Caveolin-1-overexpression affects extracellular vesicle loading and modulates tumour microenvironment in a rhabdomyosarcoma model

Introduction: Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space and have a pivotal role in cancer disease. Caveolin-1 (CAV1) is a 22 kDa protein located in strategic areas of the plasma membrane, such as caveolae and cholesterol-enriched lipid rafts. In the context of rhabdomyosarcoma (RD) CAV1-overexpression promotes tumour growth and metastatic diffusion, acting as a tumour enhancer. The present work aims to investigate if EV machinery is affected by CAV1-overexpression and if the EVs released by RD cells overexpressing CAV1 (RD-CAV1) can contribute to their increased aggressiveness. 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 (WB) and Flow Cytometry Analysis (FC). Proteomic Analysis has been performed on EV subpopulations. Migration and proliferation assays were conducted on HUVEC cells. Results: The obtained data showed that RD-CAV1 cells release more EVs with an entirely different protein profile compared to RD-Ctrl ones. WB and FC analysis revealed that RD-CAV1 small extracellular vesicles (sEVs) do not exhibit the typical exosomal markers CD63, CD81, and CD9. Proteomic analysis extended this alteration to many other proteins showing an overall reduction in protein loading and expression in these sEVs compared to the control. These findings are combined with an impairment in the RD-CAV1 intracellular vesicular trafficking, suggesting that CAV1 overexpression induces an alteration of EV biogenesis and secretion. Moreover, the treatment of HUVECs with RD-CAV1 EVs showed a significant increase in cell proliferation and migration compared to the control. Conclusion: Taken together, these data demonstrate that CAV1-overexpression critically affects RD intracellular trafficking and EV cargo-release, leading to an increase in their aggressiveness. Future studies will focus on the characterization of RD-EV lipid- and miRNA-loading and on the evaluation of RD-EV effects in other cell types, typical of tumour niche.