Extracellular vesicles from ovarian cancer tumor spheroids harbor disease-related and survival-associated proteins

Aim: Extracellular vesicles (EVs) play a pivotal role in tumor progression, influencing the tumor microenvironment. Despite significant research, the targeted analysis of EVs directly derived from primary tumors remains limited, particularly in ovarian cancer. The majority of existing studies have focused on EVs derived from peritoneal fluid (ascites), which encompasses contributions from different cell types. This study aims to isolate and characterize EVs secreted specifically by ovarian cancer spheroids derived from primary patient ascites. Methods: A three-dimensional cell culture model was employed to cultivate tumor spheroids in a defined medium, with EVs purified via differential ultracentrifugation and size-exclusion chromatography. Purified EVs were characterized by nanoparticle tracking analysis, nanoflow cytometry, and electron microscopy prior to performing high-resolution mass spectrometry. Results: This approach allowed the identification of known cancer-associated proteins, including danger molecules, which are linked to poor prognosis. Moreover, enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the ascites abundance levels of novel candidates [RAB14 (Ras-related protein Rab-14), SCAMP3 (secretory carrier membrane protein 3), and FAM3C (FAM3 metabolism regulating signaling molecule C)] correlated with patients' progression-free survival, further validating their clinical relevance. Finally, we used the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database to compare our dataset with The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data. Thereby, we revealed a signature of three TOP genes encoding proteins within our dataset (CORO1B, LAMP2, MSLN), which were differentially expressed in ovarian cancer patients compared to healthy individuals. Conclusion: This study provides the first proteomic profile of EVs derived directly from primary tumor spheroids, and paves the way for a better mechanistic understanding of EV-associated proteins and for the development of biomarkers or therapeutic strategies.