Decoding epigenetic and transcriptional landscapes: DNA methylome-transcriptome integration reveals novel drivers in...

Epigenetic alterations, particularly DNA methylation, and dysregulation of the tumor immune microenvironment (TIME) are increasingly recognized as critical factors in esophageal squamous cell carcinoma (ESCC) pathogenesis. Understanding the dynamic interplay between DNA methylation changes and TIME evolution during ESCC progression remains essential. We established a 4-nitroquinoline 1-oxide (4NQO)-induced ESCC mouse model, capturing distinct pathological stages: normal esophageal epithelium (Normal), esophageal simple hyperplasia (ESSH), intraepithelial neoplasia (IEN), and ESCC. Genome-wide DNA methylation profiling was performed using the Infinium Mouse Methylation BeadChip (285 K), coupled with transcriptome analysis via bulk RNA sequencing (RNA-seq). Immunohistochemistry (IHC) for Cd45 (leukocyte common antigen) validated immune cell infiltration.

DNA methylation profiling revealed progressive genome-wide hypomethylation during ESCC development, with hypomethylated probes significantly enriched in immune response pathways. Notably, ESSH exhibited a methylation profile similar to IEN and ESCC. RNA-seq identified escalating numbers of differentially expressed genes (DEGs). Immune deconvolution analysis and IHC of Cd45 demonstrated dynamic changes in TIME composition from ESSH onwards. Furthermore, dynamic expression clustering identified an innate immune response-related gene cluster highly expressed in ESSH. Integrative analysis yielded 495 methylated regulatory genes, significantly enriched in leukocyte cell-cell adhesion and T cell activation pathways (e.g., Ptprc/Cd45, Il12rb1, Tox), with peak activity in ESSH.

These findings highlight ESSH as a critical window where epigenetically driven immune changes facilitate ESCC progression. Targeting these early epigenetic-immune interactions may offer a novel strategy for ESCC early detection and combination therapy.