Extracellular vesicle release and protein cargo are altered by caveolin-1-overexpression and contribute to cancer dissemination in a model of rhabdomyosarcoma

Rhabdomyosarcoma (RD) is a malignant tumor arising from striated muscle cells; many studies have shown that Caveolin-1 (CAV1) overexpression cooperates with tumor growth and metastatic potential in RD cells. CAV1 is an integral membrane protein required to generate caveolae and cholesterol-enriched lipid rafts, which are pivotal structures in extracellular vesicle (EV) secretion. The present work aims to investigate if the increased aggressiveness of RD-cells overexpressing CAV1 (RD-CAV1) correlates with an altered extracellular vesicle release and cargo and if RD-CAV1 EVs contribute to cancer dissemination. Large (lEVs) and small (sEVs) extracellular vesicles were isolated from RD-ctrl and RD-CAV1 conditioned media by sequential ultracentrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Western Blot Analysis, and Flow Cytometry Analysis. Proteomic Analysis has been performed on both EV subpopulations. The obtained data show that RD-CAV1 cells release more EVs, particularly more sEVs, than RD-Ctrl cells. Western Blot analysis highlighted that sEVs exhibit the typical exosomal markers, whereas lEVs are positive for Calnexin. Interestingly, RD-CAV1 sEVs are negative for the tetraspanins CD63, CD81 and CD9, unlike the control ones. All these data suggest that CAV1 overexpression induces an alteration of EV biogenesis and secretion. Moreover, the treatment of HUVEC with RD-CAV1 EVs shows an increase in cell proliferation and migration in a dose-dependent manner. Altogether, these data demonstrate that CAV1 overexpression critically affects RD-EV release and cargo; moreover, RD-CAV1 EVs can alter the behaviour of the tumor microenvironment cells. Future studies will focus on the characterization of RD-EV cargo in terms of lipid- and miRNA-loading and the evaluation of RD-EV effects in other cells, typical of tumor niche.