Extracellular vesicle release and cargo are altered by caveolin-1-overexpression and modulate tumor microenvironment in a model of rhabdomyosarcoma

Background. Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space which have a pivotal role in cancer disease. Caveolin-1 (CAV1) is an integral membrane protein required to generate caveolae and cholesterol-enriched lipid rafts. In cancer, CAV1 has a controversial role: it is widely accepted that the loss of CAV1 correlates with early-stage tumor progression, while its over-expression and phosphorylation are associated with metastatic disease. In particular in rhabdomyosarcoma (RD), CAV1 has been shown to cooperates with tumor growth and metastatic potential. Aim. 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 the cancer dissemination. Methods. EVs 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 EV subpopulations. Results. The obtained data show that RD-CAV1 cells release more EVs compared to RD-Ctrl cells. Western Blot analysis highlighted that small extracellular vesicles (sEVs) exhibit the tipical exosomal markers, whereas large extracellular vesicles (lEVs) are positive for Calnexin. Interestingly, the tetraspanins CD63, CD81, and CD9 were not detectable in RD-CAV1 EVs, unlike RD-Ctrl EVs. 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. Conclusions. Taken together, these data demonstrate that CAV1 overexpression critically affects RD-EV release and cargo; moreover RD-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 on the evaluation of RD-EV effects in other cells, typical of tumor niche.