Acrolein target-linked toxicological perturbation and tumor-immune microenvironment heterogeneity in colorectal cancer

Abstract

Background Acrolein is a highly reactive aldehyde generated endogenously during lipid peroxidation and encountered through environmental and dietary exposures. Although acrolein has established electrophilic, genotoxic, and oxidative-stress effects, the extent to which acrolein-linked gene perturbation is represented in colorectal cancer (CRC) transcriptomes and relates to tumor immune-stromal heterogeneity remains incompletely characterized. Methods We integrated direct human acrolein target evidence from the Comparative Toxicogenomics Database (CTD) with TCGA-COAD/READ bulk RNA-sequencing profiles and a single-cell RNA-sequencing subset from GSE231559. The CTD curation yielded 179 unique direct human targets. Bulk analyses included 624 primary-tumor and 51 solid-tissue normal profiles. Single-cell analyses included six tumor and three normal libraries, yielding 24,128 retained cells assigned to eight broad marker-supported compartments. Target dysregulation burden, immune-stromal expression programs, and library-aware single-cell target localization were evaluated as transcriptomic summaries. In addition, RT-qPCR was performed in HCT 116 colorectal carcinoma cells to experimentally evaluate four measurable CTD targets (NFKBIA, GSTP1, VIM, and GPX4) after acrolein exposure. Results The bulk tumor-normal analysis identified 5,594 differentially expressed genes at FDR < 0.05 and |log2 fold change| ≥ 1. Of 179 CTD acrolein targets, 155 were measurable in the bulk dataset and 43 met the differential-expression threshold. Mean absolute normal-reference target burden was higher in tumors than normal tissue (1.96 vs. 0.75; difference 1.21; Welch p = 3.62e-42). Within primary tumors, target burden correlated negatively with T-cell and endothelial programs (rho = −0.27 for each), myeloid (rho = −0.20), fibroblast-ECM (rho = −0.19), and antigen-presentation programs (rho = −0.20), and positively with cell cycle (rho = 0.20). In single-cell data, descriptive mean target scores were highest in myeloid cells (0.455), fibroblasts (0.424), and endothelial cells (0.344). Cross-scale effects for 154 shared targets showed modest concordance (rho = 0.31, p = 6.95e-05). RT-qPCR showed vehicle-normalized relative expression of 2.18 ± 0.14 for NFKBIA, 1.83 ± 0.13 for GSTP1, 1.57 ± 0.15 for VIM, and 0.71 ± 0.09 for GPX4 after acrolein exposure (mean ± SD of three biological replicates). Conclusions Acrolein target-linked expression perturbation is detectable in CRC and is associated with immune-stromal heterogeneity across bulk and single-cell transcriptomic scales. RT-qPCR provides targeted experimental support for acrolein-responsive expression changes in NFKBIA, GSTP1, VIM, and GPX4 in HCT 116 cells. These findings support a toxicogenomic framework for hypothesis generation but do not measure acrolein exposure in patients, establish causal toxicological effects in human tumors, or demonstrate a specific intercellular signaling mechanism. Additional mechanistic, protein/adduct, dose-response, and independent-cohort validation will be required to determine the biological significance of the observed associations.