Caveolin-1 overexpression induces extracellular vesicle release and protein cargo impairment in an in vitro model of rhabdomyosarcoma

Introduction: Caveolin-1 (CAV1) is a transmembrane protein enriched in caveolae and lipid rafts, which are involved in signal transduction and intracellular trafficking processes. In rhabdomyosarcoma (RD) CAV1- overexpression promotes tumour growth and metastatic dissemination. Since extracellular vesicles (EVs), lipidbound vesicles secreted by all cells, have a well-established role in cancer disease, the present work aims to investigate if CAV1-overexpression impacts the EV machinery and if these EVs can contribute to their increased aggressiveness. Methods: Three RD cell lines were employed for the study: RD-Mock (transfected with an empty vector), RD-CAV1-F0 engineered for CAV1 overexpression, and RD-CAV1-F2 derived from the second generation of lung metastases after RD-CAV1-F0 injection in mice. EVs were isolated from conditioned media by sequential ultracentrifugation and small-EVs (sEVs) were further purified by density gradient centrifugation. For EV characterization, Nanoparticle Tracking Analysis (NTA), Western Blot (WB), Flow Cytometry (FC) and proteomic analysis were employed. Intracellular trafficking was evaluated by immunofluorescence staining, WB analysis, endosome isolation and live cell imaging. Migration and proliferation assays were conducted on HUVECs. Results: The obtained data showed that RD-CAV1 cells release more EVs compared to RD-Mock. WB and FC analyses revealed that RD-CAV1 sEVs exhibit TSG-101 and Alix but the expression of other typical exosomal markers CD63, CD81, and CD9 is much lower than in RD-Mock. Proteomic analysis extended this alteration to other proteins showing an overall reduction in protein loading and expression. These findings are combined with an increased intracellular trafficking in RD-CAV1 cells, suggesting that CAV1-overexpression induces an alteration of EV machinery and release. Moreover, RD-CAV1 sEVs significantly increased HUVEC proliferation and migration compared to the control. Summary/Conclusion: Taken together, these data demonstrate that CAV1-overexpression critically affects RD-intracellular trafficking and EV cargo and release, which might contribute to RD-CAV1 increased 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.