Small CD63 + CD81+ extracellular vesicles induce autophagy via the FOXO3a pathway in pancreatic cancer cells during starvation.

Pancreatic ductal adenocarcinoma (PDAC), the most common histological subtype of pancreatic cancer, is an aggressive malignancy expected to become the second leading cause of cancer-related deaths by 2040. A hallmark of PDACs is the highly desmoplastic and hypovascularized nature of its microenvironment, which enables PDAC cells to adapt and survive under conditions of low oxygen and nutrient deprivation through various cellular mechanisms. Among these processes, autophagy has emerged as a key response mechanism to cope with these adverse conditions. Consequently, a deeper understanding of the molecular events driving autophagy could pave the way for the development of new and more effective treatments for PDAC. In this context, we provide evidence of novel pathway mediated by extracellular vesicles (EVs) that promotes autophagy in a paracrine manner in PDAC cells in response to starvation. Our findings indicate that, under starvation conditions, EVs-associated tetraspanins, namely CD9, CD63 and CD81, mobilize towards domains-like structures within the plasma membrane of PDAC cells. Correspondingly, a specific release of small EVs (sEVs) is observed in the starved cells. Notably, sEVs derived from starved cells, but not those from cells at basal conditions, strongly induce the autophagy pathway in PDAC cells cultured with optimal nutritional media. Interestingly, this ability to induce autophagy is specific to CD63/CD81 double-positive sEVs and is effective even in non-tumoral pancreatic cells. This sEVs-mediated autophagy is, at least partially, mediated by the activation of the FOXO3a pathway. It is worth noting that, although EVs release returns to basal level after 1 h of recovery following starvation, these EVs retain a capacity to induce autophagy, suggesting a decupling between quantity and quality of secreted vesicles. Our results demonstrate that pancreatic cancer cells in nutrient-deprived environments release specific sEVs that, in turn, activate the FOXO3a pathway and autophagy flux in recipient cells.