Fig. 3: Cd177^hiPad4^hi neutrophils produce NETs and drive lung metastasis in iRFA.

A Flow cytometric analysis and quantification of Cd177^hi neutrophils in liver tumor tissues dissected from iRFA and Ctrl mice (gated on Cd11b⁺Ly6G⁺ cells, n = 4 mice). Unpaired two-sided t-text. B Immunofluorescence detection of Cd177, Pad4, and S100a8 in liver tumors. Scale bars, 50 μm and 10 μm. Quantification of Pad4⁺Cd177⁺S100a8⁺ cells shown on the right (n = 15 mice). Unpaired two-sided t-test. C Western blotting analysis in Cd177^-, Cd177⁺Mki67^-, and Cd177⁺Mki67⁺ neutrophils. Quantification shown on the right (n = 3 biological replicates). ANOVA. D ELISA-detected MPO-dsDNA levels in neutrophils (n = 3 biological replicates). P < 0.001, ANOVA. E Transwell assay assessing CT26 cell invasion in co-culture with PBS- or G484-treated neutrophils. Scale bars, 200 μm. F dsDNA levels in peripheral blood from Ctrl and iRFA mice 3 days post-treatment (n = 5 mice). Unpaired two-sided t-test. G (Left) Immunofluorescence detection of Ly6G and cit-H3 in liver tumors. Scale bars, 20 μm. (Right) Quantification of the cit-H3⁺ area (n = 15 mice), two-sided Mann-Whitney U test. H Study design of iRFA-CT26 mice treated with G484 or PBS (Upper). Liver tumor tissues were dissected 14 days post-treatment (Lower). Circles: drug administration; triangle: iRFA or Ctrl treatments; asterisks: sample collection (peripheral blood was collected on days 3 and 5, while lung and liver tissues were collected on day 14). Circles and arrows: liver tumors. I, J MPO-dsDNA levels in peripheral blood on days (I) 3 and (J) 5 following G484 or PBS treatment (Day 3, G484+iRFA: n = 8 mice; other groups: n = 9 mice). P < 0.001, ANOVA. K, Lung tissues were dissected (Circles: lung tumors). HE staining of lung tissues. Scale bars, 1 mm. L Lung metastasis area proportion and (M) lung weight (n = 9 mice). P < 0.001, ANOVA. Data are presented as mean values ± SD. E, H, M G484: GSK484. Source data are provided as a Source Data file.