Single-Cell Profiling of Tumor-Associated Neutrophils in Advanced Non-Small Cell Lung Cancer

Abstract

Jinpeng Shi,1,* Jiayu Li,1,* Haowei Wang,1,* Xuefei Li,2 Qi Wang,1 Chao Zhao,2 Lei Cheng,2 Ruoshuang Han,1 Peixin Chen,1 Haoyue Guo,1 Zhuoran Tang,1 Caicun Zhou,1 Zhemin Zhang,1 Fengying Wu1 1Department of Medical Oncology, Shanghai Pulmonary Hospital & Thoracic Cancer Institute, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 2Department of Lung Cancer and Immunology, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fengying Wu; Zhemin Zhang, Department of Medical Oncology, Shanghai Pulmonary Hospital & Thoracic Cancer Institute, School of Medicine, Tongji University, No. 507, Zheng Min Road, Shanghai, 200433, People’s Republic of China, Tel [phone], Email [email protected]; [email protected]: 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. Keywords: tumor-associated neutrophil, single-cell RNA-sequencing, non-small cell lung cancer, tumor microenvironment, heterogeneity

Keywords

• tumor-associated neutrophil
• single-cell rna-sequencing
• non-small cell lung cancer
• tumor microenvironment
• heterogeneity