COL8A1-positive cancer-associated fibroblasts are drivers of 5-fluorouracil resistance in colorectal cancer.

Acquired resistance to 5-fluorouracil (5-FU) curtails the survival benefit of chemotherapy in colorectal cancer (CRC). We asked whether a discrete subset of cancer-associated fibroblasts (CAFs) drives this phenotype. Multi-omic profiling of 24 CRC cohorts identified 10 collagen genes linked to poor outcome and 5-FU resistance; single-cell RNA-seq localized them to specific CAFs. Cellular communication modelling, in vitro proliferation, migration, apoptosis, and drug-sensitivity assays, together with CRISPR/Cas9 and siRNA perturbations, xenografts, and immunochemical analyses, examined the functional relevance of COL8A1-positive fibroblasts (COL8A1⁺Fibs) and their downstream signalling pathways. COL8A1⁺Fibs were enriched in advanced tumors and preferentially interacted with 5-FU-resistant malignant cells. Conditioned media from COL8A1⁺Fibs accelerated CRC cell growth, invasion, and 5-FU tolerance, and activated an epithelial-mesenchymal transition (EMT) programme. Secreted COL8A1 engaged integrin-β1 (ITGB1) on tumor cells; silencing ITGB1 or COL8A1 abrogated EMT induction, reduced proliferation, and restored 5-FU sensitivity in vitro and in vivo. COL8A1⁺Fibs orchestrate 5-FU resistance in CRC via a COL8A1/ITGB1-mediated EMT axis. Disrupting this stromal-tumor crosstalk represents a promising therapeutic strategy to overcome chemoresistance.