During tumorigenesis, interactions between malignant epithelial cells and surrounding stromal and immune compartments progressively reshape the tumor microenvironment (TME) toward pro-tumoral states. In triple-negative breast cancer (TNBC), tumor progression is tightly linked to microenvironmental plasticity, yet the early, coordinated events that initiate and structure TME remodeling remain insufficiently defined. Most clinical specimens represent advanced, heterogeneous disease, limiting reconstruction of the temporal sequence of niche formation. Spatially resolved transcriptomic approaches in genetically controlled in vivo systems enable systematic dissection of how tumors emerge and reorganize their microenvironment in space and time.

Genetically engineered TNBC mouse model; conditional Trp53^R172H^, Pik3ca^H1047R^, Ctnnb1^ΔEx3^ activation in luminal epithelial cells via WAP-iCre; YFP reporter, spontaneous multifocal, asynchronous tumors in immunocompetent background. Single-nucleus RNA sequencing (>58,000 nuclei); integration with high-resolution spatial transcriptomics (Open-ST); manual duct segmentation and pseudotime ordering of >100 ducts; spatial niche clustering; ligand–receptor interaction mapping (CellChat). Functional validation: CAF (Cancer-associated fibroblasts) co-culture assays; orthotopic syngeneic co-injection; extracellular matrix remodeling analysis; validation in TNBC patient-derived xenografts.

Ordering spatial transcriptomes along the luminal-to-basal trajectory reconstructed the spatiotemporal dynamics of TME remodeling from early transformation to invasive progression. Early lesions were associated with recruitment of inflammatory CAFs and macrophages, followed by transition into myofibroblastic CAFs that establish a structured tumor-stroma interface. Cancer-associated fibroblasts emerged as key regulators of tumor evolution. MyCAFs formed a circumferential niche around advanced ducts and coordinated extracellular matrix remodeling, promoting invasive phenotypes. Two temporally distinct waves of TGFβ signaling linked epithelial transformation to stromal activation and subsequent amplification of pro-invasive programs. These remodeling trajectories were conserved in patient samples and PDX models. Co-injection experiments demonstrated that CAF–tumor interactions accelerate progression toward invasive states, and pharmacologic interference with predicted signaling pathways supported therapeutic relevance.

Temporal alignment of spatially resolved TNBC lesions defines trajectories of coordinated TME remodeling that drive tumor progression. CAF-mediated reorganization of the tumor-stroma interface represents a central determinant of invasive evolution and provides a framework for identifying early TME vulnerabilities.