Single‐Cell Characterization of SPP1 ‐Expressing Macrophages and Their Predicted Interactions With T Cells in the Breast Cancer Bone Metastatic Microenvironment

BACKGROUND: Breast cancer is a highly common malignancy in women, with bone as its most frequent site of distant metastasis significantly increasing mortality risk. METHODS: This study integrated single-cell RNA sequencing datasets (GSE266330 and GSE243526) to systematically characterize the tumor microenvironment across normal tissue, primary tumor, and bone metastasis in breast cancer. RESULTS: In total, 141 056 high-quality single cells were analyzed from an integrated cohort comprising a bone-metastasis dataset (GSE266330) and a primary/normal breast dataset (GSE243526), identifying 11 major cell types. Within the bone-metastatic cohort, we identified a macrophage subpopulation highly expressing SPP1 (Mac-SPP1) that exhibited an M2-polarized signature and elevated metabolic gene-set enrichment, and that expressed transcription factors such as HOXA9 and MAF. We also identified CD4-S100A9 and CD8-GZMK T-cell subpopulations with high cytotoxicity, high exhaustion, and metabolic reprogramming. Pseudotime analysis positioned CD4-S100A9 cells at one end of the inferred CD4+ T-cell trajectory and CD8-GZMK cells at an intermediate node along the inferred CD8+ T-cell trajectory. Cell-cell communication analysis predicted that Mac-SPP1 interacts with these T-cell subsets through the SPP1-integrin (e.g., SPP1-CD44) and CD74-MIF/COPA signaling axes. CONCLUSION: This study provides a single-cell characterization of the breast cancer bone-metastatic microenvironment, identifying an SPP1-expressing macrophage subset (Mac-SPP1) and predicting its interactions with CD4-S100A9 and CD8-GZMK T cells. Because the bone-metastatic and primary/normal specimens derive from different datasets, these findings are descriptive and cannot be attributed to metastatic progression alone and require experimental validation and confirmation in cohorts that profile primary and metastatic specimens within a single study.