Bone-derived Osterix+ osteolineage cells are a source of tumor-promoting myofibroblastic cancer-associated fibroblasts in breast cancer.

Cancer-associated fibroblasts (CAFs) are major regulators of breast cancer (BC) progression and therapeutic resistance, yet the extent to which CAF heterogeneity is dictated by distinct cellular origins remains unresolved. Here, we identify bone-derived Osterix+ (Osx) osteolineage cells as a source of CAFs in BC. Using BC models in female mice and biopsies from women with BC, we show that bone-resident Osx+ cells are recruited to primary tumors. These cells preferentially differentiate into a myofibroblastic CAF subset with unique osteolineage identity (OsteoLin-myCAFs). OsteoLin-myCAFs are transcriptionally and functionally distinct from other subsets, exhibit enhanced extracellular matrix remodeling and pronounced pro-tumorigenic activity. Mechanistically, Osx drives expression of matrix-remodeling programs, including MMP13, which supports tumor growth. Cross-species analyses show a conserved 54-gene osteolineage signature in myCAFs from human BC samples, strongly associated with poor survival. Together, these findings identify a distinct bone-derived osteolineage cell that gives rise to OsteoLin-myCAFs and is linked to adverse clinical outcomes.