Non-Redundant Mechanisms of Immune Resistance to Radiotherapy Converge on Innate Immunity

Abstract

Despite evidence of preclinical synergy between radiotherapy (RT) and immune checkpoint blockade (ICB), randomized trials of RT/ICB have demonstrated limited benefit in solid tumors. We performed single-cell RNA sequencing (scRNA-seq) and CITE-seq (cellular indexing of transcriptomes and epitopes) to address the discordance between preclinical and clinical data. We hypothesized that multiple orthogonal inhibitory immune pathways restrain the local and systemic efficacy of RT beyond T-cell oriented immune checkpoints. We used the EO771 syngeneic murine model of breast cancer to characterize the immune tumor microenvironment following RT with or without ICB. RT (16 Gy x 1) was delivered using the X-RAD SmART platform with CT image guidance. Neutralizing antibodies (anti-PD-1/Ly6G/Gr-1/CD47) were delivered by intraperitoneal injections. scRNA-seq analysis were performed by Seurat and BBrowser (BioTuring). We found that adaptive ICB (anti-PD-1) reprogrammed the immune response to RT by promoting an M1-like interferon-primed state (ISG15, CXCL10) in tumor associated macrophages (TAMs) and by increasing the late recruitment of intratumoral neutrophils. Given that neutrophils may drive resistance to RT in other models, we evaluated the effect of intratumoral neutrophil depletion using anti-Ly6G or anti-Gr-1 on the antitumor efficacy of RT/ICB. Both neutrophil depletion strategies led to enhanced tumor control and improved survival in advanced EO771 tumors compared to RT/ICB alone (P<0.001). In parallel to this approach, we found that TAMs upregulated several innate immune checkpoints including SIRPα in response to RT. Disruption of the SIRPα-CD47 interaction by anti-CD47 antibodies similarly enhanced the antitumor efficacy of RT/ICB by improving tumor control and survival (P<0.001). Using scRNA-seq and unbiased clustering, we found that anti-CD47 eliminated an entire cluster of chronically inflamed TAMs, characterized by pro-inflammatory markers (IL1A, NOS2) and chemokines (CCL3, CXCL1/2/3). Anti-CD47 also reduced intratumoral neutrophils by eliminating a cluster of pathologically activated neutrophils, termed myeloid-derived suppressor cells (PMN-MDSCs) that expressed several markers of ferroptosis (TFRC, PTGS2, SLC3A2). Consistent with the potent immunosuppressive capacity of PMN-MDSCs, we found that anti-CD47 increased tumor-infiltrating lymphocytes including central memory TCF7+ T cells and CD19+ B cells. Lastly, by inference and analysis of cell-cell communication (CellChat), we found that anti-CD47 strengthened the interactions between TAMs and CD8+ T cells compared to RT/ICB alone. Our data collectively indicate that resistance to RT/ICB in the EO771 model Is driven by innate immune cells including neutrophils and chronically inflamed TAMs. Targeted disruption of the CD47-SIRPα axis is a promising approach to overcoming immune resistance by reprogramming TAMs and eliminating PMN-MDSCs.