Abstract 109: Identification of tumor-intrinsic and extrinsic mechanisms of T cell exclusion in estrogen receptor-positive (ER+) breast cancer through integration of bulk and single-cell transcriptome and spatial analysis

Abstract T cells are generally present in low numbers in estrogen receptor-positive (ER+) breast cancer (BC) potentially due to limited antigens or impaired antigen presentation, however defined mechanisms are not fully understood. This study explores the interplay between estrogen receptor signaling (ERS), antigen presentation machinery (APM), and T cell signaling (TC) using diverse datasets, including BC cell lines, TCGA, METABRIC, and clinical trial bulk transcriptomes, single-cell RNA-sequencing of ER+ primary BC, and single-cell spatial multi-plex cyclic immunofluorescence (CyCIF) of primary BC patients. Co-expression analysis of both computationally and manually curated genes in TCGA and METABRIC data unveiled distinct 29-gene ERS and 225-gene combined APM/TC modules with strong inverse correlations in ER+ BC. This inverse correlation was confirmed in independent cohorts. ERS and APM/TC modules were down- and up-regulated, respectively, in response to aromatase inhibitor therapy in ER+ BC patients from the ACOSOG Z1301B trial who demonstrated clinical benefit. Analysis of published single-cell RNA-sequencing data of 20 primary ER+ BC was performed to elucidate cell types that express individual genes in the modules. It confirmed that high ERS activity is associated with reduced T cells, and identified distinct cell types contributing to each module, showing higher ERS activity in tumor cells and higher APM/TC activity in stromal and immune cells. At the cell type level, we found distinct contributions and precise insights of individual genes. The prevalence of ERS activity in tumor cells prompted investigation to determine if the ERS and APM/TC modules were preserved as tumor-intrinsic signaling. Analysis of 54 BC cell lines indicated partially preserved module correlations in cancer cell lines and revealed that the APM/TC module is a contribution of both tumor-intrinsic (e.g., complement and type-I IFN-response), and tumor-extrinsic (e.g., lymphocyte activation) factors. CyCIF analysis of 29 ER+ BC samples was performed to bridge gene signatures with spatial biology and validated that higher ERS activity correlates with lower T cell infiltration. The spatial pattern of T cells varied from immune desert to stromally bound to fully inflamed tumors. Neighborhood analysis revealed increased frequency of CD8 T cells around a tumor cell is associated with elevated MHC I and II in broad immune cells and macrophage subsets, respectively. pTBK1+ macrophages inversely correlated with CD8 T cell frequency and MHC expression in neighboring cells, suggesting an immunosuppressive role. This study uncovers robust and tightly regulated ERS and APM/TC signaling in ER+ patients, unraveling molecular intricacies and offering potential insights for targeted therapeutic interventions. Citation Format: Kenichi Shimada, Yvonne X. Cui, Daniel Michaud, Kelly F. Zheng, Jonathan Goldberg, Ricardo Pastorello, Lukas Kania, Sandra S. McAllister, Sara M. Tolaney, Adrienne Waks, Rinath Jeselsohn, Peter K. Sorger, Judith Agudo, Jennifer L. Guerriero, Elizabeth A. Mittendorf. Identification of tumor-intrinsic and extrinsic mechanisms of T cell exclusion in estrogen receptor-positive (ER+) breast cancer through integration of bulk and single-cell transcriptome and spatial analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 109.