Over the past decade, multiple groups have classified colorectal carcinoma (CRC) subtypes based on molecular profiles to explain CRC heterogeneity. Increasing evidence suggests microbial dysbiosis plays a crucial role in CRC development and progression. Our study aimed to identify integrated tumor microenvironmental groups (iTMEg) through multi-omics subtyping based on preselected cancer-related features.

Harnessing a unique multi-omics CRC dataset comprising of 6 distinct modalities, we analyzed 138 patients with stage 0–IV CRC (without prior neoadjuvant treatment) using unsupervised consensus clustering of three of the modalities: microbial composition of tumor mucosa, tumor genes expression, and tumor mutational landscape. For further refinement, we studied immune cell fractions inferred from bulk tumor transcriptomics via xCell, spatial distributions of morphological regions within tumor tissue, and stool-derived microbial and miRNA profiles.

Through comprehensive multi-omics analysis, we identified 6 distinct iTMEgs, reflecting unique immune, stromal, and microbial compositions. iTMEg 1 (Immune Activated) exhibits an immune-active yet immunosuppressive microenvironment enriched with oral pathogens such as Fusobacterium nucleatum, Parvimonas micra; MSI-H status, and BRAF mutations. iTMEg 2 (Immune Desert) represents a CIN-driven, immune-depleted group with low microbial diversity, stromal sparseness, and canonical APC and TP53 mutations. Stromal-enriched tumors divided into two groups: iTMEg 3 (Immune Excluded), showing a desmoplastic phenotype with EMT activity, noncanonical WNT signaling, and NRAS mutations; and iTMEg 6 (Highly Immunogenic), characterized by active KRAS signaling, Bacteroides enrichment, and an immune-activated stromal microenvironment. Mucinous tumors stratified into three groups: iTMEg 1 and iTMEg 4 (Regulated Immunity), both MSI-H, but differing in immune and microbial profiles; and iTMEg 6. iTMEg 5 (Immune Suppressed), in contrast, displays a structured, immune-low profile with complex tubular morphology and PIK3CA mutations. Partial associations of iTMEgs were observed with CMS and iCMS.

Our approach offers a novel, more comprehensive perspective on the tumour microenvironment beyond existing molecular classifications. These groups suggest potential of more targeted therapeutic strategies, including immune checkpoint inhibitors for immune-active groups, stromal modulation for desmoplastic groups, and targeted therapies for CIN-driven tumors.