Single-cell profiling identifies heterogeneity of the immune microenvironment in healthy,primary and lymph node metastatic BC

ChuYu LIU¹Feng Zeng¹Shanshan Gao²Nengying Zhang¹Cai Yulan^3*

• ¹Thyroid and Breast Surgery, the Second Affiliated Hospital of ZunYi Medical University, ZunYi, Guizhou, China
• ²Department of Biomedical Sciences, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
• ³Department of Endocrinology and Metabolism, the Second Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou, China

Breast cancer (BC) exhibits extensive heterogeneity in its molecular and cellular landscapes, with metastasis, particularly to lymph nodes, driving disease progression and mortality. However, the precise characteristics of the immune microenvironment across healthy, primary,and lymph node metastatic BC remain poorly defined. In this study, we provide a high-resolution single-cell transcriptomic landscape of BC, encompassing estrogen receptor positive and human epidermal growth factor receptor 2 positive primary tumors (ER and HER2),ER positive lymph node metastases (ER_LN), alongside healthy breast tissue. Our comprehensive analysis specifically delineates the profound heterogeneity of endothelial cells (EC) within these distinct tumor microenvironments. Through single-cell transcriptomics, spatial transcriptomics and immunohistochemistry, we identify two novel tumor-enriched endothelial cell subtypes, EC4 and EC5, which exhibit subtype-specific functional adaptations and prognostic significance. EC4 cells,prevalent in BC, are characterized by antigen presentation, immune cell recruitment and anti-tumor inflammation. Similarly,EC5 cells, enriched in BC, show robust extracellular matrix (ECM) remodeling and tumor angiogenesis. Concurrently, we investigated the functional heterogeneity of the EC4 and EC5 cells within the ER, relative to those enriched in the HER2 and ER_LN. Furthermore, we reveal conserved endothelial programming mechanisms across BC subtypes, alongside distinct tumor microenvironment-driven transcriptional adaptations. Our interactome analysis highlights novel and subtype-specific communications between these EC subsets and immune cells, particularly CD8+ T cells and macrophages. Experimental evidence shows that EC with overexpressed APP can mediate the M2 polarization of macrophages, suggesting diverse immunomodulatory roles for ECs in different BC contexts. These findings offer critical insights into the complex interplay between EC and the immune microenvironment in BC progression and metastasis, identifying potential therapeutic vulnerabilities and informing future precision immunotherapeutic strategies.