Androgen-induced gene 1 correlates with extracellular vesicle-associated cholesterol metabolism and hepatic metastasis of pancreatic cancer

Figure 5 Spatial validation of immune remodeling and tumor proliferation in metastatic livers. A: Co-staining of F4/80 and T cell immunoglobulin and mucin domain-containing protein 4 in tumor-bearing liver sections. Tumor regions were outlined with red dashed lines according to increased nuclear density (4’,6-diamidino-2-phenylindole); B: Quantification of Kupffer cell density in adjacent liver tissue and monocyte-derived macrophage density within tumor regions (cells/mm²). Data represent n = 3 mice per group; C: Co-staining of F4/80 and programmed death-ligand 1in tumor regions; D: Quantification of programmed death-ligand 1 mean fluorescence intensity in F4/80⁺ macrophages within tumor areas. Each dot represents an individual F4/80⁺ cell. The total number of analyzed cells per group is indicated on the X-axis. Data represent n = 3 mice per group; E: Ki67 immunohistochemical staining of metastatic liver sections. Data represent n = 5 mice per group. For panels B, D, and E, quantitative analyses were performed by randomly selecting three non-overlapping fields per section. For panels B and E, mean value was calculated for each mouse. Statistical significance was determined using a two-tailed unpaired t-test or Mann-Whitney U test as appropriate. ^aP < 0.05; ^bP < 0.01; ^cP < 0.0001. TIM4: T cell immunoglobulin and mucin domain-containing protein 4; DAPI: 4’,6-diamidino-2-phenylindole; AIG1-KD: Androgen-induced gene 1 knockdown; sgControl: Non-targeting control single-guide RNA; PD-L1: Programmed death-ligand 1; IHC: Immunohistochemistry.