Hypoxia enhances the production and antitumor effect of exosomes derived from natural killer cells

Background: Exosomes are a subgroup of extracellular vesicles that are naturally released by almost all types of cells. However, the factors that promote the capacity of natural killer (NK) cells to release exosomes are unclear. In this study, we investigated whether hypoxia can enhance the yield of NK cell-derived exosomes and improve the immunotherapeutic effects of these cells. Methods: Exosomes from NK92 or NK92-hIL-15 cells were isolated from culture medium under normoxic (NK92-Exo and NK92-hIL-15-Exo) or hypoxic (hypoxic NK92-Exo and hypoxic NK92-hIL-15-Exo) conditions. NK92-Exo and hypoxic NK92-Exo were characterized by transmission electron microscopy (TEM), nanoparticle-tracking analysis (NTA), and western blot. Real-time cell assay, wound healing assay, flow cytometry, and western blot were then performed to assess cytotoxicity, cell proliferation, cell migration, apoptosis, and the expression levels of cytotoxicity-associated proteins. Results: After 48 hours of hypoxic treatment, NK92-Exo exhibited significantly increased cytotoxicity, enhanced inhibition of cell proliferation, and elevated levels of molecules associated with NK cell cytotoxicity. The hypoxia-treated NK92-Exo and NK92-hIL-15-Exo showed increased expression of three functional proteins of NK cells-specifically FasL, perforin, and granzyme B-as compared with their NK92-Exo counterparts exposed to normoxia. Conclusions: As an approach that supports overproduction of exosomes, hypoxic treatment of NK cells may serve as a promising therapeutic option for cancer immunotherapy.