Fig. 3

The in vivo effects of metformin pretreatment on cancer responses to cyclophosphamide (CTX). (a and b) Growth curve and survival curve of 4 T1 cancers from mice untreated or treated with metformin (225 mg/kg•day), low dose CTX (Cyclophosphamide, 20 mg/kg•day) or the combined treatment (n = 8). Mice were pretreated with metformin for 5 days before receiving CTX. (C-E) (c) H&E staining of sections of 4 T1 cancers, and quantification of (d) necrotic and (e) hemorrhagic areas, revealing enhanced cancer responses to CTX by metformin pretreatment (n = 8). “N” indicates “Necrosis”; black arrows indicate hemorrhage; “C & M” indicates the combination of CTX and metformin. Bars: 200 μm. (f) Double staining for CD31 and PARP of 4 T1 tumor sections, and quantification of (g) PARP⁺ CD31⁺ cancer cells and (h) PARP⁺ CD31⁺ vessels (n = 8). Tumor-bearing mice were treated with metformin (pretreatment for 5 days), CTX or combined treatment. White arrows indicate PARP⁺ endothelial cells (ECs); yellow triangles indicate PARP⁺ cancer cells. (i) Representative images showed increased cisplatin-DNA adducts in 4 T1 tumors from metformin pre-treated mice (225 mg/kg•day, lasting for 5 days). After that, mice were treated with intraperitoneal injections of cisplatin (CPT, 5 mg/kg, every 2 days). The right column refers to the reverse-color image of the left column. Red arrows indicate the cells with positive cisplatin-DNA adducts. Magnification:200Х. (j) Quantification of cells with nuclear cisplatin adduct in 4 T1 cancers (n = 8). (k) Quantification of PI⁺ necrotic ECs and 4 T1 cells in vitro upon combined 4-OH (activated CTX) treatment (n = 5). Quantitative data are indicated as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001