Fig. 2
Effects of metformin on the growth and metastasis of the metastatic breast cancers. (a and b) Growth curve (cm3) and measurement of weight (g) of 4 T1 tumors from BALB/c mice untreated or treated with metformin at different concentrations (0.1, 1, 25 and 225 mg/kg•day; n = 8). (c and d) H&E staining for 4 T1 primary tumor lung metastasis, and quantification of 4 T1 metastatic index (metastatic nodules per gram of the primary tumor; n = 8). Blue arrows indicate metastatic nodules in lung. (e) Representative images of in vitro colonization of 4 T1 cancer cells, which were untreated or persistently treated with 50 μM or 100 μM metformin (repeated for 5 times). Cancer cells were inoculated into each well of a 6-well culture plate. (f) Bar graphs showing reduced lung weight, and comparable weights of liver and spleen of mice untreated or treated with metformin at concentration of 225 mg/kg•day or 25 mg/kg•day (n = 6). Organ weight was normalized for body weight. (g) In vitro proliferation curve of 4 T1 cancer cells untreated or treated with metformin at concentrations of 50 or 100 μM (repeated for 5 times). (h) Representative images showing 4 T1 cancer cell migration in vitro, and quantification of migrated cancer cells per 400 X field (n = 5). (I) H&E staining of paraffin-processed tumor tissues from metformin-treated or untreated mice, showing the comparably direct invasion of cancer cells to the peritumoral muscle. Black arrows indicate muscle fibers penetrating into the tumor. Bars: 200 μm. Quantitative data are indicated as mean ± SEM. **p < 0.01; ***p < 0.001