Fig. 5

Effects of metformin administration on hypoxia and the vascular functionality. (a-d) Representative images showing more lectin-perfused CD31⁺ vessels in metformin-treated than untreated (A) 4T1 and (c) MDA-MB-231 cancers (n = 8). TRITC-conjugated lectin (Red) was intravenously injected into tail veins 15 min before the sacrifice. Tumor sections were further immunostained with anti-CD31 antibody (Green). White arrows indicate CD31⁺ vessels with lectin perfusion. Higher (B) percentage (of CD31⁺ vessels) and (d) density (per mm²) of Lectin⁺ CD31⁺ vessels in metformin-treated than untreated tumors. Bars: 100 μm. (e) Immunohistochemical staining for pimonidazole (PIMO; brown) and (f) quantification of PIMO⁺ hypoxic area in 4 T1 cancers from untreated or metformin-treated mice, revealing ameliorated tumor hypoxia by metformin (n = 8). Bars: 500 μm. (g) Representative images showing decreased vascular leakage in metformin-treated than untreated 4 T1 tumors. FITC-dextran (green) was injected through tail vein in advance. Tumor sections was counterstained with anti-CD31 antibody (Red). White triangles indicate the dextran leaking outside the vessel wall. Bars: 100 μm. Quantitative data are indicated as mean ± SEM. *p < 0.05