Proteomic and functional divergence of small and large extracellular vesicles reveal subtype-specific drivers of ovarian cancer progression

Objective: High-grade serous ovarian cancer (HGSOC) progression and therapeutic resistance are increasingly attributed to extracellular vesicle (EV) mediated communication. While small EVs (SEVs) are widely studied, the proteome and biological contributions of large EVs (LEVs) remain poorly defined. This study aimed to (i) establish long-term bioreactor-based production of extracellular vesicles (SEVs and LEVs) from ovarian cancer cell lines, (ii) define subtype-specific proteomic cargo, and (iii) compare the functional roles of SEVs and LEVs in tumor growth, metastasis, and immune modulation. Methods: OVCAR4, CaOV3, and SKOV3 ovarian cancer cell lines were expanded in T-175 flasks and gradually transitioned to serum-free CDM-HD media in Bioreactors. Cell lines were maintained for eight weeks in bioreactors to measure EV secretion. SEVs and LEVs were isolated by ultracentrifugation followed by size-exclusion chromatography (SEC). Particle number and size were quantified by nanoparticle tracking analysis (NTA), and protein concentration was measured by Nano Drop. EV identity and purity were confirmed by Western blotting (CD9, CD63, CD81, LAMP1), Imaging Flow Cytometry (ISF), and transmission electron microscopy (TEM). Proteomic profiling of SEVs and LEVs was performed for each cell line, with downstream pathway analysis using Ingenuity Pathway Analysis. Functional assays included: (i) EdU proliferation assays, (ii) migration and invasion assays, (iii) splenocyte co-culture to evaluate immune modulation, and (iv) mouse xenograft models to assess EV-dependent tumor growth and metastatic dissemination. Results: Bioreactors supported stable EV production in OVCAR4 and CaOV3 cells, while SKOV3 adapted poorly. NTA and ISF confirmed consistent EV output across eight weeks. SEVs were enriched for CD9, CD63, and CD81, whereas LEVs showed strong LAMP1 expression. Proteomics revealed distinct cargo profiles: SEVs contained proteins linked to cytoskeletal remodeling, migration, and metastatic signaling, while LEVs carried proteins involved in proliferation, metabolism, and stress responses. Functionally, SEVs enhanced migration, invasion, metastatic burden, and immune suppression, whereas LEVs promoted cell proliferation and larger primary tumors in-vivo. Conclusion: SEVs and LEVs derived from ovarian cancer cells exhibit distinct proteomic signature and exert divergent effects on tumor progression. SEVs predominantly drive metastatic potential, whereas LEVs enhance proliferative tumor growth. EV subtype specific characterization provides new opportunities for biomarker discovery and targeted therapeutic strategies in HGSOC.