Single‑cell multi‑omics analysis reveals TGF-β1-RUNX2-mediated matCAF differentiation and its role in remodeling the lymph node metastatic microenvironment in bladder cancer.

Lymph node metastasis (LNM) in bladder cancer (BCa) is associated with poor clinical outcomes and may contribute to the development of metastatic tolerance, yet the stromal mechanisms underlying this process remain incompletely understood. By integrating single-cell RNA sequencing, spatial transcriptomics, clinical cohorts, and functional models, we identified an LNM-enriched matrix-remodeling cancer-associated fibroblast (matCAF) state characterized by LRRC15 and POSTN expression and associated with adverse survival and reduced immunotherapy response. Mechanistically, Hybrid_CCND1⁺ malignant epithelial cells promoted matCAF differentiation through FOS-dependent TGFB1 transcription and TGF-β1-TGFBR1-RUNX2 signaling. Spatial analyses further revealed barrier-like accumulation of matCAFs at tumor-stroma interfaces and linked matCAF-rich neighborhoods to enhanced PI3K activity and lymphangiogenesis-related programs. Functionally, matCAF-derived POSTN engaged ITGAV/ITGB5 to activate PI3K-AKT signaling and promote tumor and lymphatic endothelial remodeling. MatCAFs were also associated with SPP1⁺ macrophage remodeling and impaired CD8⁺ T-cell function, partly through RUNX2-associated CXCL12-CXCR4 signaling. Pharmacological disruption of the RUNX2-dependent matCAF program reduced tumor burden and partially restored T-cell activity in vivo. Collectively, these findings identify matCAF-centered stromal remodeling as a potential cellular basis of the metastatic-tolerance niche in BCa LNM and provide proof-of-concept for therapeutic targeting of the matCAF program.