Single-cell transcriptome analysis reveals regulatory programs associated with tumor resistance during immunotherapy...

Colorectal cancer (CRC), a predominant cancer type globally, is one of the most common cancers worldwide. Immune-checkpoint inhibitors have robust efficacy in the treatment of patients with metastatic DNA mismatch repair (dMMR) CRC; however, some of the patients among them demonstrate resistance to immunotherapy, and the underlying molecular mechanisms remain elusive.

Using single-cell sequencing data, we identified and annotated cell populations, systematically comparing cell proportions and gene expression changes across different groups. Gene set enrichment analysis was conducted to identify significantly enriched signaling pathways in T-cell subpopulations. Furthermore, we assessed the impact of transcriptional dysregulation in stress response stated T (TSTR) and γδ T cells on tumor immunotherapy responses and drug resistance. The potential role of T-cell subset interactions in modulating drug resistance and sensitivity was investigated through comprehensive cell communication analysis. Lastly, clinical samples from 190 CRC patients were collected, with paired adjacent normal tissues. Immunohistochemistry was performed using antibodies against FOS and KLRB1.

T cells were identified as the predominant cell population influencing immunotherapy outcomes. T-cell subsets exhibited distinct functional characteristics, with notable differences in their distribution between resistant and sensitive tumor groups. Specifically, exhausted T cells (Tex), GZMK+ T, TSTR, regulatory T cells (Treg), and γδ T cells were associated with therapeutic resistance. For Tex and GZMK+ T cells, resistance was correlated with the activation of antigen processing and presentation pathways, whereas oxidative stress pathways were downregulated. In contrast, γδ T cells in the sensitive group exhibited the activation of protein folding pathways, which may contribute to anti-tumor immune responses. Transcriptional network dysregulation in TSTR and γδ T cells was observed in the drug-resistant group. Cell-to-cell communication analysis showed stronger interactions among T-cell subpopulations, with changes in key signaling pathways linked to treatment resistance. Additionally, downregulation of the CD69-KLRB1 signaling pathway was identified as a potential mechanism of drug resistance in CRC. Lastly, high expression of FOS was significantly associated with a worse prognosis, whereas high expression of KLRB1 predicted improved clinical outcomes, including prolonged overall survival and progression-free survival, and emerged as an independent prognostic factor for CRC patients.

This study highlights the pivotal role of T-cell subsets in patients with metastatic dMMR CRC who resistant to anti-PD-1 therapy, revealing that transcriptional dysregulation and impaired cell communication networks are central mechanisms underlying drug resistance. Notably, KLRB1 has been identified as a promising biomarker for immunotherapy response in CRC patients.