Extracellular Vesicles Released by Glioblastoma Cancer Cells Drive Tumor Invasiveness via Connexin-43 Gap Junctions

Abstract Background Although invasiveness is one of the major determinants of the poor glioblastoma (GBM) outcome, the mechanisms of GBM invasion are only partially understood. Among the intrinsic and environmental processes promoting cell-to-cell interaction processes, eventually driving GBM invasion, we focused on the pro-invasive role played by Extracellular Vesicles (EVs), a heterogeneous group of cell-released membranous structures containing various bioactive cargoes, which can be transferred from donor to recipient cells. Methods EVs isolated from patient-derived GBM cell lines and surgical aspirates were assessed for their pro-migratory competence by spheroid migration assays, calcium imaging, and PYK-2/FAK phosphorylation. Brain invasiveness was investigated in human cortical organoids-based assembloids and in vivo orthotopic xenografts. EV molecular features were specified by multiplex bead-based flow cytometry. Results Results unveil a self-sustaining mechanism triggering migration through autocrine release and engagement of a specific population of EVs of large size (L-EVs), isolated from either patient-derived cell lines or surgical aspirates. L-EVs act through modulation of calcium transients via Connexin 43-Gap Junctions (Cx43-GJ) and phospho-activation of PYK2. Pre-incubation with blocking antibodies targeting Cx43 hemichannels demonstrated a dose-dependent inhibition of the L-EV-mediated GBM migration. By exploiting patients’ surgical aspirates, we show that only L-EVs deriving from tumoral cells, and not those with immune origin, promote tumor migration, impacting more prominently the tumoral cells with mesenchymal subtype. Conclusions We demonstrate that L-EVs released by GBM cells, but not by the immune cells of the tumor microenvironment, represent a relevant and unique autocrine pro-migratory input for the tumor.