An integrated spatially defined roadmap of normal and cancer-associated colon fibroblasts.

Cancer-associated fibroblasts (CAFs) promote tumorigenesis and represent potential therapeutic targets, highlighting the need for precise understanding of CAF heterogeneity. In colorectal cancer (CRC), CAF subsets and nomenclature vary widely, and many studies overlook the diversity of normal colon fibroblasts that shapes tumor mesenchyme. Here, we combine large-scale single-cell RNA-sequencing analysis with spatial validation and functional assays to construct a comprehensive reference map of human normal colon fibroblasts and mesenchymal subsets, guided by the well-characterized mouse colon. We define three major fibroblast populations, subepithelial myofibroblasts (SEMFs), mucosa-associated fibroblasts (MAFs), and submucosa-associated fibroblasts (SAFs), and characterize a previously underexplored muscle-embedded interstitial fibroblast (MIF) population. This reference enabled mapping of CRC-specific changes, revealing four cancer-specific CAF subsets, including inflammatory CAFs (iCAFs), matrix CAFs (mCAFs), and two precursor populations (pre-CAFs). We also identify robust CRC CAF markers including CTHRC1, infer transcriptional regulators, and define distinct developmental trajectories driving iCAF and mCAF activation. Together, our study provides a spatially and transcriptionally resolved reference map of fibroblasts in the normal colon and CRC, enabling mechanistic studies and informing CAF-targeted therapies.