Methylation-Associated Differentiation Features Define Biological and Prognostic Heterogeneity in CMS4 Colorectal Cancer

Consensus molecular subtype 4 (CMS4) colorectal cancer (CRC) is associated with an aggressive clinical course and poor survival, yet the biological basis of heterogeneity within this subtype remains incompletely understood. DNA methylation is an epigenetic mechanism involved in transcriptional regulation, cellular differentiation, and colorectal tumorigenesis. Here, we integrated single-cell RNA sequencing (scRNA-seq), bulk data, and promoter DNA methylation data to characterize CMS4-associated cancer cell states and methylation-related features. Using the scAB algorithm, we integrated scRNA-seq with bulk CMS4 data and identified CMS4-related cells distributed across multiple patients. Single-cell analyses of cell–cell communication and transcriptional regulation revealed a CMS4-related cancer cell population characterized by macrophage migration inhibitory factor (MIF)-centered intercellular communication, enhanced caudal type homeobox 1 (CDX1) and Kruppel-like factor 5 (KLF5) regulon activity, and gene modules enriched in differentiation-related pathways. CytoTRACE analysis further stratified CMS4 cancer cells into poorly and well-differentiated states, yielding 802 differentially expressed genes (DEGs). Linking these differentiation-associated DEGs with bulk expression and promoter methylation data identified 218 methylation-associated DEGs showing significant inverse methylation expression correlations, suggesting a link between differentiation-related heterogeneity and promoter methylation. Univariable Cox regression followed by LASSO regression further prioritized eight genes for construction of the methylation and differentiation-related prognostic model (MeDiff-PM). MeDiff-PM consistently stratified overall survival in the TCGA CMS4 cohort and two independent validation cohorts, with cutoff-independent continuous Cox analyses further supporting its prognostic association across cohorts. And MeDiff-PM remained prognostically significant after adjustment for available clinical variables. High MeDiff-PM risk scores were associated with activation of P53, WNT, and ubiquitin-mediated proteolysis pathways and with consistent predicted drug response differences for compounds across three CMS4 cohorts. While individual in silico knockout analysis suggested links between MeDiff-PM genes and metallothionein-related and immune-associated transcriptional responses. Collectively, these findings indicate that methylation-associated differentiation features represent a molecular dimension of intra-CMS4 heterogeneity and provide a biologically informed framework for prognostic stratification within CMS4 CRC.