Figure 2.

Naged promote lung metastasis by capturing tumor cells via NETs. (A) Images of immunofluorescence staining (left panel) and SEM (right panel) of NETs produced by aged and non-aged neutrophils in the lungs of 2 weeks tumor-bearing mice. Red, Sytox Orange. Green, GFP). Scale bar, 50 µm. (B) SEM images of aged neutrophils in the lungs of 2 weeks tumor-bearing mice. Scale bar, 2 µm. (C) Multiplex Immunohistochemistry analysis of lung metastasis in tumor-bearing mice showing the relationship of tumor cells (green) and aged neutrophil (without red). Aged neutrophils were identified as Ly-6G⁺CD62L^lo cells (lower left panel) and tumor cells were identified as GFP⁺ cells. NETs were labeled with NE (neutrophil elastase). Scale bar, 10 µm. (D, E) Multiplex Immunohistochemistry analysis of relatively normal lung (D) and metastatic lung (E) tissues from patients with breast cancer showing the relationship of breast cancer cells (green) and aged neutrophils (without red). Aged neutrophils were identified as MPO⁺CD62L^lo cells (lower left panel). Tumor cells and pulmonary epithelial cells were identified as pan-cytokeratin⁺ cells. NETs were labeled with NE. Scale bar, 10 µm. (F) Quantification of aged neutrophils (left panel) and NETs (right panel) in relatively normal lung and metastatic lung tissues from patients with breast cancer. (G) Representative bioluminescent images and quantitative analysis of the lungs of 2 weeks tumor-bearing BALB/c mice (left panel) and nude mice (right panel) injected with luciferase-expressing 4T1 tumor cells after the injection of aged or non-aged neutrophils. Data are presented as the means±SD from one representative experiment. Similar results were obtained from three independent experiments, unless indicated otherwise. Statistical analysis was performed by two-tailed unpaired Student’s t-test (F, G). *p˂0.05, **p˂0.01, and ***p˂0.001. GFP, green fluorescent protein; NETs, neutrophil extracellular traps; ns, not significant.