Comparison of two extracellular vesicles isolation methodologies from gastric cancer cells for STn detection

• Autor(a) principal: Lopes, Tânia Isabel Meleiro
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/32802
• topic: Gastric cancer; Glycans; Extracellular vesicles; Isolation methods; Biomarkers

Gastric cancer is the 5th most common and the 3rd deadliest cancer in Portugal. The search for biomarkers and clinical approaches to improve patients’ diagnosis and stratification through less invasive and more sensitive methods is an active research topic. Overexpression of the truncated O-glycan sialyl-Tn (STn) is commonly identified in gastric carcinoma tissue, which has been correlated with poor prognosis of gastric cancer patients. In addition, cancer cells secrete high levels of extracellular vesicles (EVs) into circulation, which cargo reflects the changes occurring in the cell of origin, and therefore, represent a valuable source for biomarker discovery and detection. The aims of this study were to compare the yield and purity of two different EV isolation methodologies, the ultracentrifugation (UC) and the UC combined with size exclusion chromatography (SEC) and to access the presence and detection of STn at EV membrane. EVs were isolated from two glycoengineered cell lines that synthetize different levels of STn and two control gastric cancer cell lines negative for this glycan by UC and UC+SEC. Isolated EVs were characterized through nanoparticle tracking analysis, transmission electron microscopy and western blotting, according to the International Society of Extracellular Vesicles. The detection of STn at EV membranes was assessed using an immunogold labeling technique. Furthermore, the impact of STn in EV uptake was studied using an indirect co-culture system. UC- and UC+SEC-EV isolates were compared regarding yield and purity through the analysis of EV concentration, presence of protein contaminants and EV markers such as alix, syntenin-1, Hsp70 and CD9. Results showed that UC+SEC isolated EVs with a lower yield but higher purity, facilitating the detection of both EV markers and STn. It was also possible to observe that cells expressing STn showed to secrete more EVs and that the STn seemed to be selectively packaged in EVs. Remarkably, we were able to detect STn at the membrane of EVs secreted by gastric cancer cells. Finally, we observed a different uptake capacity of the EVs positive and negative for STn by the recipient cells. In conclusion, our results showed that the combination of UC with SEC led to purer EVs, which facilitated the STn detection. Additionally, STn was highly expressed in EVs compared to the secreting cells and it was present at the EV membrane, highlighting its potential use as EV biomarker in gastric cancer. In the same line of evidence, cells that express STn secreted more EVs, which may facilitate its detection in the clinical context. Finally, the presence of STn in EVs affected its internalization by recipient cells, suggesting a role during cancer progression.