Methylation-Guided Stratification of Colorectal Cancer Reveals Immune Subtypes with Distinct Clinical Outcomes

Background Aberrant DNA methylation is a hallmark of colorectal cancer (CRC). Yet, how DNA methylation is linked to transcriptional states, immune programs, and tissue resident microbiome within the same tumors has not been systematically analyzed. Methods We profiled genome-wide DNA methylation (Illumina MethylationEPIC) in 182 colon tumors and 76 adjacent normals from AC-ICAM, and integrated with matched transcriptomes, whole exome, microbiome, and clinical data. Tumor-specific methylation, promoter methylation–expression links, microbiome associations, and survival were analyzed and validated in TCGA-COAD. Results Tumor and normal tissues exhibited distinct DNA methylation patterns, reflecting widespread epigenetic alterations in cancer. Pathway analysis identified two major tumor pathways regulated by DNA methylation. The first involved extracellular signaling and adhesion genes, with higher methylation linked to increased proliferation and lower immune infiltration. Similarly, higher tumor methylation in nitric oxide signaling was associated with reduced adaptive immune activity and interestingly, influenced immune-related survival. These findings were also validated in the TCGA-COAD cohort. An inverse methylation–expression pattern implicated modifications of TCR signaling in naïve CD8⁺, and interferon-α/β signaling which were hypermethylated and hypomethylated in tumors compared to normal, respectively. Combining methylation and microbiome revealed connections between Akkermansia muciniphila and TGF-β and Prevotella nigrescens with MAPK signaling pathways. Finally, a methylation-based model using 43 promoters CpGs successfully identified patients with different survival outcomes, underscoring the clinical relevance of these epigenetic alterations in colon cancer. Conclusion DNA methylation shapes the molecular and immune landscape of colon cancer, altering signaling pathways and immune programs, interacting with the microbiome, and impacting patients’ survival.