Interactions between colorectal cancer cells and cancer-associated fibroblasts across desmoplastic reaction subtypes

The desmoplastic reaction (DR) at the invasive front of colorectal cancer (CRC) has recently gained increasing attention as an important prognostic indicator and as a histological hallmark that reflects tumor-stroma interactions. DR is classified into mature, intermediate, and immature patterns based on stromal morphology, each representing distinct biological states of the surrounding microenvironment. Previous clinicopathological studies have demonstrated that immature and intermediate DR are associated with unfavorable outcomes; however, the biological mechanisms underlying these prognostic differences remain insufficiently understood. Because cancer-associated fibroblasts (CAFs) constitute the predominant stromal component of DR and critically influence stromal architecture, we aimed to clarify CRC-CAF interactions by combining analyses from a well-characterized clinical cohort and a three-dimensional co-culture system incorporating patient-derived CRC organoids and CAFs.We retrospectively identified 269 consecutive patients who underwent surgical resection for CRC (pT3 or higher) between 2013 and 2018 at our institution. Clinicopathological and prognostic variables were examined in detail, with a focused analysis of 191 stage II/III patients who did not receive preoperative therapy to reduce treatment-related confounding. Primary CRC organoids and CAFs were established from resected tumors under standardized culture conditions, and the DR subtype corresponding to each CAF line was determined histologically. To further explore stromal biology, proteomic profiling was performed to characterize DR subtype-specific protein expression signatures and to investigate molecular features associated with stromal phenotypes.Immature DR demonstrated significant associations with aggressive clinicopathological characteristics, including higher T (T3 66.0%, T4 34.0% P-value : 0.0011) and N (pN1-3 62.3% P-value : < 0,001) categories, vascular invasion (positive 98.1% P-value : < 0,001), and other indicators of tumor invasiveness, whereas mature DR was linked to more favorable clinical outcomes. In three-dimensional co-culture assays, CRC organoids exhibited the greatest proliferative expansion when paired with immature CAFs, emphasizing the functional relevance of stromal immaturity in promoting tumor growth (P-value : < 0.05). Proteomic analysis revealed clear segregation among DR subtypes, reflecting distinct protein expression profiles characteristic of each subtype. Notably, in immature DR, pathways related to cell adhesion centered on integrins, actin-regulation pathways, and fatty acid metabolism were among the top enriched pathways.These findings support DR classification as a clinically meaningful prognostic factor in CRC and indicate that immature CAFs play a key role in promoting CRC cell growth.