A multi-omics dissection of INHBA⁺ CAF-mediated epithelial-mesenchymal transition and immune suppression in...

The progression of colorectal cancer (CRC) is profoundly influenced by the tumor microenvironment (TME). However, the spatial functional specialization of cancer-associated fibroblasts (CAFs) in driving malignancy and immune evasion remains poorly defined. This study aims to delineate the spatial and functional heterogeneity of specific CAF subpopulations in CRC.

We performed single-cell RNA sequencing (scRNA-seq) on CRC tissues and integrated public datasets to map the stromal landscape. Key cellular interactions inferred from scRNA-seq were validated using high-resolution Xenium in situ transcriptomics and further corroborated by public spatial transcriptomics (ST) data and immunohistochemistry (IHC).

ScRNA-seq identified two distinct, tumor-enriched CAF subpopulations: INHBA⁺ SULF1⁺ CAFs, associated with an epithelial-mesenchymal transition (EMT) signature, and INHBA⁺ BNIP3⁺ CAFs, which exhibited an immunoregulatory phenotype. Xenium spatial mapping localized these transcriptional identities to spatially segregated populations of INHBA⁺ myofibroblastic CAFs (myCAFs) and INHBA⁺ inflammatory CAFs (iCAFs), respectively. Both subsets localized to 'Invasive Tumor-Enriched Regions' alongside EMT⁺ malignant epithelial cells. Spatial analysis further revealed that INHBA⁺ myCAFs frequently clustered adjacent to SPP1⁺ macrophages, whereas INHBA⁺ iCAFs resided within specialized immunosuppressive niches. Orthogonal validation using ST and IHC confirmed that INHBA⁺ SULF1⁺ CAFs directly colocalized with the Malignant C2 (characterized by an EMT signature), while INHBA⁺ BNIP3⁺ CAFs were closely associated with CD4⁺ regulatory T cells (Tregs). These findings suggest that two spatially and functionally distinct INHBA⁺ CAF lineages coexist: one orchestrating EMT-driven invasion and the other fostering local immunosuppression via Treg recruitment.

Our study reveals a spatial division of labor among CAFs in CRC. The conserved expression of INHBA across both pro-tumorigenic lineages positions it as a central stromal regulator and a promising therapeutic target for simultaneously disrupting tumor invasion and immune evasion. These findings highlight the potential of stroma-targeted strategies in advancing CRC treatment.