Tumor Cell–Derived Microparticles Induced by Methotrexate Reprogram Neutrophil Antitumor Response via Lysosomal ROS-Mediated Degranulation

Neutrophils are one of the most important immune cells in the tumor microenvironment, and they affect the immunosuppression status by directly supporting the tumor progression or indirectly impairing T-cell antitumor response. Although recent evidence indicates that neutrophils determine the success of tumor immunotherapy, how to activate the innate immune system antitumor response still lags out. In this study, we provide evidence that the methotrexate-packaged tumor cell-derived microparticles (MTX-MP) activate neutrophil antitumor response by directly releasing tumor cytotoxic microparticles, increasing tumor-infiltrated CD8+ T cells, and promoting CD8+ T-cell antitumor response. Strikingly, mitochondrial-lysosomal membrane contacts mediate NADH translocation to lysosomal compartments. Within lysosomes, ENOX2 catalyzes NADH oxidation to generate lysosomal reactive oxygen species, which induce Ca2+ efflux via lysosomal channels. This calcium surge triggers neutrophil degranulation, thereby promoting cytotoxic microparticle release. By performing the combination of MTX-MP-activated neutrophils and OT-1 CD8+ T-cell transfer, we found that the long-term survival rate improved in OVA-expressing Lewis lung carcinoma models. IMPLICATIONS: Our findings revealed a new way by which activated-neutrophils release microparticles to kill tumor cells and provided a potential combinatorial therapeutic strategy for tumor immunotherapy.