Niche Remodeling in Metastatic Breast Cancer: LGALS9-CD44-Mediated Crosstalk Between Malignant Luminal Cells and Fibroblasts

Background The transition from intraductal to invasive Breast cancer (BC) is driven by complex interactions within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs), a key stromal component, critically influence extracellular matrix remodeling, immune modulation, and metastatic dissemination. However, the spatially resolved cellular mechanisms through which malignant epithelial subpopulations reprogram fibroblasts to promote metastasis remain incompletely understood. Methods We performed multiregional single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics on breast cancer specimens representing the tumor core, tumor border, and adjacent normal tissue. Malignant mammary luminal (ML) subtypes were identified through Large-scale chromosomal copy number variation (CNV) profiling. Fibroblast sub-clustering, pseudotime trajectory reconstruction, and cell–cell communication analysis (Cell Chat) were integrated with in vitro functional assays using primary fibroblasts and breast cancer cell lines to delineate key signaling interactions. Results Five ML clusters were identified, among which ML4—enriched at tumor borders—displayed transcriptional programs linked to ribosome activation, hypoxia, and hyper-inflammatory, and was associated with high recurrence and metastasis in TCGA-BC. Fibroblasts were classified into three spatially distinct subtypes: myofibroblasts, inflammatory fibroblasts (iFIB), and APOD⁺PTGDS⁺ fibroblasts, with iFIB predominantly localized to tumor borders. Cell–cell interaction analysis revealed hyperactivation of the LGALS9 (tumor)–CD44 (fibroblast) signaling axis in the border regions. Functional validation confirmed that LGALS9-expressing BC cells promote migration and activation of primary fibroblasts, whereas activated fibroblasts enhance epithelial–mesenchymal transition and migration of BC cells via Smad2/3 signaling. Conclusion We identify a spatially restricted malignant luminal subtype, ML4, which drives fibroblast activation through LGALS9–CD44 signaling, establishing a reciprocal, pro-metastatic feedback loop within the TME. This niche remodeling holds prognostic relevance and represents a promising therapeutic target for preventing metastasis in aggressive breast cancer.