Abstract P88: DOES EV PURITY AFFECT ITS THERAPEUTIC ACTIVITIES?

Abstract Introduction: Extracellular vesicles (EVs) are small membranous structures released by all cell types, playing a pivotal role in intercellular communication. They have garnered significant interest as biomarkers in oncology, particularly for their potential to influence cancer progression and metastasis. EVs have been shown to modulate the behaviour of recipient cells thereby impacting tumour growth and the metastatic process. EV purity is crucial for their function and potential clinical application in cancer diagnostics and therapeutics. This study compares two methods for isolating EVs from conditioned media (CM) and evaluates their size, purity, and functionality on tumor-associated microglia. Methods: EVs were isolated from the same batch of human glioma cells U251MG cultured in serum-free DMEM. The CM was divided equally and subjected to ultracentrifugation (UC) or size exclusion chromatography (SEC). Isolated EVs were characterised based on MISEV2023 guidelines. The immune-modulation effect of the EVs was tested in microglia. Results: Purity assessments showed higher protein concentration in UC-EVs compared to SEC. Both UC and SEC-EVs expressed CD81, CD63, LAMA4, and Syntenin-1, with CD81 significantly increased in UC-isolated EVs. Syntenin-1 was only present in SEC-EVs. Nano-flow cytometry showed minimal size differences despite a difference in concentration. EV-exposed microglia showed similar effects on migration, phagocytosis, filopodia formation and cytokine expression. This suggests potential implications for cancer progression and metastasis. Conclusion: Both methods isolated EVs with minimal variations which may indicate a difference in cargo that could influence downstream functions relevant to cancer. The absence of syntenin-1 in UC-EVs, suggests that the two isolation methods may have captured distinct EV populations of the same size. Additional investigations will be conducted understand the exclusive presence of syntenin-1 in SEC-EVs and its potential involvement in subsequent cellular functions, cancer progression and metastasis. Funding: This project is supported by the Singapore Ministry of Health’s National Medical Research Council (MOH-000560). Citation Format: Janice HY Tan, Ivy AW Ho. DOES EV PURITY AFFECT ITS THERAPEUTIC ACTIVITIES? abstract. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85 (15Suppl): Abstract nr P88.