Figure 2.

S100A4⁺ cells accumulate during the development of HCC, and they are a subpopulation of macrophages. (a) Schematic representation of the DEN/CCl₄-induced liver fibrosis-related HCC experiment. Groups of mice (3/group) were left untreated (control) or were treated i.p. with a single injection of 50 μg/g of DEN at 15 days old and were treated with CCl₄ twice weekly for 8 weeks 1 month later. Liver tissues were harvested at the indicated timepoints after the final injection. (b) Histological characterization of liver fibrosis and S100A4⁺ cell accumulation. Adjacent sections were stained with H&E, anti-S100A4 and Sirius Red. Representative images are shown for untreated control and CCl₄-treated mice at each time point. Scale bar, 50 μm. (c) Quantification of areas stained with Sirius Red. Statistical analysis was performed between the control and CCl₄-treated groups (n = 3). Results represent three independent experiments; mean ± SEM, n = 5 mice per group. * P < .05. (d) S100A4^+/+ GFP transgenic mice were administered DEN/CCl₄ as described above. Total cell numbers of GFP⁺ cells in the liver (calculated by multiplying the absolute liver nonparenchymal cell number by the percentage of GFP⁺ cells) of untreated (control) and CCl₄-treated mice at each time point are shown. * P < .05. (e) Double staining for S100A4 (green) and F4/80, CD11b and CD68 (red) in liver, mouse HCC and human HCC tissues. Typical double staining positive cells were indicated by arrows. Scale bar, 50 μm. (f) Percentages of double staining positive S100A4⁺ cells in HCC tissues. (g-h) Flow cytometry analysis of the phenotypes of S100A4⁺ cells in the liver of S100A4⁺/^+GFP mice treated with CCl₄ by staining GFP⁺ cells with CD11b and F4/80 antibodies. (g) Representative images of three independent experiments showed S100A4⁺ cells in the liver of CCl₄ treatment. (h) Statistic analysis of CD11b⁺S100A4⁺ or F4/80⁺S100A4⁺ cells in S100A4⁺ (GFP⁺ cells) cells.