Fig. 2: Pre-metastatic niche formation is characterized by infiltration of an Egr1⁺ neutrophil subset.

A Schematic of the pre-metastatic niche model. BALB/c mice were implanted orthotopically with CT26 (Vector or KIAA1199-OE) cells. On day 14 (pre-metastatic stage), livers were imaged by MRI and harvested for microenvironmental analyses. Scale bars: 2 mm (gross) and 2 mm (MRI). B scRNA-seq analysis showing tSNE projections (left) and cell type proportions (right) of hepatic immune and non-immune cells. Red dashed lines: neutrophils; Blue dashed lines: hepatocytes (n = 4 mice). C Subclustering of hepatic neutrophils identifies five distinct subpopulations (Neu-C0 to Neu-C4). Stacked bar plot shows their distribution. (n = 4 mice). D Pseudotime trajectory analysis (Monocle) of neutrophil subclusters (n = 4 mice). E Gene set variation analysis (GSVA) of biological pathways across neutrophil pseudotime trajectories (n = 4 mice). F Western blot analysis of neutrophils isolated from livers on day 14 following orthotopic CT26 implantation (n = 3 independent experiments). G Flow cytometry quantification of Egr1⁺ neutrophils in livers of BALB/c and C57BL/6 mice bearing Vector or KIAA1199-OE tumors (n = 3 mice, Student’s t test). H. Multiplex immunohistochemistry (mIHC) of liver sections showing representative Egr1⁺ neutrophils (white arrowheads) (n = 3 mice). Scale bars: 100 µm (left), 20 µm (right). I t-SNE projections from external scRNA-seq datasets showing Egr1⁺ neutrophil distribution in patients (Low vs. High KIAA1199) and tissue types (peritumoral liver tissue (Liver_P) vs. liver metastases (Liver_T)). Red dashed lines highlight the Egr1⁺ subset (n = 24 patients). J Spatial transcriptomic analysis of CRLM patients. Left: Egr1⁺ neutrophil distribution. Middle: Angiogenesis gene signature enrichment. Right: Spatial distance (k-nearest neighbor) from Egr1⁺ neutrophils to defined tissue zones. Red dashed lines: tumor boundary. K Representative H&E and mIHC images of mouse (top, n = 2 mice) and human (bottom, n = 2 patients) CRLM tissues. Markers: DAPI (blue), EGR1 (red), LY6G (green). Yellow arrowheads: co-localized cells. Scale bars: 100 µm. L Tube formation assay. HUVECs were co-cultured with conditioned media from dHL-60 cells transfected with EGR1 or Vector (n = 3 independent experiments; Scale bars: 20 µm.). M Schematic summary: Egr1⁺ neutrophils localize to the tumor microenvironment to promote neovascularization. FACS sequential gating strategies are shown in Supplementary Fig. 5G. All statistical tests are two-sided. Data are presented as mean ± s.d. Source data and exact p values are provided as a Source Data file.