Caveolin-1-overexpression induces deep plasma membrane rearrangement and secretion of extracellular vesicles in a model of rhabdomyosarcoma

Introduction Caveolin-1 (CAV1) is an integral membrane protein required to generate caveolae and cholesterol-enriched lipid rafts of the plasma membrane. Moreover, CAV1 binds to many other proteins, controls cholesterol homeostasis, and regulates various cell functions. CAV1 has a controversial role in cancer, it is widely accepted that loss of CAV1 correlates with early-stage tumor progression, while its re-expression and phosphorylation are associated with recurrence and metastatic disease. For example, it has been shown that CAV1 cooperates to tumor growth and metastatic potential in rhabdomyosarcoma (RD). A considerable body of evidence suggests the possibility that extracellular CAV1 may be relevant in cancer cell metastasis. The present work aims to investigate if the increased aggressiveness of RD cells overexpressing CAV-1 correlates with an altered extracellular vesicle release. Methods Vesicles were isolated by differential ultracentrifugation and density gradient separation methods from conditioned media of control and metastatic RD lines overexpressing CAV1 (RD-CAV1). Collected small (sEVs) and large (lEVs) extracellular vesicles were characterized by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy (TEM) and western blot analysis. Results Scanning and transmission electron micrograph analysis of RD cells showed that the overexpression of CAV1 induced deep plasma membrane reorganization demonstrated by developing a significant number of membrane protrusions emerging from the cell surface of RD-CAV1 compared to RD-Control cells. Furthermore, TEM analysis revealed the presence in RD-CAV1 of higher quantities of intracellular organelles, resembling multivesicular bodies, compared with RD-Ctrl cells. Physical characterization with Nanosight technology and transmission electron microscopy (TEM) showed that the applied separation protocols allowed the isolation of two EV subpopulations of about 93 nm (sEVs) and 170 nm (lEVs) in diameter, respectively. Quantification data showed that RD-CAV1 releases 3-4 fold more EVs compared to RD-Ctrl; furthermore, NTA revealed that CAV1 overexpression seems not to affect the sEV versus lEV production ratio with sEVs about 10 times more abundant than lEVs. Analysis of key vesicular markers' protein expression by western blotting confirmed the efficient separation of supernatant-derived EV sub-populations: sEVs were positive for the exosomal markers Alix, Flot-1, Syntenin-1, and TSG101; whereas lEVs were positive for the endoplasmic reticulum marker calnexin. Summary/Conclusion The reported data suggest that CAV1 could be a key regulatory factor promoting tumor microenvironment remodeling by modulating EV release in addition to the well-established structural role