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Background <p>Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer, exhibits substantial molecular heterogeneity, complicating the identification of reliable prognostic and therapeutic markers. Single-cell transcriptome analysis, combined with other omics and multi-level analyses, offers a feasible solution to address this issue.</p> Methods <p>We clustered, annotated, and identified landmark genes for single cells in LUAD dataset. Using the expression profiles of these marker genes, we classified TCGA-LUAD samples via non-negative matrix factorization (NMF) clustering. The LUAD subtypes were compared for prognosis, tumor stage, gene expression, aging and stemness scores, and immunotherapy response (assessed by TIDE). LASSO-Cox regression analysis selected key genes for a prognostic model, which was validated in training and external datasets. RT-qPCR was used to validate mRNA expression levels. Protein-level dysregulation was confirmed through proteomics and immunohistochemistry (IHC). Additionally, the functional effects of the key genes and their drug-sensitivity associations were explored in LUAD cell lines.</p> Results <p>Single cells were annotated into 12 types/subtypes, including three epithelial-associated and two fibroblast-associated subtypes. The top 100 marker genes for each cell type/subtype were identified. Based on these marker gene expressions, TCGA-LUAD samples were classified into C1 and C2 subtypes. Compared with C1 LUADs, C2 LUADs exhibited poorer prognosis, a higher proportion of late-stage tumors, higher MKI67 expression, and higher levels of aging and stemness, but lower MHC II gene expression and TIDE scores. LASSO-Cox regression analysis identified an eight-gene signature that effectively distinguished patient outcomes in both training and validation datasets. Dysregulation of these eight genes was confirmed at the mRNA level by RT-qPCR and at the protein level by proteomic and IHC analyses. Moreover, these eight genes were associated with sensitivity to 3–28 anti-cancer drugs in LUAD cell lines.</p> Conclusion <p>Subtyping based on single-cell derived marker genes and multi-level analyses provide a comprehensive understanding of LUAD biology. The distinct features of C1 and C2 subtypes highlight the need for tailored therapies. The eight-gene signature may serve as a novel prognostic, diagnostic, and therapeutic indicator.</p>

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Single-cell-marker-based subtyping and multi-level analyses uncover the prognostic effects, dysregulations and therapeutic indicative potential of an eight-gene signature in lung adenocarcinoma

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月30日

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  • Xiuzhi Zhang,
  • Fengqi Chen,
  • Wenke Sun,
  • Hanke Ma,
  • Feifei Liang,
  • Liping Dai,
  • Chunyan Kang,
  • Xiaoli Liu

摘要

Background

Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer, exhibits substantial molecular heterogeneity, complicating the identification of reliable prognostic and therapeutic markers. Single-cell transcriptome analysis, combined with other omics and multi-level analyses, offers a feasible solution to address this issue.

Methods

We clustered, annotated, and identified landmark genes for single cells in LUAD dataset. Using the expression profiles of these marker genes, we classified TCGA-LUAD samples via non-negative matrix factorization (NMF) clustering. The LUAD subtypes were compared for prognosis, tumor stage, gene expression, aging and stemness scores, and immunotherapy response (assessed by TIDE). LASSO-Cox regression analysis selected key genes for a prognostic model, which was validated in training and external datasets. RT-qPCR was used to validate mRNA expression levels. Protein-level dysregulation was confirmed through proteomics and immunohistochemistry (IHC). Additionally, the functional effects of the key genes and their drug-sensitivity associations were explored in LUAD cell lines.

Results

Single cells were annotated into 12 types/subtypes, including three epithelial-associated and two fibroblast-associated subtypes. The top 100 marker genes for each cell type/subtype were identified. Based on these marker gene expressions, TCGA-LUAD samples were classified into C1 and C2 subtypes. Compared with C1 LUADs, C2 LUADs exhibited poorer prognosis, a higher proportion of late-stage tumors, higher MKI67 expression, and higher levels of aging and stemness, but lower MHC II gene expression and TIDE scores. LASSO-Cox regression analysis identified an eight-gene signature that effectively distinguished patient outcomes in both training and validation datasets. Dysregulation of these eight genes was confirmed at the mRNA level by RT-qPCR and at the protein level by proteomic and IHC analyses. Moreover, these eight genes were associated with sensitivity to 3–28 anti-cancer drugs in LUAD cell lines.

Conclusion

Subtyping based on single-cell derived marker genes and multi-level analyses provide a comprehensive understanding of LUAD biology. The distinct features of C1 and C2 subtypes highlight the need for tailored therapies. The eight-gene signature may serve as a novel prognostic, diagnostic, and therapeutic indicator.