Metastasis-associated DNA methylation alterations persist after accounting for immune and stromal cell heterogeneity in primary colorectal tumors

Background Metastatic colorectal cancer (CRC) remains a major cause of cancer mortality, yet how epigenetic states within the tumor microenvironment (TME) relate to metastatic progression has not been fully characterized. Although aberrant DNA methylation has been implicated in cancer progression, many studies do not account for immune and stromal cell composition, which can confound bulk methylation analyses. We sought to define DNA methylation patterns in primary colorectal tumors associated with lymph node and distant metastasis, while accounting for tumor microenvironment cellular composition. Methods We analyzed DNA methylation patterns from 57 patients with stage pT3 colorectal adenocarcinoma, comparing tumors with and without concurrent metastasis. Methylation cytometry deconvolution was performed to estimate immune, stromal, and tumor cell fractions. Differentially methylated positions associated with metastatic status were identified using covariate-adjusted epigenome-wide association analyses, with genomic context enrichment to assess regional methylation patterns. Genomic context enrichment and gene annotations were used to assess biological relevance. Methylation-expression correlations were assessed in an independent TCGA-COAD cohort to evaluate whether discovery loci correspond to differences in gene expression. Results Metastatic status was not associated with large-scale differences in inferred cellular composition. Epigenome-wide analyses identified hypomethylated loci mapped to genes involved in Wnt/β-catenin and PI3K/Akt signaling, epithelial–mesenchymal transition, and immune modulation, whereas hypermethylated loci tracked to genes related to adhesion, Wnt signaling, cytoskeletal organization, and interferon signaling. Genomic context enrichment revealed CpG island enrichment among hypermethylated DMPs in the distant metastasis contrast. Among metastasis-associated CpGs, 23 showed significant negative correlations between methylation and gene expression in TCGA-COAD (FDR < 0.05), predominantly at promoter-proximal CpG islands, supporting a relationship between promoter methylation and reduced gene expression. Two candidate CpGs showed directional concordance with significance in matched TCGA contrasts. Conclusions We identify distinct epigenetic alterations associated with metastatic progression in colorectal cancer that are largely independent of bulk immune and stromal composition. Integration of methylation-expression data supports a role for promoter hypermethylation in metastasis-associated gene silencing. These findings highlight potential methylation-based biomarkers of metastatic risk and may inform future precision oncology strategies in CRC.