P4HA1⁺ collagen-modifying fibroblasts are associated with pro-tumor niches and therapeutic vulnerabilities in colorectal cancer

Cancer-associated fibroblasts (CAFs) are a major stromal component of the tumor microenvironment (TME) and actively participate in tumor progression. Although CAF heterogeneity in colorectal cancer (CRC) is increasingly recognized, how individual CAF subpopulations simultaneously coordinate extracellular matrix (ECM) remodeling, angiogenesis, and immune suppression within spatially organized niches remains poorly understood. CAF heterogeneity was systematically analyzed by integrating single-cell RNA sequencing, spatial transcriptomics, and bulk transcriptomic data. By combining cell clustering, pseudotime analysis, transcriptional regulatory network inference, spatial deconvolution, and ligand–receptor interaction analysis, we revealed the spatial distribution and cellular communication characteristics of CAF subpopulations. At the same time, machine learning-based prognostic models and transcriptional perturbation analysis were used to evaluate the clinical significance and potential therapeutic value of CAF-related transcriptional programs. We identified a prolyl 4-hydroxylase subunit alpha 1 (P4HA1)-positive collagen-modifying CAF subpopulation (P4HA1⁺ CAFs) that was enriched in tumor tissue and exhibited collagen remodeling characteristics. This subpopulation preferentially localized at the tumor-stroma interface, co-localizing with pro-angiogenic endothelial cells, immunosuppressive macrophages, and malignant epithelial cells to form a spatially organized niche with concurrent ECM remodeling, angiogenesis, and immune suppression. P4HA1⁺ CAFs were predicted to engage distinct ligand–receptor axes, including WNT5A–MCAM with endothelial cells, MDK–LRP1 with macrophages, and MDK–NCL with malignant epithelial cells. The MDK–NCL axis was associated with increased RANBP1 expression and enhanced tumor cell proliferation and invasion. Machine learning analysis further identified P4HA1, TIMP1, ARL4C, and SFRP4 as stable core genes associated with poor prognosis. Drug perturbation analysis showed that 2-methoxyestradiol and its combination with the histone deacetylase (HDAC) inhibitor JNJ-26,481,585 may partially reverse the malignant transcriptional program of P4HA1⁺ CAFs. This study characterizes a P4HA1⁺ collagen-modifying CAF subpopulation that resides within a spatially organized multicellular niche and engages distinct signaling axes to simultaneously promote angiogenesis, immune suppression, and tumor cell proliferation in CRC, highlighting its prognostic and potential therapeutic value.