Figure 3. Increase of vascular maturity contributed to metformin-induced reduction of necrosis and MVD.

4T1 tumor-bearing BALB/c mice were orally treated with metformin (1.5 mg/mL) for 21 days before extraction. The wholly extracted 4T1 tumors were fixed by 4% paraformaldehyde and cut into 40μm thick frozen section for further 3D-reconstruction of immunofluorescent signal. 3D-reconstruction of fluorescent signal of CD31 (Green) and α-SMA (indicating vascular smooth muscle cell, VSMCs) in (A) untreated 4T1 tumor (Con), (B) normal breatst tissue and (C) metformin-treated 4T1 tumor (Met). α-SMA signal co-located with CD31 signal indicate VSMCs that covered the tumor vascular lumen. (D) Quantification of ratio of co-located fluorescent α-SMA to CD31 in NB (normal breast), PNR (peri-necrotic region), TR (transitional region) and NNR (non-necrotic region). Scale bar: 100 μm. (E) Representative image (Immunological Histological Chemistry) for showing CD31⁺ vessels distributed in peri-necrotic region, transitional region and non-necrotic region of metformin-treated 4T1 tumor. Scale bar: 500 μm. (F) The color-reverse image and (G) regional distribution map of panel (E) for revealing angiogenic ability of vessels distributed in different regions of metformin-treated tumor. Scale bar: 500μm. The peri-necrotic and non-necrotic regions were surrounded by red and yellow dotted lines, respectively. (H) Quantification of percentage of necrotic area in 4T1 tumors from untreated or metformin-treated BALB/c mice (n = 10). Quantification of (I) microvessel density (MVD, No. of vessels per mm²) and (J) vascular branch density (No. of vascular branches per vessel) of different regions in 4T1 tumors from untreated or metformin-treated BALB/c mice (n = 10). Quantitative data are indicated as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates no significant (P > 0.05).