Integrated single-cell and multi-cohort analyses reveal CTCF as a key regulator of chemo-immunotherapy resistance in lung adenocarcinoma.

Background: Chemo-immunotherapy has improved clinical outcomes in non-small cell lung cancer (NSCLC), but resistance remains a major challenge. The tumor cell states and molecular mechanisms underlying treatment resistance are not fully understood. Methods: Single-cell RNA sequencing data from NSCLC patients receiving chemo-immunotherapy were integrated with public transcriptomic cohorts and functional experiments. Resistance-associated tumor cell populations were identified, and key regulatory factors were investigated through bioinformatic analyses and experimental validation. Results: Single-cell analysis identified a malignant epithelial cell subpopulation enriched in patients with stable disease, suggesting a resistance-associated tumor cell state. Genes derived from this population were associated with unfavorable prognosis and were used to construct a prognostic model with consistent performance across multiple independent cohorts. Among the candidate genes, CTCF was identified as a key regulator associated with poor survival and reduced immune infiltration. In an orthotopic lung tumor model, Ctcf knockdown enhanced the antitumor efficacy of pemetrexed combined with anti-PD-1 treatment and promoted CD8 Conclusion: Our findings identify CTCF as a critical regulator of chemo-immunotherapy resistance in NSCLC. The CTCF-RRM2/CD276 axis links tumor-intrinsic drug resistance with immune evasion and may represent a potential therapeutic target.