DIFFERENT EXTRACELLULAR VESICLES SUBPOPULATIONS CHARACTERIZE METASTATIC PROGRESSION: QUALITATIVE AND QUANTITATIVE ANALYSIS OF ISOGENIC MELANOMA CELL LINES AND THEIR SECRETED FACTORS

The survival and proliferation of metastases is a consequence of the pre-metastatic niche (PMN) evolution, an abnormal, tumor growth-favoring microenvironment devoid of cancer cells. Among tumor derived secreted factors, extracellular vesicles (EVs) are key players in PMN establishment and facilitate organotropic metastasis. Compared to normal melanocytes, melanoma cells produce a large quantity of EVs, that can be detected in the plasma of melanoma patients. For this reason, a full characterization of secreted vesicles subpopulations and of their cargo is necessary to understand how EVs affect PMN formation. In this study, we demonstrated for the first time that EVs secreted by isogenic primary tumor and metastatic melanoma cell lines are quantitatively and qualitatively different, suggesting that diverse EVs subpopulations characterize metastatic progression. We also set a deep quantitative proteomics protocol to analyze the proteome of these cells and of their EVs and soluble secreted factors. WNT5A was found as an important component of primary tumor secreted EVs; on the contrary, we observed a specific APOE and Fibronectin sorting to EVs in in metastasis versus primary tumor cell. Finally, we observed that increased levels of RAB27A protein in metastatic cells do not correlate with an increased EVs secretion. Our preliminary results demonstrate that EVs secreted by RAB27A-KD cells maintain cancer cells clonogenic ability and that low levels of RAB27A expression correlate with higher cells motility. These findings suggest a paracrine activity of a RAB27A -independent EVs subpopulation in tumor-PMN communication to promote cancer progression.