Fig. 3

Immune cell distributions in mice after various treatments. a Measurment of tumor-infiltrating immune cells (B cell: CD19⁺ stained; CD4⁺ T cell: CD4⁺ stained; CD8⁺ T cell: CD8⁺ stained; M1 type macrophage: F4/80⁺MHC II⁺CD206⁻ stained; M2 type macrophage: F4/80⁺MHC II⁻CD206⁺ stained) in orthotopic HCC (*, p < 0.05; **, p < 0.01, ^# p > 0.05, two-tailed unpaired Student’s t test). Median with 95% confidence interval. b Tumor-infiltrating M1-type macrophages, M2-type macrophages, CD8⁺ T cells, CD4⁺ T cells, and B cells in formalin-fixed, paraffin-embedded tumor tissues were quantified using multiplex immunofluorescence staining after various treatments. Representative spectrally unmixed composite images (20 × magnification) are shown (yellow, M1-type macrophages; orange, M2-type macrophages; green, CD4⁺ T cells; red, CD8⁺ T cells; blue, B cells; grey, cell nucleus). Bar = 50 μm. c M1/M2-type macrophage ratio (**, p < 0.01; ^# p > 0.05, two-tailed unpaired Student’s t test) and imaging of the whole specimen area; each dot represents one acquisition field (80 fields / 5 mice tumor sections)