Fig 2: Shp2 deletion downsizes the KC pool and induces hepatic recruitment of bone marrow-derived macrophages. (A) Experimental scheme to generate a mutant mouse line Shp2ΔK (Shp2F/F:Clec4f-cre+/-). (B) Left: Representative immunoblotting for Shp2 protein in hepatocytes and macrophages isolated from 2-month-old mouse livers of various genotypes. Right: Relative Shp2 protein levels in isolated macrophages of WT and Shp2DK livers. (C) FACS analysis to quantify the percentages of KCs (Clec4f+ CD11b+ F4/80high), non-KC macrophages (Clec4f- CD11b+ F4/80high), and monocytes (CD11b+ Ly6C+) in CD45+ cells in mouse livers at 2 months. Data are presented as means ± SD (n = 6). (D) Representative immunostaining (left) and quantification (right) of total macrophages (Mac, F4/80+), KCs (F4/80+ Clec4f+), and non-KC macrophages (F4/80+ Clec4f-) in liver sections. Magnification, ×200; scale bar: 50 μm. (n = 5 per group) (E) FACS analysis to quantify the ratios of caspase-3/7 cells in KCs. (F) FACS analysis to show the ratios of Ki67+ cells in KCs and non-KC macrophages (n = 6). (G) A heatmap built with RNA-seq data shows the different gene expression profiles in NPCs isolated from WT and Shp2ΔK livers at age 2 months. (H) Volcano plot for up-regulated and down-regulated expression of chemokines and adhesion molecules in NPCs of Shp2ΔK livers, relative to WT controls. (I) Representative FACS analysis and quantification to show the ratios of Clec4flow KCs in total KCs in WT and Shp2ΔK livers. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 3: Loss of Shp2 in KCs promotes metastatic liver tumor progression. (A) Representative macroscopic views and H&E staining of tumor-bearing WT and Shp2ΔK mouse livers 16 days after intrasplenic injection of Mouse Colon Cancer Cells 38. (B) Tumor burdens were evaluated by liver weight to body weight (LW/BW) ratios, maximal diameters, and number of tumor nodules. Data are presented as means ± SD. (C) FACS analysis to determine the ratios of various immune cell subsets in CD45+ cells in mouse livers 10 days after injection of Mouse Colon Cancer Cells 38. Data are presented as means ± SD (n = 8 per group). (D) FACS analysis to quantify the ratios of Ki67+ cells in non-KC macrophages. (E) Representative immunostaining of KCs (F4/80+ Clec4f+) and non-KC macrophages (F4/80+ Clec4f−) in liver sections. Magnification, ×100; scale bar: 100 μm. (F) Immunostaining of F4/80+ or CD8+ cells in tumor areas of liver sections. Magnification, ×100; scale bar: 100 μm. (G) Representative macroscopic views and H&E staining of liver sections 6 weeks after oncogene transfection. Magnification, ×100; scale bar: 200 μm. (H) Tumor burdens were evaluated by LW/BW ratios, maximal diameter, and number of tumor nodules. Data are presented as means ± SD (n = 10). ∗P < .05; ∗∗P < .01. Maxi, maximum.

Fig 4: Representative flow cytometric images to show macrophages, KCs, and monocytes. (A) PCR was performed to determine the genotypes of Shp2flox and Shp2WT alleles as well as the Clec4f-cre transgene. (B) FACS analysis to quantify ratios of KCs in total macrophages in livers. (C) Representative immunostaining of F4/80+ cells in isolated macrophages in the liver. (D) Representative flow cytometric images to show macrophages (CD11b+ F4/80high) in CD45 cells in the liver. (E) FACS analysis to quantify ratios of macrophages in CD45+ cells in livers. (F) Representative flow cytometric images to show KCs (Clec4f+ macrophages) and non-KC macrophages (Clec4f- macrophages) in total macrophages in the liver. (G) Representative flow cytometric image to show monocytes (CD11b+ly6Chigh) in CD45+ cells in the liver. (H) Annexin-V and propidium iodide staining for cell apoptosis induced by 1 μM (μmo/L) of SHP099. (I) Quantitative reverse-transcription PCR to determine expression of Ccl2 and Ccl7 mRNA in NPCs in mouse livers at 2 months (n=5 per group). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; FSC-A, forward scatter-area; Mac, macrophage.