LAUSR

Multiple studies have demonstrated synergy between immune checkpoint blockade (ICB) and radiotherapy (RT) in preclinical murine models; however, randomized trials of RT/ICB have been inconsistent in patients with solid tumors. To better understand this discordance, we hypothesized that there are non-redundant inhibitory immune pathways that restrain the efficacy of RT beyond T-cell oriented immune checkpoints. To this end, we performed scRNA-seq and CITE-seq analysis of the EO771 syngeneic murine model of breast cancer to characterize the immune landscape following RT±ICB. We found that ICB reprograms the immune response to RT by shifting tumor-associated macrophages (TAMs) from a lipid-associated phenotype (APOE, FABP5) to an M1-like interferon-stimulated state (CXCL10, ISG15). However, ICB also promoted the late recruitment of intratumoral neutrophils, which drive resistance to RT in other contexts. To evaluate whether neutrophils may be limiting antitumor immunity to RT/ICB, we depleted intratumoral neutrophils using two separate antibodies, anti-Ly6G and anti-Gr-1. Compared to RT/ICB alone, both neutrophil depletion strategies enhanced tumor control and prolonged survival in advanced EO771 tumors (P<0.001). Given that indiscriminate neutrophil depletion is not a viable therapeutic strategy, we tested alternative immune targeting approaches to alter the TAM response to RT/ICB. By scRNA-seq, we found that RT strongly upregulated several innate immune checkpoints on TAMs (e.g., SIRPα, SLAMF3/7, LRP1). Accordingly, we disrupted the SIRPα-CD47 interaction with anti-CD47 antibodies and characterized the impact on response to RT/ICB. Anti-CD47 significantly improved tumor regression and survival when combined with RT/ICB (P<0.001). We then used scRNA-seq and CITE-seq to understand why disruption of SIRPα-CD47 improved antitumor responses to RT/ICB. We found that anti-CD47 depleted an entire cluster of chronically inflamed TAMs, expressing pro-inflammatory markers (IL1A, NOS2) and chemokines (CCL3, CXCL1/2/3). Furthermore, anti-CD47 reduced the recruitment of intratumoral neutrophils and depleted a cluster of pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), expressing WFDC17, PTGS2, S100A8/9. Lastly, anti-CD47 enhanced the recruitment of tumor-infiltrating lymphocytes including central memory TCF7+ T cells and CD19+ B cells. By inference and analysis of cell-cell communication (CellChat), we found that anti-CD47 strengthened the interactions between myeloid cells and T cells compared to RT/ICB alone. Collectively, our data indicate that innate immune cells, in particular neutrophils and chronically inflamed TAMs, promote resistance to RT/ICB in the EO771 model. These data suggest that inhibition of CD47-SIRPα is a promising therapeutic strategy to overcoming immune resistance through the elimination of PMN-MDSCs. Citation Format: Anthony T. Nguyen, Tahir B. Dar, Jolene Viramontes, Satchel Stevens, Julie K. Jang, Emily Y. Ko, Diana J. Lu, Eric M. Chung, Samuel C. Zhang, Katelyn M. Atkins, Mitchell Kamrava, Howard M. Sandler, Jlenia Guarnerio, Simon Knott, Zachary S. Zumsteg, David M. Underhill, Stephen L. Shiao. Non-redundant mechanisms of immune resistance to radiotherapy converge on innate immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6409.