Abstract
Background/Objectives: Colorectal cancer (CRC) exhibits significant molecular heterogeneity, as reflected in Consensus Molecular Subtype (CMS) classification, and demonstrates extensive crosstalk with the microbiome. However, the role of the microbiome in determining subtypes of CRC, and CMS4 in particular, which represents an aggressive, stromal-rich variant associated with poor prognosis, remains poorly understood. Here, we reveal the role of the tumor microbiome in shaping the tumor microenvironment (TME) and its impact on CMS4 determination. Methods: A total of 25 CRC tissues were analyzed using RNA sequencing and classified with CMScaller to identify significantly enriched microbial species. Functional studies were performed using these CMS-specific microbial species and CMS2 organoids co-cultured with stromal (18Co) and immune (THP-1) cells. Results: 16S rRNA profiling of matched CRC tissues showed that Bacteroides fragilis was significantly enriched in CMS4 tumors (linear discriminant analysis score = 4.7). Functional studies revealed that exposure to enterotoxigenic Bacteroides fragilis (ETBF) induced CMS4-like features, including enhanced growth and gene expression patterns resembling those of primary CMS4 tumors. Conclusions: These findings suggest that ETBF contributes to the development of CMS4 and may facilitate the acquisition of aggressive phenotype associated with this CRC subtype.