Objective To investigate the expression of neutrophil extracellular traps (NETs) marker proteins citrullinated histone H3 (CitH3) and myeloperoxidase (MPO) in early-stage lung adenocarcinoma, and to analyze the dynamics of neutrophil infiltration, subpopulation heterogeneity, and the relationship between NET activity and tumor progression. Methods Paired tumor and adjacent normal tissue specimens were collected from patients with pathologically confirmed early-stage lung adenocarcinoma after surgical resection at the Second Affiliated Hospital of Hainan Medical University between October and December 2024. Immunofluorescence staining was performed to detect CitH3 and MPO expression. Image J software was used for quantitative analysis of fluorescence intensity and density of double-positive cells. Single-cell RNA sequencing data of cancerous and matched normal tissues were obtained from patients with early-stage lung adenocarcinoma at the same hospital from October 2024 to April 2025. Patients were stratified into groups with versus without high-grade components, and well-differentiated versus moderately/poorly differentiated groups according to pathological subtypes. Unsupervised clustering was applied to identify cell populations. The ratio of observed to expected values was used to analyze tissue distribution preference. Subset clustering was performed based on neutrophil marker genes, and a NETs gene set score was calculated to assess activity signatures. Results Paired tumor and normal tissues from 3 patients with early-stage lung adenocarcinoma (2 males and 1 female; median age 68.0 years, range 60.0-70.0 years) were included for immunofluorescence staining. Another 14 patients with early-stage lung adenocarcinoma were enrolled, including 6 males and 8 females, with a median age of 62.5 years (range 41.0-73.0 years). Three of these 14 patients also provided paired normal tissues. Single-cell RNA sequencing was conducted on tumor tissues from all 14 patients and paired normal tissues from the 3 patients. Among the 14 invasive non-mucinous adenocarcinomas, 7 samples were well-differentiated and 7 were moderately/poorly differentiated; 10 samples had no high-grade components and 4 had high-grade components. Immunofluorescence showed that NETs expression was significantly higher in tumor tissues than in normal tissues (P<0.001). Single-cell sequencing revealed an immunosuppressive tumor microenvironment in early lung adenocarcinoma, characterized by an early depletion of neutrophils followed by a sharp increase in neutrophil abundance as disease progressed. Two neutrophil categories were identified: resident subsets (c0/c4) and tumor-associated subsets (c1/c2/c3/c5). Resident subsets decreased while tumor-associated subsets accumulated with increasing malignancy. Among them, c3-Neut-LGALS3 and c2-Neut-CKLF were associated with progression in the high-grade group. The c0-Neut-AZIN1-AS1 subset was the major source of high NETs expression, and its activity was negatively correlated with tumor malignancy. Conclusion NETs-related proteins (CitH3 and MPO) are highly expressed in early-stage lung adenocarcinoma tissues. The microenvironment of early-stage lung adenocarcinoma exhibits immunosuppressive characteristics, and neutrophil subpopulation composition is remodeled with tumor progression, with the c0-Neut-AZIN1-AS1 subpopulation serving as a key source of high NETs expression.

Copyright ? the editorial department of Chinese Journal of Clinical Thoracic and Cardiovascular Surgery of West China Medical Publisher. All rights reserved