ciências da Saúde, Coimbra, Portugal

3 Univ Coimbra, Faculty of Pharmacy, Laboratory of Pharmaceutical Chemistry, Azinhaga de Santa Comba, Pólo III - Pólo das Ciências da Saúde, Coimbra, Portugal

4 Univ Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal

5 Clinical Academic Center of Coimbra (CACC), Praceta Professor Mota Pinto, Coimbra, Portugal

6 Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Laboratory of Oncobiology and Hematology and University Clinics of Hematology and Oncology, Faculty of Medicine

7 Hematology Service, Centro Hospital e Universitário de Coimbra (CHUC), Coimbra, Portugal

Context/aims: Cancer remains one of the most life-threatening diseases worldwide despite the number of therapies currently available. Both steroids and oximes have been associated with antitumor activity. Moreover, previous studies by our group demonstrated that steroidal oximes were also very potent against several types of cancer. Thus, we decided to design and synthesize new steroidal oximes and evaluated their potential antitumor activity against lung and prostate cancer cells, aiming to find alternative cytotoxic compounds for cancer treatment.

Methods: Compounds OX1, OX2 and EP2OX and their parent compounds P1, OL2 and EP2, were synthesized, and their cytotoxicity evaluated in H1299 and PC3 cell lines through SRB assay after the treatment of the cells with the compounds (1-75 µM). Cell viability, cell death profile, alterations on cell cycle and mitochondrial membrane potential were assessed by flow cytometry.

Results: OX1 and EP2OX decreased H1299 and PC3 cell proliferation in a dose-dependent manner but OX2 did not have the same effect. Overall, the parent compounds decreased cell proliferation in a less pronounced way, proving that the introduction of an oxime group was beneficial for the cytotoxicity displayed. Moreover, the best compound was EP2OX in both cell lines with IC 50 values of 1.1 and 2.02 µM in H1299 and PC3, respectively. OX1 was also quite active with IC 50 values of 18.69 µM (H1299) and 29.95 µM (PC3). Flow cytometry studies showed that EP2OX was able to decrease cell viability in H1299 and PC3 cells by causing apoptosis and/or necrosis, which was accompanied by a blockage at phases G 2 /M and S, depending on the cell line and concentration. Considering the mitochondrial membrane potential, EP2OX induced mitochondrial dysfunction, which might also be related to apoptosis.

Conclusions: Our results show that EP2OX possesses a beneficial antitumor effect from the introduction of the oxime group, which is mediated by apoptosis/necrosis. This effect encourages further studies on its mechanism of action and selectivity in order to discover new molecules for cancer treatment.

Funding: Foundation for Science and Technology (FCT), Portugal (Strategic Projects UID/NEU/04539/2019, UIDB/04539/2020, UIDP/04539/2020); COMPETE-FEDER (POCI-01-0145-FEDER-007440); PhD Fellowship from FCT and European Social Funding of Ana R. Gomes (UI/BD/150865/2021).

Dual immunohistochemistry p16 and Ki67 (CINtec®PLUS) in cervical screening – increasing its profitability to detect higher-risk patients?

José Ruivo, Inês Ferreira, Clara Cabral, Salomé Pessoa, Ilda Simões, Magda Mendes, Catarina Ferreira, Paula Agapito, Rui Caetano Oliveira

Centro de Anatomia Patológica Germano de Sousa

Context/aims: The screening of cervical cancer encompasses several methods, from cytology and HPV genotyping. The dual immunohistochemistry for p16 and ki67 (CINtec®PLUS) allows us to assess the viral genome integration into the cell. The co-test is indicated in women with abnormal cytology results and HPV-positive, but it is not always positive. This work aims is to characterize our population of patients with CINtec®PLUS and correlate with cytology features and HPV genotype to try to assess better predictive status for CINtec®PLUS positivity.

Methods: Retrospective study from January-April/2023. Only patients with results for the following three modalities were considered: cytology, HPV detection (genotyping/COBAS) and CINtec®PLUS. Histology and age status were not considered. Cytology results were grouped: negative, ASC-US and positive for lesion (ASC-H/LSIL/HSIL). Correlation was performed with the Chi-square test (p<0.05).

Results: 201 cases were obtained. For CINtec®PLUS 153 cases were negative (76.1%) and 48 cases negative (23.9%). Cytology results: 108 (53.7%) were negative, 48 (23.9%) had ASC-US and 45 (22.4%) were positive for lesion.

HPV infection was detected in 132 patients (65.7%), with the following genotypes: HPV 16 in 21 (10.4%), HPV 18 in 9 (4.5%), HPV 31 in 13 (6.5%), HPV 55 in 11 (5.5%), HPV 52 in 6(3%), HPV 53, 58 and 66 in 8 patients each (4%); in the COBAS methology a high-risk HPV was present in 24.

A correlation was observed between CINtec®PLUS and HPV positive (p<0.001) and between CINtec®PLUS and epithelial lesion on cytology (p<0.001). There was a higher rate of CINtec®PLUS positivity for HPV 16/53/58 (p=0,006), with a lower rate for HPV 31/51/66 (p=0.006). When HPV is described only as positive for high-risk subtypes there is a lower frequency of CINtec®PLUS positivy, but without statistical significance.

Conclusions: Our results support CINtec®PLUS performance in patients with cytological lesions and HPV positivity. The efficacy of the CINtec®PLUS can be increased by performing in specific HPV subtypes such as 16, 53, 58, reinforcing the role for genotyping rather than low vs. high-grade HPV positivity results.

Serological responses to acute exercise modulate in vitro growth and bioenergetics of human lung carcinoma cells in a cohort-dependent manner

Carlos M. Soares 1,2 , João P. Moura 3 , Ana Pedrosa 1 , Luís Rama 1 , Ana M. Teixeira 1 , Ana M. Urbano 2,3,4

1 Research Unit for Sport and Physical Activity (CIDAF), Faculty of Sport Sciences and Physical Education, University of Coimbra, Portugal; 2 Molecular Physical Chemistry R&D Unit, University of Coimbra, Portugal; 3 Department of Life Sciences, University of Coimbra, Portugal; 4 Center of Investigation in Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, Portugal

Context/aims: There is extensive evidence from observational studies of a link between physical activity and lower cancer risk and mortality. This link may result, at least partially, from alterations in serum composition that compromise its ability to stimulate cellular proliferation. To investigate this hypothesis, several in vitro studies compared the viability of cultured human cancer cells exposed to human serum conditioned by an acute exercise intervention (exercise-conditioned serum) to that of cultures exposed to serum obtained before the intervention (control serum). Despite significant study heterogeneity (e.g., cohort type, exercise intervention, cell line and viability assay), our meta-analysis of the results showed that viability in cultures exposed to exercise-conditioned serum was lower than that of control cultures, with a large overall effect size that became more pronounced with the intensity of the exercise [1]. Our aim was to strengthen and expand those observations.

Methods: A549 human lung cancer cells were exposed to serum collected from two distinct cohorts – master athletes and volunteers who did not meet the WHO guidelines on physical activity for health – obtained before and after an acute exercise intervention, and their growth characteristics (SRB, clonogenic and migration assays) and bioenergetics (colorimetric assay) were analyzed.

Results: Our results showed that, compared to control serum, serum conditioned by acute exercise decreased proliferation, clonogenic potential (in terms of colony size) and lactic acid fermentation in A549 cells. There was also suggestion that it improved cell adhesion to the substratum.

Conclusions: Altogether, our results provided further evidence that serological responses to acute exercise shift cultured human cancer cells to a less transformed phenotype, ultimately supporting a causal relationship between physical activity and cancer risk and mortality. They also showed that serum-mediated effects of acute exercise can depend on cohort type.

Study funded by CIMAGO (10/22) and FCT (UIDB/00070/2020; UIDP/00070/2020), Portugal.

[1] Soares et al (2021) Exerc Immunol Rev 27, 24–41.

Comparison of sealing ability of two Endodontic Thermoplastic Obturation techniques.

Siri Paulo 1,2,3 , Inês Quadros 1 , José Martinho 1,2,3 , Catarina Carvalho 1,2,3 , Ana S. Pires 2,3,4 , Ana Margarida Abrantes 2,3,4 , Manuel Marques Ferreira 1,2,3

1 Institute of Endodontics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2 Coimbra Institute for Clinical and Biomedical Research (iCBR), Area of Environment, Genetics and Oncobi-ology (CIMAGO), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 3 Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal; 4 Institute of Biophysics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Context/aims: The aim of the present study is to compare the sealing ability of two different warm gutta-percha obturation techniques: continuous wave of condensation and a core-carrier system through the evaluation of the apical microleakage.

Methods: 68 single-rooted mandibular and maxillary extracted human teeth were prepared using rotary NiTi files in a reciprocating motion. The specimens were randomly divided into four groups: negative control group (CN) – obturated specimens completely covered in nail varnish; positive control group (CP) – prepared, non-obturated specimens; group A - obturated with GuttaCore Pink carriers and group B - obturated with the continuous wave of condensation technique using Calamus Dual. The last 3 groups (CP, A and B) were covered with nail varnish except for the 2 apical millimeters. To evaluate the apical microleakage the specimens were incubated in 99m Tc, a radioisotope widely used in nuclear medicine. The scintigraphic images were captured by a gamma camera with quantitative results. The obtained values were statistically analyzed at a significance level of 0.05, or with a confidence level of 95% (Kruskal-Wallis test).

Results: The total counts represent the amount of infiltrated radioisotope. The negative control group presented an average of the total counts of 181,5 ± 69,44, as for the positive control group the counts were 40183,25 ± 12127,57. This result led to validate the evaluation method (p<0,001). Group A and B presented total counts of 18799,29 ± 3246,95 and of 17910,8667 ± 4522,97, respectively. There was no significant difference between group A and group B in terms of apical leakage for a confidence level of 95%. (p=0,928).

Conclusions: Both obturation techniques showed apical leakage through the infiltration of the 99m Tc significantly inferior when compared to the positive control group. This suggests that both techniques are valuable in terms of preventing apical leakage, within the limitations of this evaluation method. The difference between the counts of groups A and B was not statistically significant. However, further studies with other evaluation methods are needed to clarify which method provides the most adequate, tridimensional and hermetic seal.

In vitro cell viability of B-doped a-C:H coatings for Orthodontics

Duarte Ferreira 1,3 , António Fróis 2,3 , Cristina Santos Louro 2 , Ana Cristina Santos 2,3,4

1 Faculty of Sciences and Technology, University of Coimbra, Portugal; 2 CEMMPRE, Department of Mechanical Engineering, Faculty of Sciences and Technology, University of Coimbra, Portugal; 3 Biophysics Institute, Faculty of Medicine, University of Coimbra; Coimbra Institute for Clinical and Biomedical Research/Centre for Innovative Biomedicine and Biotechnology (iCBR/cibb) - Area of Environment Genetics and Oncobiology (CIMAGO), Coimbra, Portugal

Context/aims: Technological advancements have significantly impacted the health field, particularly the development of biomaterials for biomedical applications. In Dentistry, Orthodontics has extensively explored the use of materials such as nickel-titanium (Ni-Ti) and stainless-steel alloys, due to their unique properties. However, concerns like corrosion and friction associated with these metallic alloys prompted research into their coating to enhance biocompatibility, corrosion resistance, and to reduce friction.

This study focuses on the development of carbon-based coatings (Diamond-Like Carbon - DLC) to improve the properties of biomaterials, specifically stainless steel 316L(AISI). The aim is to assess the biocompatibility of these coatings by simulating oral conditions, immersing samples in artificial saliva for 30 days. Cell viability analysis, using the MTT assay, was performed after incubation with macrophages, fibroblasts, and their co-culture trying to provide insightful results.

Methods: Hydrogenated amorphous carbon (a-C:H) was deposited onto the stainless-steel surface through reactive magnetron sputtering, using a carbon target and reactive gas (CH 4 ). The samples were then immersed in artificial saliva at 37°C (30 days). The resulting sterile eluates were incubated in vitro with the cells, assessing their viability through the MTT colorimetric assay.

Results: MTT results were statistically analysed using the RStudio, suggesting that the DLC1 coating (boron-doped DLC sample deposited with CH 4 at a flow rate of 1.5 SCCM) is promising. However, a higher number of experiments will increase statistical robustness.

Conclusions: This study indicates that the design with co-culture of macrophages & fibroblasts is crucial for a more real simulation of oral cavity features. The obtained results are promising for predicting in vivo behaviour in a future experimental animal model. The research underscores the significance of DLC coatings in improving biocompatibility, corrosion resistance, and friction reduction of biomaterials for orthodontic applications.

Acknowledgements: Foundation for Science and Technology (FCT), Portugal (SFRH/BD/143905/2019 grant, and UIDB/00285/2020 and LA/P/0112/2020 projects); COMPETE-FEDER-POCI programs CENTRO-01-0247-FEDER-039880 and PTDC/EMD-EMD/21402020. A special thank you to Centro Hospitalar da Universidade de Coimbra (CHUC), to iCBR-FMUC Vivarium , to the Biophysics Institute of FMUC, to University of Aveiro, and to RI.TE.

Potential of nanotechnology in combination with natural extracts for therapeutic efficacy in dermatological diseases: a review

Cecília Nascimento 1,2 , Lillian Barros 1,2 , Tayse da Silveira 1,2

1 Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Portugal; 2 Laboratório Associado para a Sustentabilidade e Tecnologia em Regiões de Montanha (SusTEC), Instituto Politécnico de Bragança, Portugal

Context: Dermatological diseases, such as atopic dermatitis, skin cancer, and autoimmune dermatological disease (e.g. psoriasis), have a huge prevalence, impacting quality of life in population. It is important to search for effective treatments that provide better conditions - physical, emotional, and social. Compounds extracted from natural products are an excellent alternative to synthetic drugs and are increasingly preferred by patients due to good therapeutic efficacy coupled with low secondary effects. In this context, nanotechnology has been the subject of growing research for drug encapsulation, due to its advantages as compared to administration in the free form (increase bioavailability, controlled release, and reduced side effects).

Methods: Therefore, the objective of this review is to collect data from literature and critically analyze the potential of nanotechnology to be used with natural extracts to provide benefits for therapeutic efficacy in the treatment of skin diseases.

Results: According to the results found from in vitro and in vivo analysis, the use of natural extracts encapsulated in nanoparticles amplifies their therapeutic effects. For example, in in vivo experimental models for psoriais (mice), administration of solid lipid nanoparticles loaded with resveratrol, curcumin and capsaicin extracted from Vitis vinifera L., Curcuma longa L., Capsicum annum L., respectively, demonstrated high anti-inflammatory potential, suppressing the production of interleukin-6. In another study, transfersomes loaded with glycyrrhizic acid (a saponin), demonstrated a reduction in atopic dermatitis symptoms. Quercetin, present in fruits and vegetables, encapsulated in nanostructured lipid carriers demonstrated promising effects in reducing skin permeabilization for psoriasis or skin cancer.

Conclusions: These findings highlight the growing importance as well as the beneficial role of nanoparticles loaded with natural extracts for the treatment of dermatological diseases. In the future, it is necessary to optimize this synergy for innovative dermatological treatments, unlocking better therapeutic results and applications to improve efficacy and quality of life.

Acknowledgements: The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2020). L. Barros also thanks FCT through the institutional scientific employment program–contract for her contract. T. Silveira, thanks to Fundação BPI La Caixa, within project titled ‘AquaeVitae - Água Termal Como Fonte de Vida e Saúde” - “PROMOVE – O futuro do Interior” call 2020.

New calcium silicate-based cement for Endodontics in an animal model - the important contribution of imaging methods

Nuno Braz 1,2,3 , Bruna Santos 1,2,3 , Sofia Brás 1,2,3 , Benedetta Marin 1,4 , Fabiana Ribeiro 5 , Pedro Encarnação 5 , Ana Luísa Silva 5,6 , Pedro Correia 5,6 , Filipe Castro 6 , João Veloso 5,6 , Claúdia Brites 7,8 , João Miguel Santos 7,8 , Ana Cristina Santos 1,2

1 Institute of Biophysics, Faculty of Medicine of the University of Coimbra, Coimbra, Portugal; 2 iCBR-Coimbra Institute for Clinical and Biomedical Research –Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB -Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 3 Faculty of Sciences and Technology of the University of Coimbra, Coimbra, Portugal; 4 Dipartimento di Ingegneria dell’Informazione DEI, Università Degli Studi di Padova,Italy; 5 Universityof Aveiro, Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, Aveiro, Portugal; 6 RI-TE Radiation Imaging Technologies, Lda, Ílhavo, Portugal; 7 Institute of Endodontics, Faculty of Medicine of the Universityof Coimbra, Portugal; 8 Centre for Innovation and Research in Oral Sciences (CIROS).

Context/aims: Obturation of root canals is a fundamental step for the long-term success of endodontic treatment. One of the main requirements of the materials used for this purpose is their biocompatibility, not triggering nor potentiating an inflammatory or foreign body reaction ofthe periradicular tissues. Recently, new endodontic sealing cements, based upon calcium silicate, have been introduced in the market, after showing in vitro the ability to induce biomineralization, being considered bioactive materials. The aim of this project is to evaluate the biocompatibility of the new AH Plus ® Bioceramic Sealer cement, in an animalmodel (BALB/c (+) mice).

Methods: Comparison is made with 2 other cements used in the clinic: AH Plus Jet ® (gold standard) and TotalFill ® BC Sealer™. All materials were implanted s.c. in the dorsum of the mice, to assess their in vivo biocompatibility and bioactivity. Signs of inflammatory response were studied using 2 imaging techniques: microPET (with a novel prototype) and optical microscopy. The acute reaction (8 days after implantation) has been evaluated.

Results: The comparative histopathological analysis of the materials vs control, allowed to ascertain the AH Plus ® Bioceramic Sealer good biocompatibility. Histopathological features were compared with functional images acquired with the easyPet.3D (after i.p. administration of [ 18 F]-FDG). It was possible to infer the potential of this minimally invasive method. Thelongitudinal feature of the in vivo design, enabled to evaluate the evolution of the inflammatory response, following a correct detection of the corresponding reaction/position of the implanted materials.

Conclusions: easyPET.3D is able to detect signs of slight inflammatory reaction caused by the materials’ implantation. The new material showed reduced signs of acute inflammation, according to histopathological analysis of the collected tissue samples. The results obtained in this study, carried out in mice - due to the dimensions of the easyPET.3D - are superimposable to the studies of equivalent materials carried out in rats (already published by the group). Studies of chronic reaction (30 days) are in course.

Should we continue to report IMMP2L gene deletions detected by array-CGH?

Susana Isabel Ferreira 1 , Mariana Val 1 , Luís Miguel Pires 1 , Alexandra Mascarenhas 1 , Joana Barbosa Melo 1,2,3 , Isabel Marques Carreira 1,2,3

1 Laboratório de Citogenética e Genómica, Faculdade de Medicina da Universidade de Coimbra, Coimbra, Portugal; 2 CIBB- Centro de Inovação em Biomedicina e Biotecnologia, Universidade de Coimbra, Portugal; 3 iCBR CIMAGO – Centro Investigação em Meio Ambiente Genética e Oncobiologia, Faculdade de Medicina da Universidade de Coimbra, Portugal

Context/aims: IMMP2L gene has been reported as a candidate gene for Tourette syndrome, autism spectrum disorder (ASD) and additional neurodevelopmental disorders such as attention-deficit hyperactivity, speech-language delay and various developmental delays. As array Comparative Genomic Hybridization (array-CGH) has become a global technique for the diagnosis of genomic imbalances in patients with the referred phenotypes, several IMMP2L gene deletions have been reported. However, there is some controversy in the literature concerning this association because some studies do not support it, and also because deletions have been observed in healthy subjects, what might suggest incomplete penetrance.

Methods: We have realized Agilent 180K oligonucleotide array-CGH in more than 4000 blood samples from patients with ASD, intellectual disability, language delay or global developmental delay.

Results: We detected 34 patients, 24 males and 10 females, with IMMP2L gene partial deletions, most of them involving the first exons of the gene. Inheritance determination was only possible in 7 patients, being maternally inherited in 5 cases and paternal in 2.

Conclusions: IMMP2L gene encodes a protein involved in processing the signal peptide sequences used to direct mitochondrial proteins to the mitochondria. In mice heterozygous for an inactive Immp2l gene, increase of ischemic brain damage and apoptosis of cerebellar neurons have been detected, and this observation lead to the notion that IMMP2L alterations may predispose to cognitive/behavioral disturbances in humans. Zhang and collaborators conducted a comprehensive meta-analysis to quantify the association of IMMP2L deletions with ASD using 5,568 cases and 10,279 controls and found no evidence of association.

We struggle with the question if we should continue reporting aCGH detected IMMP2L gene deletions, since most of them are inherited from normal progenitors, and considering the controversial literature reports. We also question the value of this diagnosis for the clinical management and genetics counseling of the diagnosed patients, and how to deal with genetics counseling in subsequent pregnancies of a couple with an affected child and a carrier normal progenitor.

Integrative Analysis of Primary Glioblastomas Cultures through Array Comparative Genomic Hybridization and Cytogenetics

Alexandra Mascarenhas 1 , Domingos Roda 2 , Cláudia Pais 1 , José Luís Alves 3 , Joana Barbosa Melo 1,4,5 , Isabel Marques Carreira 1,4,5 , Ilda Patrícia Ribeiro 1,4,5

1 Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2 Algarve Radiation Oncology Unit – Joaquim Chaves Saúde (JCS), Faro, Portugal; 3 Centro Hospitalar e Universitário de Coimbra, CHUC, EPE, Serviço de Neurocirurgia, Coimbra, Portugal; 4 University of Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) and Center of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, Coimbra, Portugal; 5 CIBB – Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal and Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal.

Context/aims: Glioblastomas are a diverse group of primary brain tumors with complex genomic alterations that contribute to their heterogeneous nature and different clinical outcomes. Important advances have been made in understanding the molecular biology of glioblastomas. Several relevant biomarkers with a potential clinical interest have been identified using various techniques. This study focuses in a comprehensive approach, combining Array Comparative Genomic Hybridization (aCGH) and cytogenetic analyses, to undertand the genomic landscape of glioblastomas. This Cytogenomic analysis enables high-resolution profiling of genomic alterations of these tumors.

Methods: This study aimed to perform a cytogenomic characterization of both, a commercial glioblastoma cell line, U87 from the ATCC, and four established primary cell cultures from glioblastoma tumor samples. The characterization was assessed by karyotype and aCGH using sex-matched healthy controls.

Results: U87 is a hypertriploid cell line, with a modal chromosome number of 79. The most common rearrangements in this cell line are translocations, some in the centromere/near-centromeric regions. Numerical and structural chromosomal abnormalities were found in both U87 cell line and one primary cell culture. In the other three primary cell lines the most frequently rearrangements observed were numerical, namelly trisomy or tetrasomy of chromosome 7 and monosomies of chromosomes 10, 14 and the loss of the Y chromosome.

Conclusions: Cytogenomic analyses of tumour cell lines have been helpful to find genetic alterations that seem to have a major role in cancer development and progression, thus having the potential to identify biomarkers with clinical and therapeutic value, such as partial or complete chromosome 7 gain and chromosome 10 loss in glioblastoma diagnosis criteria. Recent studies have advocated for testing of these markers as part of prognostic stratification due to the amplification of the EGFR locus in chromosome 7p and loss of the PTEN locus in 10q. These preliminary findings can be useful to define the genetic profile of glioblastomas and to attempt a correlation with the stage of the disease.

NRXN1 intragenic deletions in patients with neurodevelopmental disorders

Mariana Val 1 , Susana I. Ferreira 1 , Luís M. Pires 1 , Alexandra Mascarenhas 1 , Isabel M. Carreira 1,2,3 , Joana B. Melo 1,2,3

1 Laboratório de Citogenética e Genómica, Faculdade de Medicina da Universidade Coimbra, Coimbra, Portugal; 2 iCBR-CIMAGO – Centro de Investigação em Meio Ambiente, Genética e Oncobiologia, Faculdade de Medicina da Universidade Coimbra, Coimbra, Portugal; 3 CIBB – Centro de Inovação em Biomedicina e Biotecnologia, Faculdade de Medicina da Universidade de Coimbra, Coimbra, Portugal

Context/aims: Neurexin 1 ( NRXN1 ) is a presynaptic neuronal adhesion molecule that binds postsynaptic neuroligins forming a neurexin/neuroligin complex at synapses which plays an important role in synaptic formation and function. A wide spectrum of phenotypes associated with heterozygous intragenic microdeletions of NRXN1 have been reported including autism spectrum disorder (ASD), intellectual disability (ID), seizures, schizophrenia and bipolar disorder. The highly variable expressivity and incomplete penetrance of deletions in this gene make genetic counseling difficult.

Methods: The cases referred to aCGH (Agilent, 4x180K) with NRXN1 intragenic deletions were searched in our laboratory database. Indication for array-CGH testing included psychomotor developmental delay, ID, ASD and dysmorphic features.

Results: Thirty-three probands with NRXN1 intragenic deletions were identified. Of those, 4 overlapped two or three exons ( NM_001330078 ), ranging in size from 64 to 325kb, while the deletions in the remaining 29 patients involved only an intron and the size ranged from 11 to 148kb. An 11kb deletion in intron 17 was recurrent in our cohort (18/29 cases). One exonic and 5 intronic NRXN1 deletions were inherited; in the other cases inheritance was not established. aCGH identified an additional variant of unknown significance (VUS) in 2 patients with NRXN1 exonic deletions. In 10 cases with an intronic NRXN1 deletion, 13 additional reportable CNVs were detected - 12 VUS and 1 classified as likely pathogenic.

Conclusions: Rare exonic NRXN1 deletions were first identified in patients with ASD and developmental delay (DD)/ID; in our cohort, despite the small number of exonic deletions, all the patients presented with ASD, and one of them also revealed psychomotor developmental delay. Recurrent intronic NRXN1 deletions have been reported previously. However, there is not enough evidence to suggest that all intronic deletions in this gene are benign. Additional studies including inheritance determination and accurate phenotypic characterization of patients would be useful to better understand the incomplete penetrance and variable expression of intragenic NRXN1 deletions.

Detection of a germline pathogenic variant in BRCA1 gene in a patient with ovarian cancer: a clinical case

Regina Arantes 1 , Beatriz Teixeira 2 , Marta Souto 1 , Márcia Martins 3 , Osvaldo Moutinho 4 , Rosário Pinto Leite 1

1 Genetics/Andrology Laboratory, Hospital Center of Trás-os-Montes and Alto Douro, Portugal; 2 MSc student in Clinical Laboratory Genetics, Faculty of Medicine of University of Coimbra, Portugal; 3 Genetic Consultation, Hospital Center of Trás-os-Montes and Alto Douro, Portugal; 4 Department of Woman and Child, Hospital Center of Trás-os-Montes and Alto Douro, Portugal

Context/aims: Hereditary breast and ovarian cancer (HBOC) is a genetic syndrome usually caused by germline pathogenic or probably pathogenic variants in BRCA1 and BRCA2 genes, with an autosomal dominant inheritance, incomplete penetrance and variable expressivity. Carriers of these variants have an increased risk to develop various tumors, particularly breast (45-65%) and ovary (20-50%).

The autors present an ovarian cancer patient with a germline pathogenic variant in BRCA1 gene, as well as its implications for the family.

Clinical case: An 87-year-old woman with ovarian cancer was referenced by oncology consultation for tumor genetic testing. Next Generation Sequencing panel for breast/ovarian genes was performed in a a formalin-fixed paraffin-embebbed (FFPE) sample obtained by an ovarian biopsy. Two pathogenic variants were detected: a variant in BRCA1 gene (c.791_794delGTTC (p.S264fs*33)), with 78% allelic fraction and a variant in TP53 gene (c.365_366delTG (p.V122fs*26)), with 68% allelic fraction.

Since pathogenic variants in both BRCA1 and TP53 genes can be responsible for two distinct tumor genetic syndromes (HBOC syndrome and Li-Fraumeni syndrome, respectively) if of germline origin, an additional germline testing was performed. The same pathogenic variant in BRCA1 gene from the FFPE sample (c.791_794delGTTC (p.S264fs*33)) was deteted in the blood sample, with 50.98% allelic fraction (heterozygous).

A family study was requested by genetic consultation. The proband has two healthy daughters, ages 60 and 63. The 60-year-old daughter’s germline testing shows no pathogeneic variant, whereas the 63-year-old daughter’s shows the same pathogenic variant BRCA1 gene form the mother.

Discussion and conclusion: The proband and her daughter have been diagnosed with HBOC syndrome with the identification of the germline pathogenic variation c.791_794delGTTC (p.S264fs*33) in BRCA1 gene.

Family study is crucial to detect carriers of germline pathogenic variant in BRCA1, which enables improved screening, medical surveillance and early detection of breast and ovarian cancer, should they arise in the future.

Essential thrombocythemia with 3p deletion: Importance of cytogenetic study

Marta Souto 1,3 , Rita Tenreiro 2 , Catarina Pinto 1,3 , Manuel Cunha 2 , Osvaldo Moutinho 3 , Rosário Pinto-Leite 1,3

1 Laboratório de Genética, Centro Hospitalar Trás-os-Montes e Alto Douro (CHTMAD); 2 Serviço de Hematologia, CHTMAD, Vila Real, Portugal; 3 Departamento da Mulher e da Criança, CHTMAD, Vila Real, Portugal.

Introduction: Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm characterized by a consistently increased platelet count in the absence of a secondary cause. Chromosome abnormalities are present in less than 10%, but no specific aberration has been defined to date.

The authors present an ET patient with chromosome 3p deletion.

Clinical case: 81-year-old man referred to Haematology consultation to investigate thrombocytosis that had persisted for over six months. In initial blood count, hemoglobin was 13.9, leucocytes were 9.99 (neutrophils were 7.2), and platelets were 578 G/L. A sequential analysis of JAK2 V617F, CALR , and MPL mutations was performed (all negative). A bone marrow examination was carried out in the absence of a definitive diagnosis. The myelogram displays a numerous morphologically normal megakaryocytes and bone marrow biopsy exhibiting enlarged megakaryocytic line (CD61+) with heterogeneous morphology and Grade 1 interstitial reticulin fibrosis. A chromosome 3p deletion was detected in medullary karyotype: 46,XY,del(3)(p14p12)[7]/46,XY[13]. An ET diagnosis was established and cytoreduction therapy was initiated with 500 mg/d of Hydroxyurea.

Discussion and Conclusion: Cytogenetic abnormalities in ET are linked to advanced age, increased leukocyte count and JAK2 mutation. Nevertheless, survival is significantly shorter in patients with abnormal karyotype compared to those with normal karyotype. In the present case, the patient has an old advantage age, presented an interstitial deletion in chromosome 3, involving the bands 3p14 to 3p12, leukocyte counts are within the limit, and there isn’t a JAK2 mutation.

In hematologic malignancies, 3p deletions are uncommon, and their occurrences are similar throughout the morphologic subgroups of hematologic diseases. The prognostic impact of 3p deletions could not be analyzed, due to the lack of information regarding survival in most of the published cases.

Highlighting the importance of cytogenetic study as part of the diagnostic workup of ET at diagnosis, enabling correlations with disease progression and survival.

Case study of a child with attention deficit hyperactivity disorder. Assessment and intervention

Helena Carvalho 1,2 , António Nabais 3 , Cristina De Sousa 4 , João Ferreira 5 , Olga Valentim 1,6,7

1 Escola Superior de Enfermagem de Lisboa; 2 Hospital de Cascais Dr.José Almeida; 3 Escola Superior de Enfermagem de Coimbra; 4 Escola Superior de Saúde Atlântica; Atlântica-Instituto Universitário; 5 Faculdade de Medcina da Universidade de Coimbre; 6 Centro de Investigação, Inovação e Desenvolvimento em Enfermagem de Lisboa (CIDNUR); 7 Centro de Investigação em Tecnologias e Serviços de Saúde, CINTESIS&RISE

Context/aims: Manuel (fictitious name), male, is 7 years old and lives with his mother and two siblings. In 2020, his older sister died, and M. began to show changes in his behavior, sleep patterns, and ability to concentrate, with repercussions at school. In 2022, he went to the emergency room due to increased energy, agitation, self and heteroaggression, and reported hearing voices and having “visions of his sister”. He was diagnosed with attention deficit hyperactivity disorder (ADHD). The aim of this study is to present a description of the experience within the scope of specialized care in Nursing in Mental and Psychiatric Health (NMPH), focusing on the implementation of the Nursing Process with a child with ADHD.

Methods: This is a descriptive case study and data was collected during a nursing consultation. Observations, anamnesis, clinical data on the patient were used, along with a family assessment according to the Calgary model. Needs were identified based on Jean Watson’s Transpersonal Care Theory, culminating in the presentation of a specialized NMPH care plan using the CIPE - 2015 nursing classification system. An investigative process was used to analyze ADHD.

Results: Manuel was referred to the nurse by the child psychiatrist with the aim of exploring play therapy and expressive mediators to facilitate the expression of feelings and promote a healthy attachment. Initially, he had difficulties bonding with his mother and identifying friends. He expressed a desire to spend more time with his father. After adjusting to the therapy and psychoeducation, he showed a feeling of security and had a greater ability to trust, establish more meaningful relationships and manage his emotions.

Conclusions: The study underscores the significance of interdisciplinary collaboration among professionals. The systematic and individualized intervention, guided by the nursing process, effectively organized care for Manuel and his mother, resulting in substantial health improvements, especially emotional well-being and interpersonal skills.

Psychological nature of alexithymic personality type

Sergii Tukaiev 1 , João Ferreira 2 , Mykola Makarchuk 3

1 serhii.tukaiev@usi.ch, Università della Svizzera italiana, Faculty of Communication, Culture, and Society, Institute of Public Health, Via Giuseppe Buffi 13, 6900, Lugano, Switzerland; Taras Shevchenko National University of Kyiv, Educational and Scientific Centre “Institute of Biology and Medicine”, 64/13, Volodymyrs’ka St., Kyiv, Ukraine, 01601; 2 naquelepordosol@gmail.com, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

3 makarchuk_mykola@knu.ua, Taras Shevchenko National University of Kyiv, Educational and Scientific Centre “Institute of Biology and Medicine”, Department of Human and Animals Physiology, 64/13, Volodymyrs’ka St., Kyiv, Ukraine, 01601.

Introduction: The alexithymia construct is characterized by impairment of emotional processing and reduced interaction between different brain areas during various experimental conditions. The aim of the current study was to establish the psychological nature and mechanisms of the occurrence of alexithymia by analyzing its connection with the properties of the nervous system, mental states, and characteristics of the emotional sphere of personality.

Methods: 232 volunteers, first-third year students from the Taras Shevchenko National University of Kyiv aged 18 to 24 years (Mage = 19, SD = 1.13) participated in this study. To determine the level of alexithymia the 26-item Toronto Alexithymia Scale (TAS-26) was used. In order to measure individual typological characteristics, emotional sphere and mental states of the respondents, the following psycho-diagnostic methods were used: Temperament Diagnostics Test by J. Strelau; Eysenck’s Personality Inventory; Taylor Manifest Anxiety Scale; UN Scale (Questionnaire for express diagnostics of neurotization level by L. Wasserman); State-Trait Anxiety Inventory (STAI) by C. Spielberger and Y. Hanin; Syndrome of Emotional Burnout by V. Boyko (SEB) and Maslach Burnout Inventory (MBI), Leonova`s the Degree of Chronic Fatigue Syndrome Test; Lemur-Tessier-Fillion Psychological Stress Measure (PSM-25); Boston the Social Stress Test “Lifestyle Analysis”; Technique of Differential Diagnostics of Depression by V.A. Zhmurov; Aggressive Behaviour Test by E. Illyn and P. Kovalev; Aggression Test by A. Assinger (assessment of aggressiveness in the relationship); Diagnostics of Emotional Response to Environmental Stimuli by V. Boyko. The Mann-Whitney test was carried out to compare the data of independent samples. To determine the type of distribution, the Kolmogorov-Smirnov test was used. Based on the normality of the sample, Pearson’s correlation coefficient and Spearman’s rank correlation coefficient were applied.

Results: Alexithymic personality type was found in 43 volunteers (TAS-26 total score ≥74, alexithymia group). A control group consisted of 113 subjects with low alexithymia score (TAS-26 total score ≤62, non-alexithymia group). 85 participants formed intermediate group (TAS-26 total score 62>and<74). The main factors related to alexithymia were a weak nervous system, low stress resistance, such characteristics of the emotional sphere, as high level of trait anxiety, depression, neuroticism, indirect verbal aggression, low levels of aggression; with mental states as, chronic fatigue, depression, psychological stress. The emotional exhaustion and reduction of personal achievements (MBI), the Resistance Phase (SEB) were the most pronounced within the alexithymia group.

Conclusions: The obtained results confirm the adaptation theory of the alexithymia development, according to which, due to the weakness of the nervous system and high trait anxiety, the persons adapt to stressful situations by avoiding and suppressing negative emotions, which eventually creates the impossibility of their verbal description and expression.

SNHG1: redefining the landscape of Hepatocellular Carcinoma through LncRNAs

Tiago S. Fonseca 1 , Rui M. Martins 1,2,3 , Nuno Bonito 2 , Anabela P. Rolo 4 , Carlos M. Palmeira 1,4 , José G. Tralhão 1,3,5

1 Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2 Department of Surgery, Instituto Português de Oncologia (IPO), Coimbra, Portugal; 3 Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 4 CNC - Center for Neuroscience and Cell Biology, Coimbra, Portugal; 5 Department of Surgery, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal.

Context/aims: Hepatocellular carcinoma (HCC) poses a significant global health concern, due to increasing incidence and poor prognosis. Current diagnostic methods, reliant on alpha-fetoprotein (AFP) and imaging, exhibit limitations, hindering early detection and effective treatment. This narrative review explores the intersection between HCC and SNHG1, a long non-coding RNA with potential as a molecular biomarker, emphasizing the urgency for improved diagnostic and therapeutic tools.

Methods: Based on extensive research from 2022 to 2023, utilizing the Medline/Pubmed database, this review details the most updated evidence on the involvement of SNHG1 in HCC. The inclusion criteria yielded 63 English-written review and research articles and 1 book, forming the foundational bibliography.

Results:

SNHG1: • Induces the methylation of p53, while deregulating the expression of important pro-apoptotic genes, like BAX and FAS, thus promoting cellular proliferation and facilitating invasion of HCC cells; • Functions as a competitive endogenous RNA, inhibiting a network of miRNAs, impairing their regulatory functions, and promoting HCC progression; • Contributes to metabolic reprogramming in HCC cells, deregulating fatty acid and iron metabolism processes crucial for cancer cell ferroptosis; • Regulates the intra-tumoral infiltration by immune cells, and inhibits the expression of immune-infiltration genes, decreasing the cytotoxic and regulatory T cell responses, leading to immune escape of HCC cells; • Contributes to sorafenib resistance and is involved in the sensitivity to various chemotherapeutic agents; • Significantly correlates with larger tumor size, higher histological grande and advanced disease stages; • Yields potential diagnostic utility, with higher sensitivity than AFP and a significant correlation with a lower 5-year overall survival in HCC patients.

Conclusion: SNHG1 plays a crucial role in liver carcinogenesis and impacts the prognosis of HCC patients. This review supported the ongoing development of an upcoming clinical study to validate SNHG1’s diagnostic and prognostic potential in HCC patients, emphasizing the need for additional research and clinical studies.

CLEC7A variants and HIV-related opportunistic fungal infections: systematic review

Leandro Augusto Silva 1 , Teresa Gonçalves 2,3 , Rui Soares 1,2,3

1 Instituto Português de Oncologia de Coimbra Francisco Gentil, EPE; 2 Faculty of Medicine of University of Coimbra, Portugal; 3 CNC – UC Centre for Neurosciences and Cell Biology, University of Coimbra

Context/aims: Opportunistic fungal infections still have a significant impact in morbidity and mortality in HIV (Human Immunodeficiency Virus) patients. This systematic review examines the relationship between CLEC7A (C-Type Lectin Domain Containing 7A) gene, encoding for dectin-1 receptor, a crucial receptor for the recognition of fungi, and its genetic variants, influencing susceptibility to these infections among this group of patients.

Methods: Databases and related literature were thoroughly reviewed to identify studies investigating CLEC7A and dectin-1 variants and their correlation with susceptibility to opportunistic fungal infections in HIV patients. This study focused on human-based research, examining studies with genotypic frequencies of both normal and variant forms of the CLEC7A gene, including those with at least a control and a fungal-infected patient group and evaluating the statistic correlation between CLEC7A variants and clinical manifestation of opportunistic fungal infections.

Results: Following the methodology, the information available was scarce. Results suggest inconsistency in how the rs16910526 variant impacts susceptibility to opportunistic fungal infections, with one study showing no effect on the occurrence of oropharyngeal candidiasis, Pneumocystis jiroveccii pneumonia and cryptococcosis. However, an apparent association with an increased risk of Pneumocystis jiroveccii infection in HIV patients was demonstrated in another study. Another variant, rs16910527, showed a potential protective effect against oropharyngeal candidiasis.

Conclusions: Given the limited reports and inconclusive or low statistical evidence in some findings, additional research is necessary. Further studies in future could clarify whether these genetic variants should be part of risk assessments for opportunistic fungal infections in HIV patients.

Cocaine and quercetin: experimental animal model in brain imaging – PET functional and histopathological studies

Bruna Santos 1,2,3 , Mariana Marques 1,2,3 , Glória Figueiredo 1,2 , Sofia Brás 1,2,3 , Carolina Alves 1,2,3 , Nuno Braz 1,2,3 , Fabiana Ribeiro 4 , Pedro Encarnação 4 , Ana Luísa Silva 4,5 , Pedro Correia 4,5 , João Veloso 4,5 , Filipe Castro 5 , Ana Cristina Santos 1,2

1 Institute of Biophysics, Faculty of Medicine of the University of Coimbra, Coimbra, Portugal; 2 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 3 Faculty of Sciences and Tecnhology of the University of Coimbra, Coimbra, Portugal; 4 University of Aveiro, Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, Aveiro, Portugal; 5 RI-TE Radiation Imaging Technologies, Lda, Ílhavo, Portugal.

Context/aims: This study addresses the global concerns surrounding cocaine, the second most used drug with 21.6 million users in 2020. Cocaine addiction imposes significant socio-economic costs and negatively affects physical and mental health by disrupting the limbic system. Despite benzodiazepines managing withdrawal symptoms, they carry addiction risks. Seeking a safer alternative, quercetin, a flavonoid in fruits and vegetables, shows promise with neuroprotective properties. This study aims to investigate the impact of different low-dose cocaine exposures on brain metabolism and evaluate the effectiveness of a five-day quercetin therapy. Using distinct protocols and a microPET prototype, it analyzes cellular damage and explores genetic influences on dependence vulnerability in cocaine consumption dynamics using two mouse strains.

Methods: This study used two mouse strains (BALB/c & B6) to investigate metabolic and histological changes from varied cocaine exposure and quercetin therapy impact. Using a PET imaging system ( easy PET.3D) and [ 18 F]-FDG radiopharmaceutical, metabolically active brain regions post-cocaine and quercetin were identified. Histological analysis involved careful brain collection and optical microscopy.

Results: Results showed low doses of cocaine induced metabolic and structural changes, with C57BL/6J mice being more sensitive. Quercetin restored metabolic activity in BALB/c (+) mice across exposure levels but had limitations in C57BL/6J mice. Histologically, both strains exhibited cocaine-induced cell damage, with quercetin mitigating severity after consumption cessation, though not fully reversing structural damage.

Conclusions: While quercetin couldn’t entirely reverse all histological damage, it demonstrated a remarkable ability to restore metabolic activity in crucial brain regions. This suggests that quercetin treatment could potentially promote the recovery of neuronal pathways, improving overall recovery and mitigating cognitive and behavioral deficits associated with cocaine addiction.

Acanthus mollis leaves: toxicity, in vitro & in vivo anti-cancer activity and imaging evaluation of DIBOA & an ethanolic-extract

Patrícia Matos 1,2,3,4 , Bruna Santos 4,5,6 , Sofia Brás 4,5,6 , Mariana Marques 4,5,6 , Glória Figueiredo 4,5 , Fabiana Ribeiro 7 , Pedro Encarnação 7 , Pedro Correia 7,8 , João Veloso 7,8 , Ana Cristina Santos 4,5 , Maria Teresa Batista 3 , Artur Figueirinha 1,2

1 Faculty of Pharmacy, University of Coimbra, Portugal; 2 LAQV, REQUIMTE, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal; 3 CIEPQPF, Research Center for Chemical Processes Engineering and Forest Products, University of Coimbra, Portugal; 4 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal; 5 Institute of Biophysics, Faculty of Medicine, University of Coimbra, Portugal; 6 Faculty of Sciences and Technology of the University of Coimbra, Coimbra, Portugal; 7 University of Aveiro, Institute for Nanostructures, Nanomodelling and Nanofabrication (i3N), Department of Physics, Aveiro, Portugal; 8 RI-TE Radiation Imaging Technologies, Lda, Ílhavo, Portugal

Context/aims: Neoplastic pathologies often arise from dysregulated inflammatory processes. Chronic inflammation heightens cancer incidence, indicating a shared signalling pathway. Building on prior findings of the anti-inflammatory activity of an Acanthus mollis leaves ethanolic extract and its major phytochemical DIBOA, this study aimed to evaluate the anticancer potential using in vitro and in vivo assays and the safety.

Methods: This research explores the safety and anti-cancer potential of two samples: an ethanolic extract of Acanthus mollis leaves and DIBOA against colon cancer (WiDr cancer cell line). Their cytotoxicity for WiDr cells (human colon cancer) has been studied with the MTT assay (Microculture Tetrazolium Technique). BALB/c (+) mice were used to perform histopathological studies after administration of the samples to ensure their biological safety. Nude BALB/c mice were chosen to do in vivo anti-cancer assays (non-orthotopic model, subcutaneous injection of WiDr cells). The nude mice underwent weekly monitoring (weight and tumour measurement) and imaging studies using the easyPET.3D system with the radiopharmaceutical 18 F-FDG.

Results: Toxicity for WiDr cells was verified for the ethanolic extract and the DIBOA. This extract and the compound suppressed LPS-induced inflammatory responses, including NO production and downregulation of proinflammatory cytokines in RAW 264.7 macrophages. These results suggest the compound’s pivotal role in the apoptotic cascade regarding WiDr colon cancer cells, emphasizing the importance of evaluation using an in vivo model. Histopathological analyses of the collected organ samples of BALB/c (+) mice demonstrated normal features, proving the non-toxic behaviour of DIBOA and the extract. However, the tested doses induced tumour necrosis on nude BALB/c mice with no significant tumour reduction.

Conclusions: This study provides valuable insights regarding the studied samples’ safety and in vivo efficacy, enhancing understanding of their potential applications in cancer research.

Acknowledgements: This work was funded by Ph.D. grant ref. SFRH/BD/139413/2018FCT (Fundação para a Ciência e Tecnologia) and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano). European Union funded this research (FEDER funds project iPET CENTRO-01-0247-FEDER-039880 and project PTDC/EMD-EMD/2140/2020 (easyPET-CT)) and PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the projects UIDB/50006/2020 | UIDP/50006/2020 and UIDB/00102/2020. A special thank you to Centro Hospitalar da Universidade de Coimbra (CHUC), to iCBR-FMUC Vivarium , to the Biophysics Institute of FMUC, to University of Aveiro, and to RI.TE