OBJECTIVE: To investigate the molecular and cellular features associated with immunotherapy resistance in colorectal cancer (CRC) and identify potential biomarkers. METHODS: The single-cell RNA sequencing data from 14 pre-treatmant CRC samples (11 complete respondence and 3 stable disease) were collected from GSE236581. Clustering, pseudotime trajectory analysis, and intercellular communication analysis were performed to explore the tumor microenvironment and malignant cell characteristics. Bulk transcriptome datasets TCGA-COAD and GSE39582 were collected to assist in screening and validating key genes involved in tumorigenesis and prognosis. Key findings were validated in independent cohorts and via immunohistochemistry and T cell cytotoxicity experiments. RESULTS: Unsupervised clustering identified 21 cell clusters and 8,810 malignant epithelial cells. Differential gene expression analysis revealed 7,648 genes significantly differing between groups, with 568 upregulated in stable disease samples, enriched in ECM organization, ERK1/2 signaling, and metabolic reprogramming. Exploratory pseudotime analysis of cross-sectional pre-treatment samples revealed differences in inferred cellular state distributions in stable disease cells, accompanied by reduced leukocyte chemotaxis‑related signatures. Immune profiling highlighted enhanced CD8 + T-cell cytotoxicity in responders, while stable disease TME contained immunosuppressive macrophages. Intercellular communication analysis revealed stronger malignant cell-CD8 + T-cell interactions in stable disease samples. ASPHD1, associated with poor survival, was upregulated in CRC tissues and may serve as a candidate biomarker associated with immunotherapy response. CONCLUSION: Our findings highlighted the molecular and cellular features associated with immunotherapy resistance in CRC, involving malignant cell adaptations and TME remodeling. ASPHD1 was identified as a candidate gene linked to immunotherapy response in CRC. Given the exploratory nature of this work, these findings and the functional significance of ASPHD1 warrant further validation through larger cohorts and in-depth experimental studies.
Wang et al. (Mon,) studied this question.