Extracellular vesicle release, loading, and phenotype are affected by caveolin-1 overexpression in a model of rhabdomyosarcoma

Extracellular vesicles (EVs) are lipid-bound nanoparticles known to be involved both in physiological cell communication and in cancer spread. Caveolin-1 (CAV1) is a transmembrane protein located in strategic areas associated with the process of vesiculation, such as caveolae and cholesterol-enriched lipid rafts. CAV1-overexpression has been identified as a tumour enhancer by promoting tumor growth and metastatic spread in rhabdomyosarcoma (RD). The present work investigates whether CAV1-overexpression can affect RD-EV release, loading, and phenotype and whether RD-EVs can contribute to the increased cancer aggressiveness. Three RD cell lines were employed for the study: RD-Mock, RD-CAV1F0 and RD-CAV1F2. RD-EVs, isolated from the conditioned media by a sequential ultracentrifugation protocol, were characterized by Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), Western Blot Analysis (WB), and Flow Cytometry Analysis (FC). Moreover, the large (lEV) and small (sEV) EV subpopulations were characterized by proteomic analysis, while the lipidomic study was conducted on both cell bodies and EVs. WB and FC analyses showed that RD-CAV1 cells and EVs are positive for TSG-101 and Alix, but do not exhibit other typical sEV markers such as CD63, CD81, and CD9. Proteomic analysis confirmed these data and showed an overall reduction in sEV protein loading. These findings, combined with the preliminary data obtained by the treatment of THP-1 cells with RD-derived EVs, suggest that these vesicles might be able to elude the immune system, thus encouraging cancer spread. The lipidomic analysis demonstrated a clear clustering of RD-CAV1F0-F2 and RD-Mock samples; moreover, RD-CAV1 cells and EVs exhibit high levels of many lipid species that correlate with an aggressive cancer phenotype. In addition, cholesterol levels are significantly higher in RD-CAV1 cell bodies and sEVs compared to the control. Lastly, the treatment of HUVECs demonstrated the ability of RD-CAV1-derived sEVs to increase their proliferation and migration, thus potentially contributing to cancer dissemination. All these data demonstrate that CAV1-overexpression critically affects RD-EV loading in both protein and lipid composition, which may have a role in enhancing the tumor spread. Future studies will be conducted to complete the characterization of RD-EV miRNA-loading and to evaluate the mechanisms by which Cav-1 can introduce such deep alterations when overexpressed.