https://orcid.org/0000-0001-7418-2116
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Abstract
Purpose
Neutrophils act as a non-negligible regulator in the initiation and progression of malignancies, playing bifacial roles in the process. Thus, to understand the heterogeneity of tumor-associated neutrophils (TANs) comprehensively in advanced non-small cell lung cancer (NSCLC) at single-cell resolution is necessary and urgent.
Materials and Methods
We applied single-cell RNA-sequencing (scRNA-seq) to portray the subtype-specific transcriptome landscape of TANs in advanced NSCLC using nine freshly obtained specimens. The scRNA-seq data were further processed for pseudo-time analysis to depict the developmental trajectory of TANs. Meanwhile, the interplay between TANs and other cell types within tumor microenvironment (TME) was revealed by intercellular interaction analysis.
Results
Seven distinct TAN subtypes were defined, of which, the N3 cluster was considered inflammatory phenotype expressing genes encoding multiple chemotactic cytokines, and correlated with inferior overall survival, indicating that N3 might be a pro-tumorigenic TAN subtype. N1 and N5 clusters were considered to be well differentiated and mature neutrophils based on CXCR2 expression and pseudo-time patterns, and both accounted for relatively high proportions in lung adenocarcinoma. In addition, genes related to neutrophil differentiation were discovered. We also found that TAN subtypes interacted most closely with macrophages through chemokine signaling pathways within TME.
Conclusion
Our study refined TAN subtypes and mapped the transcriptome landscape of TANs at single-cell resolution in advanced NSCLC, collectively indicating the heterogeneity of TANs in NSCLC. Neutrophil differentiation- and maturation-related genes were also discovered, which shed light on different functions of TAN subclones in tumor immune escape, and may further provide novel targets for immunotherapy.
Graphical Abstract
Abbreviations
DEG, differentially expressed gene; DNA, deoxyribonucleic acid; dNLR, derived neutrophil-to-lymphocyte ratio; ECOG PS, Eastern Cooperative Oncology Group performance status; EGFR, epidermal growth factor receptor; fDC, follicular dendritic cells; GO, Gene Ontology; HDN, high-density neutrophil; HSP, Heat Shock Protein; HVG, highly variable gene; IFN, interferon; KEGG, Kyoto Encyclopedia of Genes and Genome; LDN, low-density neutrophil; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MP, mononuclear phagocyte; NAN, normal adjacent tissue-associated neutrophil; NLR, neutrophil-to-lymphocyte ratio; NSCLC, non-small cell lung cancer; OS, overall survival; RNA, ribonucleic acid; scRNA-seq, single-cell RNA-sequencing; TAN, tumor-associated neutrophils; TGF, transforming growth factor; TME, tumor microenvironment; UMAP, Uniform Manifold Approximation and Projection.
Data Sharing Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Acknowledgments
Abstract of this paper was presented at the ESMO Immuno-Oncology Congress 2022 as a poster presentation with interim findings. The poster’s abstract was published in “Poster Abstracts” in Annals of Oncology: https://www.esmoiotech.org/article/S2590-0188(22)00251-9/fulltext.
Ethics Approval and Informed Consent
This study was performed in line with the principles of the Declaration of Helsinki. The Ethical Committee of Shanghai Pulmonary Hospital Affiliated to Tongji University approved this study (No. K18-089-1). Informed consent was obtained from all individual participants included in the study.
Disclosure
Fengying Wu serves as the associate editor-in-chief of Lung Cancer: Targets and Therapy. The other authors have no conflicts of interest to declare for this work.