Abstract PS3-06-05: A Single-Cell Atlas of the Breast Cancer Microenvironment Reveals Subtype-Specific Immune States and Therapeutic Vulnerabilities

Abstract Background Breast cancer (BC) is the most commonly diagnosed cancer and a leading cause of cancer-related mortality among women worldwide. Despite advancements in detection and treatment, challenges persist due to the disease's molecular heterogeneity and racial disparities in outcomes. Methods We performed a meta-analysis of single-cell RNA-seq (scRNA-seq) data from 376 samples with various stages of BC. The dataset comprises 1.2 million tumor, immune, and stromal cells across the major subtypes: Triple-Negative (TN), Luminal A (LA), Luminal B, and HER2-enriched. Results Comparative analysis across subtypes revealed distinct immune phenotypes consistent with the “hot” versus “cold” tumor paradigm. In localized disease, TNBC displayed the highest immune infiltration, while LA tumors exhibited immune exclusion. This difference diminished in metastatic lesions. Ligand–receptor analysis highlighted that LA tumors had a higher frequency of anti-inflammatory chemokine signaling than TNBC.In localized LA tumors, we observed enrichment for anti-inflammatory macrophages (MC1), whereas metastases contained more pro-inflammatory macrophages (MC0). The MC0/MC1 ratio correlated with a gene program enriched in SPP1 expression—previously linked to bone metastases. Additionally, we found that estrogen receptor (ER) expression declined in LA metastases, suggesting a loss of ER signaling during disease progression. However, HER2 expression remained high in ER-positive LA cells, comparable to clinically defined HER2-enriched tumors, indicating a subset of HER2+ LA subset.To quantify ER-dependent transcriptional programs, we constructed an ER score based on genes enriched in either ER+ or ER− tumor cells. A high ER+ score was associated with improved overall survival, greater response to endocrine therapy (ET), and reduced immune infiltration. This ER+ program also correlated with fibrotic fibroblasts and MC1 macrophages, pointing to an immune-excluded TME. Notably, part of the ER+ score genes showed chromosomal amplification in ER+ population. Conversely, genes enriched in ER− tumors including MYC were linked to poor prognosis, ET resistance, and an inflamed TME.In TNBC, immune checkpoint expression diverged by disease stage. Primary TNBCs exhibited high PD1 and LAG3 expression, corresponding with improved response to immune checkpoint inhibitor (ICI) therapy in combination with chemotherapy. In contrast, metastatic TNBC showed predominant CTLA4 signaling, suggesting stage-specific immune escape mechanisms. In parallel, we identified EMT-associated tumor programs associated with metastatic progression and poor survival, partly driven by chromosomal amplifications observed in metastatic samples. These programs showed elevated TROP2 expression, nominating them as potential candidates for TROP2-targeted therapies. Conclusion This study presents the first in-depth single-cell analysis of transcriptional heterogeneity within LA and TNBC tumors. In LA we identified two major tumor cell profiles: ER+ and ER-. These were strongly associated with differences in patient survival and response to ET. MYC was identified as a potential driver of ER- cells and may contribute to therapeutic resistance. The distinct profiles also corresponded with specific TME characteristics: ER+ tumors were linked to immune exclusion and fibrotic stroma enriched in anti-inflammatory macrophages, whereas ER- tumors showed increased immune infiltration and a pro-inflammatory stromal profile. In TNBC, we mapped the IC landscape across disease stages—findings that may guide future ICI trial designs. Finally, EMT-programs with high TROP2 expression nominate a subset of TNBC tumors as potential candidates for targeted therapies such as Sacituzumab govitecan. Citation Format: L. P. Zisman Schachter, A. Pinhasi, K. Yizhak. A Single-Cell Atlas of the Breast Cancer Microenvironment Reveals Subtype-Specific Immune States and Therapeutic Vulnerabilities abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-06-05.