Complex I Inhibition combined with TLR Activation in the Breast Tumor Microenvironment Educates Cytotoxic Neutrophils [bulk RNA-seq]

Neutrophils have emerged as diverse regulators of tissue states, displaying functions in both the resolution and promotion of tissue inflammation. While neutrophils have widely been associated with tumor promotion, immune suppression, and poor patient outcome, we provide evidence to support direct tumor cytotoxic properties of neutrophils. Using various models of murine breast cancer; we establish that TLR-mediated engagement, combined with complex I inhibition, within the breast tumor microenvironment, acting either directly or indirectly on neutrophils, primes these innate immune cells to acquire direct tumor killing properties, both in vitro and in vivo, and independently of CD8+ T cell immunity. TLR engagement stimulates emergency granulopoiesis, increasing levels of neutrophils in the circulation and infiltrating into tumors without inducing the formation of a pro-metastatic niche. Mechanistically, we show that systemic administration of various TLR agonists, while increasing systemic inflammation, elevates NFkB signalling in neutrophils, to contribute to their tumoricidal functions. Moreover, using bulk- and single-cell RNA sequencing, along with proteomics approaches, neutrophils that are trained to acquire these anti-tumorigenic functions, acquire enhanced secretory granule production and increased expression NADPH-oxidase machinery. Concomitantly, these tumoricidal neutrophils increase production of toxic levels of reactive oxygen species, which can be overcome with myeloperoxidase inhibitors or overexpression of ROS scavengers. Taken together, we describe a new class of neutrophils that possess direct and intrinsic tumoricidal functions, which can be exploited to eradicate immune cold breast tumors which otherwise are refractory to standard immunotherapies, including immune checkpoint blockade.