Integrated single-cell and bulk tissue analyses reveal distinct macrophage subtypes and a candidate prognostic signature in colorectal cancer: implications for tumor immune characterization

Abstract

Colorectal cancer (CRC) represents a major global health burden, marked by high morbidity and mortality rates that place a considerable strain on healthcare systems. This study leveraged integrated bioinformatic analyses, single-cell RNA sequencing, and clinical sample validation to investigate the role of macrophage-related genes (MRGs) in CRC, with the goal of deepening our understanding of the complex interplay within the tumor microenvironment. Differential expression analysis comparing CRC tumor and normal tissues in the TCGA-COADREAD cohort identified 1,962 differentially expressed genes (DEGs). In parallel, a predefined set of 1,719 MRGs was curated from public databases and the literature to define the macrophage-related biological context. By integrating the bulk transcriptomic DEGs, single-cell macrophage subtype-specific genes, and the predefined MRG set, we identified eight hub macrophage-related DEGs (MRDEGs) implicated in CRC. Functional enrichment analysis of these eight MRDEGs revealed significant roles in immune-regulatory processes, including leukocyte chemotaxis, eosinophil chemotaxis, chemokine receptor binding, and the chemokine signaling pathway. From these eight hub MRDEGs, we selected CCL24 and MMP12 via LASSO-Cox regression to construct a prognostic risk model. Immune infiltration analysis using CIBERSORT revealed significant differences ( p < 0.05) in the abundance of nine immune cell types (including M0/M2 Macrophages, CD8+ T cells, T follicular helper cells, regulatory T cells (Tregs), memory B cells, monocytes, eosinophils, and neutrophils) between the risk groups defined by this macrophage-related signature. Drug sensitivity analysis further revealed that the high-risk group was significantly less responsive to sorafenib ( p < 0.05). The model provides candidate molecular clues for further investigation of the CRC immune microenvironment and prognostic stratification related to macrophage biology. We further performed experimental validation of the differentially expressed genes using clinical CRC samples and assessed their expression at both the transcriptional and protein levels. This study provides a candidate prognostic stratification model that requires further validation in independent cohorts and prospective studies, offering a macrophage-related prognostic clue for future investigations.