Spatial transcriptomics and functional mapping of the suppressive TME in primary breast tumor and breast-to-liver metastases.

Abstract

e13119 Background: The tumor microenvironment (TME) of primary breast tumors (PTs) and breast-to-liver metastases shows heterogeneous infiltration and interactions between tumor, immune, and stromal cells. Hepatic metastases exhibit a more immunosuppressive TME with fewer cytotoxic T cells (CTLs) relative to PTs, which may contribute to increased tumor growth and resistance to systemic therapies. To investigate the immune cell infiltration and immune-tumor interactions in PT and liver metastases, we utilized whole transcriptome analysis and immunofluorescence-based protein expression as part of spatial TME profiling. Methods: We identified 13 chemo-naïve PTs and 3 breast-to-liver metastases. Formalin-fixed paraffin-embedded (FFPE) samples were used for hematoxylin and eosin (H&E) staining and spatial transcriptomics (ST) profiling with the 10X Genomics Visium CytAssist and Visium HD platforms. The H&E slides were manually annotated in collaboration with a board-certified pathologist, and ST data were deconvoluted using breast-specific scRNA-sequenced references and canonical marker labeling. Results: Our analysis of the 13 PTs and 3 breast-to-liver metastases revealed significant heterogeneity in tumor clusters, immune cell infiltration, gene set enrichments, and gene signatures between primary and visceral TMEs. Unsupervised clustering of hormone receptor-positive (HR+) HER2-negative (HER2) samples demonstrated considerable intratumoral and intertumoral variability, with differences in the expression of tumor markers (EPCAM, ER, PR, ERBB2, KRT14, KRT18), hypoxia-related signatures (HIF1a), and pro-angiogenic drivers (VEGF). Breast-to-liver metastases showed higher expression of pathways involved in proliferation, angiogenesis, and anti-apoptosis compared to primary tumors. Hepatic metastases were enriched for HALLMARK gene sets related to proliferation and metabolic pathways, including PI3K/AKT/mTOR, mTORC1 signaling, E2F targets, MYC targets, G2M checkpoint, mitotic spindle, glycolysis, oxidative phosphorylation, and fatty acid metabolism when compared to primary tumors. In contrast, primary tumors were enriched for immune-related pathways (TNF-α, interferon-γ, inflammatory response, IL-6 JAK/STAT3), epithelial-mesenchymal transition (EMT), myogenesis, and angiogenesis gene signatures, which are consistent with their metastatic potential. Cellular profiling of liver lesions revealed higher myeloid suppressor cells (TAMs and MDSCs): CD8 + ratio with shorter cell-cell distances relative to PTs suggesting a key role in immune evasion, tumor progression, and therapeutic resistance. Conclusions: ST data from primary and metastatic breast cancer samples revealed notable tumor heterogeneity, characterized by highly active metabolic and proliferative pathways, along with a higher abundance of myeloid cells.