Figure 5. Metformin inhibited angiogenesis in peri-necrotic region by impeding HIF-1α-induced expressions of pro-angiogenic factors.

4T1 tumor-bearing mice were untreated (con) or persistently treated metformin (1.5 mg/mL) for 21days. Before extraction, mice were sequentially injected with pimonidazole and TRITC-conjugated lectin. Staining for CD31 (Green), TRITC-conjugated lectin (Red) and pimonidazole hydrochloride (Violet) in the peri-necrotic region of 4T1 tumors from metformin-treated BALB/c mouse. The necrotic region was surrounded by yellow dotted line. Scale bar: 100 μm. (B) Quantification of percentage of hypoxic area (indicated by hypoxyprobe positive area) in 4T1 tumors from untreated or metformin-treated BALB/c mice (n = 10). (C) Percentage of perfused-lectin⁺ CD31⁺ vessels in all CD31⁺ vessels of the whole tumor area (Ave indicates average perfusion level), peri-necrotic region (PNR), transitional region (TR) and non-necrotic region (NNR, n = 10). Mice were untreated or persistently treated with metformin. (D) Increased relative mRNA expressions (to normoxia) of various angiogenic factors of 4T1 cancer cells cultured in the hypoxic condition (1% O²). mRNA level was detected by real-time quantitative PCR. Quantitative data are indicated as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates no significant (P > 0.05).