Microbial succession driven by Alistipes depletion shapes the mesenchymal and immune-excluded consensus molecular subtype 4 colorectal cancer landscape

Figure 1 Workflow of the study. Tumor, adjacent tissue, and fecal samples from 11 patients with colorectal cancer were analyzed. Gene expression profiles were compared with The Cancer Genome Atlas data and Consensus Molecular Subtypes (CMS) templates by principal component analysis, and classified by CMS using CMSclassifier. Differential expression, immune infiltration, and microbiome features were examined across subtypes, with host-microbiota associations further explored to identify CMS4-specific microbial signals. CMS: Consensus Molecular Subtypes.

Figure 2 Analysis of batch effect and the result of Consensus Molecular Subtypes classification. A: Scatter plot indicating no obvious batch effect between these samples and those samples in The Cancer Genome Atlas; B and C: Heat maps comparing the Consensus Molecular Subtypes classification results; D: Diagram showing the results of Consensus Molecular Subtypes classification for the 11 samples. PC: Principal component; RF: Random forest; SSP: Segmented spectral projection; CMS: Consensus Molecular Subtypes; NA: Not available.

Figure 3 Differential expression and enrichment analysis. A: Volcano plots of differentially expressed genes between tumor (cancer tissue) and adjacent (cancer-adjacent para-carcinoma) tissues; B: Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment of differentially expressed genes; C: Upset plot showing shared enriched pathways across Consensus Molecular Subtypes classifications. CMS: Consensus Molecular Subtypes.

Figure 4 Immune infiltration patterns and functional immunological features across Consensus Molecular Subtypes classifications. A: Z-score normalized enrichment scores of 24 immune cell types estimated by single-sample gene set enrichment analysis; B: Heatmap comparing infiltration levels of representative immune cells among Consensus Molecular Subtypes (CMS1)-CMS4; C: Boxplots showing subtype-specific differences for five significantly different immune cell types; D: Correlations between top five subtype-specific marker genes and immune infiltration within each CMS; E: Violin plots of programmed death-ligand 1 response probability, cytolytic activity score, immune exclusion score, and immunophenoscore across CMS classifications. CMS: Consensus Molecular Subtypes; NK: Natural killer; ssGSEA: Single-sample gene set enrichment analysis; PD-1: Programmed cell death protein 1; CYT: Cytolytic activity score; IPS: Immunophenoscore.

Figure 5 Microbiome diversity, composition, and host-microbe associations across Consensus Molecular Subtypes classifications. A: Stacked bar plots showing the relative abundance of dominant microbial taxa across Consensus Molecular Subtypes (CMS) classifications and sample types; B: Heatmap illustrating correlations between selected host genes and microbial phyla in cancer tissue, cancer-adjacent para-carcinoma, and F samples; C: Mean relative abundances of the top 20 differential species across CMS1-CMS4; D: Species-level differences (cancer tissue vs cancer-adjacent para-carcinoma) showing top 20 species with the largest mean abundance shifts per CMS classification; E: Shannon diversity indices comparing α-diversity of tumor (cancer tissue), adjacent (cancer-adjacent para-carcinoma), and fecal (F) microbiomes by CMS classification. CA: Cancer tissue; CP: Cancer-adjacent para-carcinoma; CMS: Consensus Molecular Subtypes.

Figure 6 Microbial features and functional pathways associated with Consensus Molecular Subtypes 4 subtype. A: Sankey diagram visualizing Kyoto Encyclopedia of Genes and Genomes orthology-pathway-function connections showing modules enriched in Alistipes indistinctus; B: Species-level differences between tumor and adjacent tissue (cancer tissue vs cancer-adjacent para-carcinoma) highlighting the top species with the largest mean abundance shifts; C: Mean relative abundances of the top 20 species identified in Consensus Molecular Subtypes 4 samples; D: Heatmap showing sample-level abundance (log₁₀-transformed) of the top 20 species in Consensus Molecular Subtypes 4 fecal samples; E: Heatmap illustrating Alistipes indistinctus proteins mapped to colorectal cancer-related Kyoto Encyclopedia of Genes and Genomes pathways. CMS: Consensus Molecular Subtypes; CRC: Colorectal cancer; KEGG: Kyoto Encyclopedia of Genes and Genomes.