Dissecting colorectal cancer heterogeneity and microbiota interactions using murine and patient-derived organoids

Alterations in gut microbiota composition and metabolism are tightly linked to colorectal cancer (CRC) initiation, progression, and therapy response, yet underlying mechanisms remain unclear. We established an integrated platform combining organoid biobanks, germ-free orthotopic models, and microbial communities to dissect the interplay between oncogenic signaling, microbiota composition, microbial metabolism, and therapy response in CRC. We generated a comprehensive murine CRC organoid biobank encompassing distinct genotypes, histological subtypes, and tumor stages, together with a complementary collection of patient-derived organoids (PDOs). To probe microbial influence in vivo , we developed the first germ-free orthotopic CRC model enabling colonoscopy-guided engraftment of tumor organoids under germ-free (GF) conditions. Parallel transplantation into specific pathogen-free (SPF) and GF mice allows direct assessment of microbiota-dependent effects on tumor progression. We also established stool-derived in vitro communities (SDICs) from oncogenic and wild-type backgrounds. Cultured anaerobically in physiologically relevant media, SDICs preserve their native metabolic output, enabling functional assessment of microbiota-derived metabolites on organoid viability and therapy response without artifacts from direct bacteria-organoid co-cultures. Comparative analyses of SPF and GF mice revealed comparable survival after carcinoma organoid transplantation, whereas GF mice showed significantly prolonged survival after adenoma transplantation, indicating tumor growth and progression are slowed without microbiota. To dissect this host-microbiota interplay, we analyzed SDIC-derived metabolites and found that supernatants from wild-type SDICs were more effective than those from oncogenic SDICs in reducing tumor organoid viability. Notably, wild-type SDIC metabolites also sensitized tumor organoids to radiation therapy, highlighting microbiota-mediated modulation of therapeutic response. These findings demonstrate that epithelial oncogenic mutations not only drive tumorigenesis but also remodel the gut microbiota, depleting protective taxa and enriching tumor-promoting species. Consistent with this, 16S rRNA and metagenomic analyses confirmed host genotype dictates gut microbiota composition. To provide a translational bridge to murine models, we transplanted PDOs orthotopically and showed that both PDOs and their xenografts faithfully recapitulated patient-specific histology, molecular features, and therapy responses, establishing robust preclinical models to target colorectal cancer heterogeneity. Together, these findings identify microbiota-derived metabolites as critical regulators of CRC evolution and therapy response, and establish a modular framework for microbiota-informed precision oncology.