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Updated: Aug 17, 2026
Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
Integration of single-cell transcriptomics and genomic mutation analysis identifies an immunotherapy-resistant tumor
Zhengang Qiu1, Hui Zhao2, Qiong Lyu3
1Department of Oncology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China.
Background:
Immunotherapy resistance in lung adenocarcinoma (LUAD) remains a critical clinical challenge, and the mechanisms underlying resistance-associated intratumoral heterogeneity are poorly characterized.
Methods:
We performed single-cell RNA sequencing of LUAD patients receiving neoadjuvant immunotherapy (responders vs. non-responders), integrating inferCNV, GSVA, and differential expression analyses. Cluster-specific genes were validated across seven independent cohorts (TCGA-LUAD, GSE13213, GSE26939, GSE29016, GSE30219, GSE31210, GSE42127). A multi-algorithm machine learning framework was used to construct a prognostic model, and the immune microenvironment was characterized using TCIA scoring, seven infiltration algorithms, and ESTIMATE. ARNTL2 function was assessed by CCK-8 and Transwell assays in A549 and H1299 cells.
Results:
Non-responders showed significant enrichment of epithelial cells, depletion of cytotoxic T/NK cells, and elevated copy number variation burden versus responders (p < 0.0001). A resistance-enriched malignant subcluster (Cluster 2) exhibited hyperproliferative and metabolic reprogramming signatures with upregulated KRT17, S100A2, and CST6, which showed tumor-specific overexpression, adverse prognostic value, and genomic amplification across cohorts. CoxBoost combined with survivalSVM achieved optimal predictive performance (C-index = 0.686), yielding robust risk stratification (HR: 2.54-10.51, all p < 0.05). Low-risk patients showed greater immune infiltration and higher TCIA immunophenoscores. ARNTL2 was an independent prognostic factor (HR: 2.07-4.64) strongly correlated with risk score (r = 0.69), and its knockdown suppressed proliferation and invasion in both LUAD cell lines (all p < 0.05).
Conclusion:
This study identifies a resistance-associated malignant subcluster in LUAD, constructs a validated CoxBoost + survivalSVM prognostic model with robust immune stratification, and establishes ARNTL2 as a core oncogenic driver and therapeutic target.
Insights
This study reveals a specific cancer cell subtype driving immunotherapy resistance in lung adenocarcinoma (LUAD). Identifying this subtype and targeting ARNTL2 offers a new strategy for improving LUAD treatment outcomes.
Area of Science:
- Oncology
- Immunology
- Genomics
Background:
- Immunotherapy resistance in lung adenocarcinoma (LUAD) is a significant clinical hurdle.
- Mechanisms of resistance-driven intratumoral heterogeneity in LUAD are not well understood.
Purpose of the Study:
- To identify mechanisms of immunotherapy resistance in LUAD.
- To develop a prognostic model for LUAD patients.
- To investigate the role of ARNTL2 in LUAD progression and resistance.
Main Methods:
- Single-cell RNA sequencing of LUAD patients undergoing neoadjuvant immunotherapy.
- Integration of inferCNV, GSVA, and differential expression analyses.
- Machine learning for prognostic model construction and immune microenvironment characterization.
- Functional assessment of ARNTL2 in LUAD cell lines.
Main Results:
- Non-responders exhibited enriched epithelial cells, depleted cytotoxic T/NK cells, and higher copy number variation.
- A resistance-associated malignant subcluster (Cluster 2) displayed hyperproliferative and metabolic signatures with upregulated KRT17, S100A2, and CST6.
- A validated prognostic model (CoxBoost + survivalSVM) stratified patients by risk and immune infiltration.
- ARNTL2 was identified as an independent prognostic factor, correlating with risk score and suppressing LUAD cell proliferation and invasion upon knockdown.
Conclusions:
- A novel resistance-associated malignant subcluster in LUAD was identified.
- A robust, validated prognostic and immune stratification model was developed.
- ARNTL2 was established as a key oncogenic driver and potential therapeutic target in LUAD.
