# The effect of cold ischemia time on hypoxia, EMT, and apoptosis pathways in normal colon mucosa

## TL;DR

This study shows that the time between tissue removal and freezing affects gene activity in colon tissue, potentially mimicking cancer traits and affecting research results.

## Contribution

The study identifies specific gene expression changes due to cold ischemia time, linking them to cancer-related pathways like hypoxia, apoptosis, and EMT.

## Key findings

- 44 genes were differentially expressed after 60 minutes of cold ischemia compared to immediate freezing.
- Prolonged cold ischemia altered pathways related to apoptosis, hypoxia, EMT, and cancer progression.
- Two gene modules were identified, one downregulating apoptosis-related genes and another linked to apoptosis and metabolism.

## Abstract

Cold ischemia time (CIT), the interval between tissue excision and preservation, is a critical preanalytical variable that profoundly impacts gene expression profiles. Variability in CIT can lead to inconsistent transcriptomic results, making study interpretation challenging and undermining reproducibility in biomedical research. Our study aimed to evaluate the impact of CIT on the expression of cancer-related genes, particularly these involved in hypoxia, apoptosis, and epithelial-to-mesenchymal transition (EMT). We performed RNA sequencing on 54 normal colon mucosa samples from nine patients undergoing colorectal cancer surgeries, freezing samples at predefined intervals ranging from 0 to 60 minutes. A total of 44 differentially expressed genes (DEGs) (p < 0.05) were identified when comparing samples frozen immediately (T0) with those frozen after 60 minutes (T5). These DEGs were further analyzed through functional and pathway enrichment analyses and weighted gene co-expression network analysis (WGCNA). The enrichment analysis revealed significant alterations in pathways associated with apoptosis, hypoxia, EMT, and cancer progression, including p53 and HIF-1 signaling. WGCNA highlighted two co-expressed gene modules: ME2, which showed downregulation of apoptosis-related genes, and ME4, linked to apoptosis and cellular metabolism. Our findings highlight CIT as a critical preanalytical variable, showing that prolonged ischemia can induce transcriptomic changes that may mimic malignancy, and potentially confound research outcomes. To minimize such effects, we recommend keeping CIT under 30 minutes.