Fig. 6

Downregulation of PDGF-B by the metformin treatment. (a) Immunoblotting for cancer cell-derived angiogenic factors using mouse angiogenesis arrays, and (b) quantification of grey intensity of each factor (independent of 3 experiments). Cancer cells were treated with metformin (100 μM) for 48 h. (c) Column chart for showing the transcriptional levels of angiogenesis-related factors. FPKM indicates Fragments Per Kilobase of transcript per Million fragments mapped. Met indicates metformin; Met1, Met2 and Met3 indicate different reports of data of independent tumors in metformin group. (d) Heatmap analysis of the effect of metformin on transcriptional levels of top 9 angiogenesis-related genes of 4T1 tumors (n = 3). The fold change ranges from − 0.5 to 0.5. “Sig.” indicates “Significance”. (e) Double immunostaining for CD31 and PDGF-B in 4T1 orthotopically implanted tumors (Left). shRNA-PDGF-B- and shRNA-Control-transfected 4T1 tumor-bearing mice received control or metformin treatment (225 mg/kg•day). Quantification of PDGF-B fluorescent intensity and microvessel density (Right; n = 8). (f) Immunostaining for CD31 and α-SMA in 4T1 cancers, revealing increased percentage of patent microarteries (n = 8). White arrows indicate the compressed lumen of arteries distributed in 4T1 cancer stroma. (g-h) Survival curve for analyzing the association of PDGF-B mRNA expression in primary breast tumor with survival probability of patients with breast cancer (g). PDGF-B mRNA levels was positively correlated with mRNA level of CD31, but not PCNA and Ki-67. Data was obtained from TCGA dataset. 1064 samples were included. FPKM: Fragments Per Kilobase of transcript per Million fragments mapped. Quantitative data are indicated as mean ± SEM. “ns” indicates not statistically significant; *p < 0.05; **p < 0.01; ***p < 0.001