Fig. 3

NETs optimized adhesion of HCC and further promoted metastasis potential by raising invasiveness with minimal cytotoxicity. a Representative fluorescence images (middle panel, 2 h post-injection) and quantification (right panel) of early trapped HCC cells in the lung and liver in LPS-induced NET model. C57BL/6 mice were subjected to systemic LPS and/or DNase 1 abrogation, and subsequently intraspleen/intravenous injected with Dil-labeled Hepa1-6 cells (n = 5 each). Scare bar: 50 μm. b Increased adhesion of Dil-labeled HepG2/MHCC97H cells within PMA-induced NETs in vitro, but not with intact neutrophils or NETs plus DNase 1 abrogation (left panel). Representative fluorescence image of HCC cells trapped within NETs in vitro was shown (right panel). Scare bar: 20 μm. c NET detection in HCC embolus and representative fluorescence image. Scare bar: 50 μm. d Little cytotoxicity on HepG2/MHCC97H cells with NET treatment or DNase 1 abrogation revealed by TUNEL assay. e Enhanced invasiveness of HepG2/MHCC97H cells with NETs under different conditions in a Transwell system as indicated, which was abrogated by DNase 1. Quantification of invading cells through Matrigel-coated PET membrane was shown. f Little alteration of in vitro proliferation capacity of HepG2/MHCC97H cells with NET treatment or DNase 1 abrogation in CCK8 assay. g Hepa1-6 subcutaneous tumor growth increased in the presence of NET-producing neutrophils from LPS-treated C57BL/6 mice in vivo (n = 5 each group). h Increased angiogenesis in NET-Hepa1-6 subcutaneous tumors compared to hepa1-6 alone. Representative images of CD31 staining were shown. Scare bar: 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance. Data were presented as means ± SEM