892 Non-redundant mechanisms of resistance to immunotherapy and radiotherapy converge on innate immunity

BackgroundAlthough immune checkpoint inhibitors (ICIs) have demonstrated activity in triple-negative breast cancer (TNBC),1 2 it is unclear how to best treat patients who progress on ICI or are ineligible for treatment. Given clinical evidence of synergy between ICI and radiotherapy (RT),3 4 we used single-cell RNA sequencing (scRNA-seq) to understand how ICI reprograms the immune response to RT and to identify novel pathways of immune resistance that restrain the local and systemic efficacy of combination therapy.MethodsC57BL/6 mice bearing orthotopic EO771 tumors, a syngeneic model for TNBC, were treated with ICI (anti-PD-1/CD47) or neutralizing antibodies (anti-Ly6G/Gr-1) by intraperitoneal injections. Tumors were treated with ablative RT (16 Gy x 1 fraction) using the X-RAD SmART platform with CT image-guidance. scRNA-seq and CITE-seq analyses of the immune microenvironment were performed by Seurat and BBrowser (v3.0, Bioturing).ResultsUsing scRNA-seq, we found that anti-PD-1 reprogrammed the immune response to RT by shifting TAMs from a lipid-associated phenotype (APOE, FABP5) to an M1-like interferon-primed state (ISG15, CXCL10). Notably, anti-PD-1 also promoted the late-stage recruitment of intratumoral neutrophils. Given that neutrophils promote resistance to RT in other models,5 we eliminated neutrophils from tumors using two separate antibodies, anti-Ly6G and anti-Gr-1. Compared to ICI-RT alone, neutrophil depletion improved local tumor control and overall survival in mice with advanced tumors (P