This application is being submitted in response to NOT-CA-19-028. Our goal is to develop an ex vivo model of human neutrophil responses in the tumor microenvironment (TME) that incorporates gene editing as a tool to delineate the effects of neutrophil responses at the genetic level. In the awarded R01, we proposed a model in which damage-associated molecular patterns (DAMPs) and other products of necrosis are released in the TME and promote recruitment of neutrophils and induce neutrophil extracellular traps (NETs), a distinct pro- inflammatory and injurious mode of neutrophil death that accelerates metastasis in tumor-bearing mouse models. We found that ascites mitochondrial DNA (a DAMP that activates neutrophils) and neutrophil elastase levels (a product of primary neutrophil granules released during degranulation and NETs) correlated with worse PFS in patients with newly diagnosed metastatic epithelial ovarian cancer (EOC). We also observed that circulating neutrophils from patients with newly diagnosed metastatic EOC were morphologically mature and not intrinsically suppressive, but acquired a suppressor phenotype (as defined by ? 1 log10 reduction of anti- CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. This neutrophil suppressor phenotype was dependent on a number of neutrophil effector functions, including complement signaling, and is distinct from granulocytic myeloid-derived suppressor cells (MDSC) and tumor-associated N2 neutrophils. Taken together, our results support a model in which DAMPs recruit neutrophils to the EOC microenvironment where they can drive tumor progression through NETs and induce suppressor neutrophils that are a barrier to anti-tumor immunity. Obstacles to the study of neutrophils in the TME are that mature neutrophils are terminally differentiated, short-lived, and not amenable to genetic modification. Genetically modified mice and myeloid cell lines are widely used systems for evaluation of neutrophil functions, but don't permit evaluation of the role of specific genes in primary human neutrophils in clinically relevant models of the TME.
The Specific Aim of the supplement is to use an established CRISPR-Cas9 editing platform to test the role of specific neutrophil effector functions that drive the suppressor phenotype in EOC ascites, an authentic component of the human TME. Accomplishment of this aim will establish the foundation for applying CRISPR-Cas9 editing technology to probe human neutrophil responses at the single gene level in the TME, and will advance an ex vivo model system expected to be a valuable resource for immuno-oncology research.
Novel models are needed to investigate the role of specific genes in primary human neutrophils in the tumor microenvironment of patients with metastatic cancers. We propose in this supplement to use an established CRISPR-Cas9-editing platform to test the role of specific neutrophil functions in suppressing anti-tumor immunity in the ovarian cancer microenvironment.
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