Caveolin-1-overexpression increases the release of extracellular vesicles with altered protein-lipid cargo, thereby modulating tumor development.

Caveolin-1 (Cav-1) is a 22 kDa transmembrane protein located in key areas of the phospholipid bilayer, such as caveolae and cholesterol-enriched lipid rafts. In the rhabdomyosarcoma (RD) context, Cav-1 overexpression has been identified as a tumor enhancer, promoting tumor growth and metastasis. Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space for cell-to-cell communication and are directly involved in cancer progression and dissemination. The present work investigates whether Cav-1 overexpression can affect the release and loading of RD-CAV1-derived EVs and whether RD-EVs can contribute to increased cancer aggressiveness. Three RD cell lines were used for the study: RD-Mock (transfected with an empty vector), RD-CAV1F0 engineered for Cav-1 overexpression, and RD-CAV1F2 derived from second-generation lung metastases after RD-CAV1F0 injection into mice. EVs were isolated from the conditioned media of the above three cell lines by a sequential ultracentrifugation protocol and characterized by nanoparticle tracking analysis (NTA), Western blot (WB), and flow cytometry (FC) analyses. Proteomics was performed on the small-EV (sEV) subpopulation, while lipidomics was performed on both cell bodies and EVs. The data showed that RD-CAV1 cells release more EVs with a different protein pattern than RD-Mock. WB and FC analyses showed that RD-CAV1 sEVs are positive for TSG-101 and Alix but do not express other typical sEV markers such as CD63, CD81, and CD9. The proteomic analysis confirmed these data, showing an overall difference in protein loading and expression in RD-CAV1-derived sEVs compared to the control. These findings are associated with increased intracellular vesicular trafficking of RD-CAV1 and impairment of the autophagic process. In addition, the lipidomic analysis revealed an apparent clustering of RD-CAV1F0-F2 and RD-Mock samples, which, together with the higher amount of cholesterol in RD-CAV1F2 cells and EVs, confirmed the ability of Cav-1 overexpression to induce profound changes in EV protein and lipid composition. Finally, treatment of HUVECs demonstrated the ability of RD-CAV1-derived sEVs to increase their proliferation and migration, potentially contributing to cancer dissemination. Taken together, these data demonstrate that Cav-1 overexpression critically affects the release and cargo of RD-EVs, both in terms of protein and lipid composition, which could have implications for the tumor niche. Future studies will be conducted to complete the characterization of RD-EV miRNA cargo and to evaluate the mechanisms by which Cav-1 can induce such deep changes when overexpressed.