Hypoxia-driven changes in EV composition are autophagy-dependent

• Published in: Journal of extracellular vesicles Vol. 7; p. 184
• Main Authors: Zonneveld, Marijke I, Keulers, Tom G H, Peeters, Hanneke, Libregts, Sten F H M, Wauben, Marca H M, Rouschop, Kasper M A
• Format: Journal Article
• Language: English
• Published: Abingdon John Wiley & Sons, Inc 01-01-2018
• Subjects: Angiogenesis; Autophagy; CD63 antigen; CD9 antigen; Endothelial cells; Flow cytometry; Hypoxia; Medical research; Phagocytosis; Ribonucleic acid; RNA; Tumor microenvironment; Tumors
• ISSN: 2001-3078

Background: Hypoxia is an important component of the tumour microenvironment (TME), correlating with increased angiogenesis, migration and treatment resistance. Tumour cells respond to changes in their TME through the secretion of extracellular vesicles (EV). How hypoxia influences EV communication with the TME remains unclear. Here, we investigated the impact of hypoxia on EV secretion, composition and function. Hypoxia is a potent trigger for autophagy and recently, endosomal proteins involved in exosome biogenesis were shown to interact with members of the autophagy machinery. Therefore, we investigated the role of autophagy in the secretion of EV. Methods: HT-29, U87 and MDA-MD-231 cells were exposed to moderate (0.2% O2) or severe (0.02% O2) hypoxia. The involvement of the autophagy machinery was assessed by inducible knockdown cell lines for ATG7, ATG12 and Lc3B. EV were isolated using a density gradient or size-exclusion chromatography. EV were analysed by high resolution flow cytometry, Western blot and next-generation sequencing. To assess angiogenic potential, endothelial cells were exposed to EV and monitored for tubule formation. Results: Using high resolution flow cytometry, we observed that the total number of secreted EV was not altered during hypoxia. Nevertheless, depending on the severity of hypoxia, we observed that gross protein content was either increased (0.2% O2) or decreased (0.02% O2) compared to controls. In contrast, the gross RNA content did not seem to be affected. Immunoblot analysis revealed distinct patterns for CD9, CD63 and Flotillin-1, suggesting composition changes dependent on oxygen level. Strikingly, an emphatic decrease in EV markers was observed by Western blot, as well as in total EV numbers in autophagy-deficient cells under hypoxia. Functionally, EV from hypoxic cells increased endothelial tubule formation. Summary/Conclusion: Collectively, these results indicate that hypoxia alters EV composition and that autophagy is required for EV secretion during hypoxia.