Investigate the heterogeneity of colorectal cancer patients at the single-cell level prior to and subsequent to immunotherapy

BackgroundImmune checkpoint blockade (ICB) has improved outcomes for a subset of colorectal cancer (CRC) patients, yet the cellular determinants that drive heterogeneous treatment responses remain insufficiently understood. The extent to which ICB reshapes immune populations across tumor, blood, and adjacent normal tissues at single-cell resolution is largely unexplored.MethodsWe reanalyzed spatiotemporal single-cell RNA-sequencing data from CRC patients treated with Pembrolizumab or Sintilimab, profiling immune and stromal compartments across pre- and post-treatment tumor, blood, and normal mucosa. Major cell types and transcriptional programs were characterized using Seurat and Harmony. Treatment-associated cellular remodeling, differential gene expression, and response-related signatures were assessed in T cells and B cells. Cell-cell communication networks were inferred using CellChat.ResultsICB produced pronounced remodeling of tumor-infiltrating lymphocytes, particularly within T cell and B cell lineages. Responders (CR/PR) exhibited robust cytotoxic T-cell activation characterized by up-regulation of GNLY, GZMB, and CXCL13, whereas non-responders (SD) uniquely showed persistent overexpression of HSPA1B, a stress-response gene associated with tumor progression and immune suppression. Responders (CR/PR) exhibited robust cytotoxic T-cell activation, with increased expression of GNLY, GZMB, and CXCL13. In contrast, non-responders (SD) exhibited persistent overexpression of HSPA1B, a stress-response gene linked to tumor progression and immune suppression. B cells from responders demonstrated strong induction of TXNIP, a regulator of redox balance and inflammasome activity. This suggests a potential association between ICB treatment and a pro-inflammatory B-cell phenotype. Cell-cell communication analysis revealed that responders selectively enhanced CXCL, IL16, and CD22 signaling pathways. This indicates coordinated activation of a T-B-myeloid communication axis, which was absent in SD patients.ConclusionsThis integrative spatiotemporal single-cell analysis reveals that effective ICB responses in CRC are associated with coordinated activation of cytotoxic T cells, TXNIP-associated transcriptional changes in B cells, and strengthening of CXCL-centered immune communication networks. In contrast, HSPA1B-high transcriptional states are associated with treatment resistance. These findings provide mechanistic insights into response heterogeneity and highlight potential candidate targets, such as HSPA1B and CXCR4-associated pathways for improving immunotherapy efficacy in CRC.