CAVEOLIN-1 OVEREXPRESSION ALTERS EXTRACELLULAR VESICLE SECRETION AND CARGO IN AN IN VITRO MODEL OF RHABDOMYOSARCOMA: A POSSIBLE LINK TO MALIGNANCY

Caveolin-1 (Cav-1) is a 22 kDa transmembrane protein located in strategic areas of the phospholipid bilayer, such as caveolae and cholesterol-enriched lipid rafts. In the context of rhabdomyosarcoma (RD), Cav-1 overexpression was found to be a marker of poor differentiation acting as a tumour enhancer and promoting tumour growth and metastatic diffusion. Recently, extracellular vesicles (EVs), lipid-bound vesicles secreted by cells into the extracellular space for cell-to-cell communication, have emerged to be crucial in cancer progression and dissemination. The present work aims to investigate whether EV machinery is affected by Cav1-overexpression in the RD cell model and whether RD-CAV1-derived EVs can contribute to the increased cancer aggressiveness. Three RD cell lines were employed for the study: RD-Mock (transfected with an empty vector), RD-CAV1F0 engineered for Cav-1 overexpression, and RD-CAV1F2 derived from the second generation of lung metastases after RD-CAV1F0 injection in mice. EVs were isolated from the conditioned media of the three cell lines by sequential ultracentrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Western Blot Analysis (WB), and Flow Cytometry Analysis (FC). Proteomic analysis was performed on the two large- (lEV) and small- (sEV) EV subpopulations. Migration and proliferation assays were conducted on HUVEC cells while THP-1 was employed as a human monocytic cell line to evaluate the cytokine activation after RD-derived sEV treatment. The obtained data showed that RD-CAV1 cells release more EVs with a completely different protein profile compared to RD-Mock ones. WB and FC analyses revealed that RD-CAV1 sEVs are positive for TSG-101 and Alix, but do not exhibit other typical sEV markers such as CD63, CD81, and CD9. Proteomic analysis extended this alteration to many other proteins showing an overall reduction in protein loading and expression in RD-CAV1-derived sEVs compared to the control. These findings are combined with an increased RD-CAV1 intracellular vesicular trafficking and an impairment of the autophagic process. Moreover, the treatment of HUVECs and THP-1 cell lines demonstrated the ability of RD-CAV1-derived sEVs to modulate the tumour niche, thus potentially contributing to cancer dissemination. Taken together, these data demonstrate that Cav1-overexpression increases RD-intracellular trafficking resulting in the alteration of EV release and cargo, which might have a role in increasing cell aggressiveness. Future studies will be conducted to complete the characterization of RD-EV lipid- and miRNA-loading and to evaluate the mechanisms by which Cav-1 can introduce such deep alterations when overexpressed.