Dissecting immunosuppressive microenvironment in chemotherapy-resistant colorectal cancer through single-cell transcriptomic analysis

BACKGROUND: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with chemotherapy resistance representing a critical barrier to effective treatment. The tumor microenvironment (TME) plays a pivotal role in mediating resistance through complex immunosuppressive mechanisms that remain incompletely understood at cellular resolution. METHODS: We performed comprehensive single-cell RNA sequencing analysis on 13,380 cells from chemotherapy-sensitive and resistant CRC specimens. Following rigorous quality control and dimensional reduction, we conducted unsupervised clustering, cell type annotation, differential expression analysis, functional enrichment assessment, trajectory inference, and cell-cell interaction network reconstruction. In vitro validation was performed using chemotherapy-resistant cell lines (HCT116-OxR and SW480-5FUR) with quantitative RT-PCR and ELISA assays. RESULTS: Single-cell analysis revealed profound TME remodeling in resistant tumors characterized by: (1) significant compositional shifts with reduced cytotoxic T cell and NK cell populations and increased regulatory T cells and M2-polarized macrophages; (2) systematic suppression of T cell activation pathways (normalized enrichment score = -2.8, FDR < 0.001) with concurrent upregulation of immunosuppressive programs including hypoxia response, angiogenesis, and metabolic adaptation; (3) enhanced inhibitory cell-cell communication networks featuring elevated PD-L1/PD-1, CTLA-4, TGF-β, and IL-10 signaling; (4) progressive T cell trajectory transitions from cytotoxic effector to exhausted phenotypes; (5) coordinated upregulation of immune checkpoint molecules (PD-L1, TIM-3, LAG-3), inhibitory cytokines (TGF-β1, IL-10), and metabolic enzymes (IDO1, ARG1). In vitro validation confirmed that resistant cell lines exhibited 2.3-5.1-fold transcriptional upregulation and significantly enhanced secretion of immunosuppressive factors (PD-L1, TGF-β1, IDO1, IL-10; all p < 0.001). CONCLUSIONS: This comprehensive single-cell atlas reveals the multi-dimensional immunosuppressive landscape of chemotherapy-resistant CRC, identifying coordinated cellular, molecular, and spatial mechanisms driving immune evasion. Our findings provide candidate biomarkers for predicting treatment resistance and highlight therapeutic vulnerabilities for developing immunotherapy-based combination strategies to overcome chemotherapy resistance in CRC.