519 Targeting myeloid inflammation to reprogram the immunosuppressive microenvironment of triple-negative breast cancer

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Background Myeloid inflammation is associated with worse outcomes across solid cancers including breast cancer. Although immune checkpoint inhibitors (ICIs) and stereotactic body radiotherapy (SBRT) are synergistic in preclinical and clinical models, combination therapy can also induce significant myeloid inflammation, which may restrain antitumor efficacy. To this end, we used single-cell RNA sequencing (scRNA-seq) to identify targetable pathways of myeloid inflammation that may limit antitumor immunity. We hypothesize that targeting myeloid function can generate novel therapeutic vulnerability to ICI/SBRT. Methods We used the syngeneic orthotopic EO771 murine model of triple-negative breast cancer, which is resistant to PD-1/PD-L1-based ICI. C57BL/6 tumor-bearing mice were treated with ICI (anti-PD-1 ± anti-CD47) and SBRT (16 Gy x 1 using X-RAD SmART irradiator). We magnetically isolated CD45+ leukocytes and CD4/CD8+ tumor-infiltrating lymphocytes (TILs) from treated EO771 tumors. Tumor-associated macrophages (TAMs, CD11b+Ly6G-) and tumor-associated neutrophils (CD11b+Ly6G+) were further enriched from the CD45+ population by flow sorting. We performed high-dimensional profiling of the tumor microenvironment (TME) using multiparametric flow cytometry and quantitative PCR (qPCR) on isolated TILs. scRNA-seq analysis and inference of intracellular communication networks were performed by Seurat, BBrowserX (Bioturing), and CellChat Results By scRNA-seq, we found that thrombospondin-1 (THBS1), an inflammatory matricellular protein, was significantly upregulated by TAMs and TANs following SBRT ± ICI (P<0.001). Given that THBS1 signals into CD47, we found that disruption of THBS1-CD47 ligation using anti-CD47 antibodies enhanced the antitumor response to ICI/SBRT improving survival and tumor weights (P<0.05). We found by scRNA-seq that anti-CD47 reprogrammed the TME by depleting inflammatory TAMs and TANs. Consistent with these observations, targeting THBS1-CD47 significantly decreased the expression of multiple neutrophil chemokines and inflammatory cytokines including CXCL3, CCL3, and TNFa by qPCR (all P<0.05). Given that T cell function is a key variable underlying treatment efficacy to ICI, we then interrogated the recruitment of T cell to the TME. By multiparametric flow cytometry, we found that targeting THBS1-CD47 enhanced the recruitment of stem-like, effector-phase TCF7+CD8+ TILs (P<0.05). Furthermore, treatment with anti-CD47 also decreased the frequency of immunosuppressive Tim3+ regulatory T cells with an effector-like phenotype (P<0.05). Conclusions Although ICI/SBRT are synergistic through interferon signaling, combination therapy is also associated with enhanced myeloid inflammation, which is potently immunosuppressive. Targeting THBS1-CD47 signaling induces therapeutic vulnerability to ICI/SBRT by mitigating inflammatory myeloid signaling and promoting stem-like effector T cell recruitment. Targeting myeloid inflammation is a novel strategy to reprogramming the TME in tumors that are refractory to ICI.

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