The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

Small extracellular vesicles (sEVs) are critical mediators of cell communications and are influenced by cellular stress. This study investigates the role of P2X4 and P2X7 purinoreceptors in the biogenesis and release of sEVs from triple-negative mammary cancer cells under hypoxia. In this study, we used CRISPR/Cas9 knockdown models from the triple-negative mammary cancer cell line 4T1 and selective pharmacology, to knock-down or inhibit either P2X4 or P2X7 receptors. We analysed intracellular endo-lysosomal compartments with transmission electron microscopy and epifluorecence imaging. The release and characterization of sEVs was analysed by nanoparticle tracking analysis, proteomics and western blotting. The incorporation of sEVs into recipient cells was followed by epifluorescent imaging and flow cytometry and consequences on cancer cell invasiveness was studied using transwell assays. We found that P2X4 but not P2X7 regulates sEV release. P2X4 silencing increased the number of multivesicular bodies (MVBs), enhanced sEV production and selectively promoted CD9-positive sEV subpopulations under hypoxia. Proteomic analysis revealed altered sEV protein content in P2X4-deficient cells, including enrichment in epithelial markers and autophagic proteins (LC3-II, p62), and depletion of the endosomal sorting complexes required for transport (ESCRT) components required for endo-lysosomal fusion. This profile indicates a shift toward autophagy-dependent, unconventional sEV secretion via amphisomes. Additionally, sEVs from P2X4-deficient cells integrated more efficiently into recipient cancer cells but decreased their invasive capacity, suggesting functional consequences of altered sEV cargo. In contrast, intervention of P2X7 had no impact on sEV release. Overall, our findings identify the P2X4 receptor as a key regulator of hypoxia-induced sEV secretion and composition through modulation of endo-lysosomal trafficking and autophagy, with potential implication for tumour progression and intercellular signalling.