Connexin46-enriched extracellular vesicles facilitate cargo delivery and promote angiogenesis

Abstract Extracellular vesicles (EVs) are constantly released from cells. Their contents vary depending on conditions and cell type, influencing their effects on recipient cells. They have been implicated in several diseases, being currently recognized as key mediators of intercellular communication in cancer because they carry bioactive cargo that modulates the tumor microenvironment and promotes tumor progression. We previously showed that MCF-7 breast cancer cells overexpressing Cx46 release more EVs containing Cx46. To investigate the role of Cx46 in EVs and, considering that hypoxia has pro-angiogenic effects, we evaluated whether EVs released under hypoxia were enriched in Cx46, modulated angiogenesis, and whether Cx46 facilitated transfer of cargo. Chemical hypoxia induced in MCF-7 breast cancer cells increased Cx46 expression and its incorporation into EVs. These Cx46-enriched EVs enhanced tube formation parameters in human umbilical vein endothelial cells. Similar results were obtained using EVs derived from Cx46-overexpressing MCF-7 cells in the absence of hypoxia, suggesting that the pro-angiogenic effect did not depend solely on hypoxia. Cx46-enriched EVs showed increased and faster cargo transfer to recipient MCF-7 cells, which was significantly reduced by clofazimine, a drug that inhibits Cx46 gap junction channel function and has other off-target activities. Treatment with clofazimine also correlated with inhibition of the pro-angiogenic effect induced by Cx46-enriched EVs. Taken together, our findings provide new insights into the mechanisms by which changes in the tumor microenvironment and Cx46-enriched EVs contribute to breast cancer progression.