A metabolic state in the cancer-associated fibroblast differentiation continuum of colorectal cancer

Background The metabolic heterogeneity of cancer-associated fibroblasts (CAFs) in the colorectal cancer (CRC) microenvironment remains poorly understood. Methods We profiled 86,142 cells from two independent CRC cohorts (GSE132465, GSE144735) using single-cell RNA-sequencing and validated findings in a public Visium dataset (GSE285505, 3,493 spots) and an external spatial cohort (GSE267401, 4 sections). Unsupervised clustering identified CAF subtypes, regulon analysis (SCENIC) mapped transcriptional drivers, and spatial deconvolution (cell2location) localized CAF populations in tissue. Findings Unsupervised clustering of 13,003 CAFs identified a metabolically distinct subpopulation (meCAF, 4.1% of CAFs) that scored highest for glycolysis (Δ = +0.60, P < 1e-4), lactate metabolism (Δ = +0.46, P < 1e-4) and hypoxia among nine metabolic pathways compared across all three CAF subtypes. SCENIC analysis inferred HIF1A as the regulon with the highest meCAF specificity (ΔAUC = +0.111, 95% CI 0.081 to 0.134, P = 2.4e-07 by patient-paired Wilcoxon test, 23/23 patients concordant). Spatial profiling confirmed meCAF enrichment at the tumor-stroma interface with spatial concordance with lactate metabolism (Spearman ρ = 0.29–0.75). meCAF abundance was inversely correlated with distance to the cancer epithelium (ρ = −0.317, p < 3e-82), accompanied by a metabolic activity gradient. In TCGA-COADREAD (n = 410), the meCAF signature varied significantly across consensus molecular subtypes (ANOVA p = 1.9e-18), with the highest scores in CMS1 (immune subtype) and the lowest in CMS2 (epithelial subtype). Interpretation These findings reveal a metabolically distinct CAF subpopulation operating within spatially restricted periepithelial micro-niches, with its clinical association to the immune-rich CMS1 subtype suggesting that stromal metabolic state may relate to the immune context of CRC.