Tumor-Myeloid Cell Crosstalk Pathways Associated with Abscopal Responses in Breast Cancer

Abstract

To identify predictive biomarkers for combined radiotherapy (RT) and immune checkpoint inhibitor (ICI) therapy, which can induce systemic anti-tumor immune responses (i.e., "abscopal" responses) in breast cancer. Four Trp53-/- Balb/c breast cancer syngeneic allograft models with low tumor mutational burden and resistance to dual ICI (anti-PD1 and anti-CTLA4, BioXcell) were used. Two lines exhibited abscopal responses to RT (8Gyx3) plus ICI combination therapy (i.e., "abscopal models"), whereas the other two did not (i.e., "non-abscopal models"). We performed spatial transcriptomics analysis (GeoMx whole transcriptome assay) of pan-cytokeratin positive (panCK+) tumor and tumor-adjacent CD45+ immune cell segments in orthotopically implanted tumors 10 days after initiating treatment with IgG control, RT alone, ICI alone, or RT/ICI combination (N>5 per group). Genes selectively induced by RT/ICI in panCK+ and CD45+ segments in abscopal models and were not induced in non-abscopal models were identified using two-tailed t-tests and FDR correction for multiple testing (FDR<5%). In vitro analyses of RT-induced secreted inflammatory signaling were conducted by exposing supernatant from irradiated (8Gy) tumor cells to a RAW264.7 macrophage cell line expressing an interferon-inducible luciferase reporter construct, followed by luciferase signal quantification (Invivogen). Hierarchical clustering of immune-related genes in CD45+ immune segments from breast cancer models with abscopal responses to RT/ICI revealed global differences in tumor-adjacent immune profiles at baseline and after RT/ICI treatment. Abscopal responsive breast cancer models at baseline showed an enrichment of myeloid cell subtypes expressing complement protein C1q, Ccl8, and Csf1r in tumor-adjacent CD45+ immune cells, and CD8+ T cells expressing Ccl5. 10 days after treatment with RT/ICI, tumor-adjacent CD45+ immune cells in abscopal models were enriched in Cxcl10, Irf7, and FcgRIV, whereas these genes were not induced in non-abscopal models. CyTOF analyses confirmed the induction of inflamed professional antigen presenting cells (CD11c+CD80+IA/IE+) by RT/ICI in abscopal models. Within panCK+ tumor segments, RT/ICI in abscopal models selectively induced Isg15 and Zbp1, both of which are involved in secreted inflammatory signaling. Consistently, RT-induced secreted inflammatory signaling from tumor cells to macrophages evaluated in vitro was significantly greater in abscopal models compared to non-abscopal models. Breast cancer models with abscopal responses to RT/ICI show increased Isg15- and Zbp1-dependent secreted inflammatory crosstalk that activates Cxcl10 and Irf7 expression in tumor-adjacent myeloid cells, which warrants further investigation as a potential predictive biomarker for combined RT/ICI therapy in breast cancer.