Fig 1: Il-1β induces Ly6ghigh neutrophil NETosis in the lung metastatic niche. (A) Heatmap of the scRNA-seq data showing the expression of cytokine genes at different time points during lung metastasis. (B and C) Representative immunofluorescence micrographs (B) showing NET formation by FACS-sorted Ly6ghigh and Ly6glow neutrophils (n = 6) after treatment with Il-1β, Cxcl2, and Ccl6 for 6 h in vitro. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The statistical data are presented in (C). (D) Representative immunofluorescence micrographs showing NET formation at the MACRO stages of lung tissue with PBS, rIl-1β, anti-IgG, and anti-Il-1β antibody treatment, respectively [4T1-LM3 (BALB/c) model, n = 5]. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies NET formation. (E) Representative bioluminescence imaging and hematoxylin and eosin (H&E) staining images at the MACRO lungs from mice treated with PBS, rIl-1β, IgG, or anti-Il-1β antibody [4T1-LM3 (BALB/c) model, n = 5]. The bar graph on the right shows the quantitative data of lung metastasis burden. (F) Violin plots showing the expression of Il1b in different cell clusters in the lung tissues based on scRNA-seq data from Fig. 1D. (G) Representative immunofluorescence micrographs demonstrate NET formation in sorted Ly6ghigh neutrophils (n = 6). Neutrophils were treated with CM-MΦ or CM-MΦ that had been neutralized with an anti-Il-1β antibody. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). (H and I) Mice were treated with anti-IgG control, anti-F4/80 antibody, or anti-F4/80 antibody combined with rIl-1β until the macrometastatic stage [4T1-LM3 (BALB/c) model, n = 6]. (H) Il-1β levels in the lungs were detected by ELISA. (I) Representative immunofluorescence images show NET formation. NETs were stained for Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies NET formation (I). (J) Macrophages were treated with CM-Neu, NETs (5 μg/ml), NETs (10 μg/ml), or NETs (10 μg/ml) combined with deoxyribonuclease (DNase) I (n = 3). The expression of Il1b was determined by qPCR. The data with error bars are presented as the mean ± SD; statistical significance was determined by 2-way ANOVA (C) and 1-way ANOVA test (D, E, and G to J). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; Ccl11, c-c motif chemokine ligand 11; Ccl12, c-c motif chemokine ligand 12; Ccl17, c-c motif chemokine ligand 17; Ccl2, c-c motif chemokine ligand 2; Ccl22, c-c motif chemokine ligand 22; Ccl3, c-c motif chemokine ligand 3; Ccl4, c-c motif chemokine ligand 4; Ccl5, c-c motif chemokine ligand 5; Ccl6, c-c motif chemokine ligand 6; CCL6; c-c motif ligand 6; Ccl9, c-c motif chemokine ligand 9; CM-MΦ, macrophage-derived conditioned medium; CM-Neu, neutrophil-derived conditioned medium; Cxcl12, c-x-c motif chemokine ligand 12; Cxcl14, c-x-c motif chemokine ligand 14; Cxcl16, c-x-c motif chemokine ligand 16; CXCL2, c-x-c motif chemokine ligand 2; Cxcl2, c-x-c motif chemokine ligand 2; Cxcl3, c-x-c motif chemokine ligand 3; Cxcl9, c-x-c motif chemokine ligand 9; DAPI, 4’,6-diamidino-2-phenylindole; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting; H3cit; citrullinated histone H3; Il12a, interleukin 12a; Il13, interleukin 13; Il18, interleukin, 18; Il1a, interleukin 1α; Il1b, interleukin 1β; Il-1β, interleukin-1β; Il2,interleukin 2; Il33, interleukin 33; Il4, interleukin 4; Il6, interleukin 6; Ly6g, lymphocyte antigen 6 complex locus g; MACRO, macrometastatic lung; MICRO, micrometastatic lung; MPO, myeloperoxidase; NETs, neutrophil extracellular trap; NK, natural killer; NL, normal lung; Ppbp, pro-platelet basic protein; Neu, neutrophil; PRE, premetastatic lung; qRT-PCR, quantitative real-time polymerase chain reaction; rIl-1β, recombinant interleukin-1β; scRNA-seq: single-cell RNA sequencing; SD, standard deviation.