Figure S2.
Neutrophil identification and characteristics. (A) Uniform Manifold Approximation and Projections (UMAPs) (left) and expression of neutrophil lineage markers (right) by the major immune populations identified from the scRNA-seq of the BM and spleen (top), and HCC tumors (bottom) of NRAS/AKT HCC mice. (B) Dot plot of the differently expressed genes by each neutrophil cluster in the BM and spleen (top), and HCC tumor (bottom). (C) RNA-seq analyses were performed on SiglecFhi and SiglecF– TANs purified from NRAS/AKT-HCC tumors. A gene set defining SiglecFhi TANs was generated from their top 100 differentially expressed genes. The distribution of this gene set among TAN subsets. (D) Representative FACs plots and percentage of SiglecFhi neutrophils detected in the BM, spleen, blood, and tumor of NRAS/AKT HCC mice. (E) CXCR2, CXCR4, CD62L, and dcTRAIL-R1 expression within SiglecF− and SiglecFhi TANs of NRAS/AKT HCC mice. (F) Functional characterization of BM neutrophils, SiglecF− and SiglecFhi TANs from NRAS/AKT HCC mice. (G) Representative FACs plots and percentages of SiglecFhi TANs of RIL175 orthotopic HCC mice fed with MCD diet (MCD + RIL175; left) or Chow diet (CD + RIL175; right) (n = 3/group). (H) Representative immunofluorescence images and proportion of SiglecFhi TANs (myeloperoxidase [MPO]: red; SiglecF: green; nucleus: blue) in NRAS/AKT HCC tumors after 2 wk of treatment with αLy6G or isotype control mAbs. Scale bar represents 100 µm. (I) Expression levels of genes in the Siglecf-Hi–like TAN signature across various cell types in the tumor of HCC patients (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA007744). Each symbol represents one mouse. Data are mean ± SEM and represent two independent experiments (D–H). **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (F) and unpaired Student’s t test (H).

Neutrophil identification and characteristics. (A) Uniform Manifold Approximation and Projections (UMAPs) (left) and expression of neutrophil lineage markers (right) by the major immune populations identified from the scRNA-seq of the BM and spleen (top), and HCC tumors (bottom) of NRAS/AKT HCC mice. (B) Dot plot of the differently expressed genes by each neutrophil cluster in the BM and spleen (top), and HCC tumor (bottom). (C) RNA-seq analyses were performed on SiglecFhi and SiglecF– TANs purified from NRAS/AKT-HCC tumors. A gene set defining SiglecFhi TANs was generated from their top 100 differentially expressed genes. The distribution of this gene set among TAN subsets. (D) Representative FACs plots and percentage of SiglecFhi neutrophils detected in the BM, spleen, blood, and tumor of NRAS/AKT HCC mice. (E) CXCR2, CXCR4, CD62L, and dcTRAIL-R1 expression within SiglecF− and SiglecFhi TANs of NRAS/AKT HCC mice. (F) Functional characterization of BM neutrophils, SiglecF− and SiglecFhi TANs from NRAS/AKT HCC mice. (G) Representative FACs plots and percentages of SiglecFhi TANs of RIL175 orthotopic HCC mice fed with MCD diet (MCD + RIL175; left) or Chow diet (CD + RIL175; right) (n = 3/group). (H) Representative immunofluorescence images and proportion of SiglecFhi TANs (myeloperoxidase [MPO]: red; SiglecF: green; nucleus: blue) in NRAS/AKT HCC tumors after 2 wk of treatment with αLy6G or isotype control mAbs. Scale bar represents 100 µm. (I) Expression levels of genes in the Siglecf-Hi–like TAN signature across various cell types in the tumor of HCC patients (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA007744). Each symbol represents one mouse. Data are mean ± SEM and represent two independent experiments (D–H). **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (F) and unpaired Student’s t test (H).

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