Integrative transcriptomic analysis reveals tumor-adjacent mucosal remodeling in colorectal cancer

Tumor-adjacent mucosa is commonly used as a normal reference in colorectal cancer (CRC) transcriptomic studies, but its relationship with distant normal mucosa and tumor tissue remains unclear. Here, we performed bulk RNA sequencing of paired distant normal, adjacent, and tumor tissues from 25 patients with CRC and used these three tissue states to construct an ordered normal-adjacent-tumor (N-A-T) expression continuum. Although adjacent mucosa was globally closer to distant normal mucosa and showed no significant differential expression by pairwise analysis, a limma-based linear trend model identified widespread monotonic transcriptional changes along the N-A-T continuum. Tumor-directed genes were enriched in translation, RNA processing, cell-cycle activity, and proliferation-related pathways, whereas genes decreasing along this axis were associated with oxidative metabolism and epithelial homeostasis. A trend gene-based N-A-T score positioned adjacent mucosa between normal and tumor tissues in external bulk cohorts and was associated with proliferative and immune-low molecular states in CRC. Single-cell RNA-seq analysis localized this signal mainly to epithelial cells and associated higher scores with proliferative and stem-like epithelial states. In conclusion, CRC-adjacent mucosa is not fully equivalent to distant normal mucosa but carries coordinated tumor-directed transcriptional changes mainly associated with epithelial-cell programs.