Objective: N6-methyladenosine (m6A) methylation regulators are critical for cancer progression, but published data on the mechanism of m6A modification in tumor microenvironment (TME) cell infiltration of colorectal cancer (CRC) remains limited. This study aimed to investigate the correlation between m6A modification patterns and CRC TME heterogeneity and explore their prognostic significance and guiding value for immunotherapy.

Materials and Methods: RNA expression profiles and clinical data of CRC were retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Unsupervised clustering was performed to evaluate m6A modification patterns of 23 m6A methylation regulators in 983 CRC samples, and their associations with TME cell infiltration characteristics were systematically analyzed. Gene Ontology (GO) and gene set variation analysis (GSVA) explored underlying mechanisms, while principal component analysis (PCA) constructed an m6A score to quantify modification patterns.

Results: m6A methylation regulators showed high genetic and expression heterogeneity in CRC, leading to three distinct modification patterns. These patterns closely matched three immunophenotypes (immune rejection, immune inflammation, and immune desert) and exhibited distinct biological functions. Univariate and multivariate Cox regression indicated m6A score as an independent prognostic factor (HR=1.010, 95% CI: 1.002-1.019; HR=1.009, 95% CI: 1.000-1.017). Low m6A scores correlated with higher tumor mutation load, PD-L1/CTLA-4 expression, and poor survival. In the CTLA-4 immunotherapy cohort, high m6A scores were associated with significantly better immune response and clinical benefit (p=3.4e-06).

Conclusions: m6A methylation modification patterns are key drivers of TME heterogeneity and complexity in CRC. Exploring the relationship between m6A modification patterns and TME aids in formulating CRC immunotherapy strategies and provides valuable prognostic guidance.