Fig. 7

Effects of PDGF-B knockdown on metastasis, chemosensitization and vascular maturity and functionality. (a) Double staining for CD31 (green) and α-SMA (red) of 4T1 tumor sections. shRNA-PDGF-B- and shRNA-Control-transfected 4 T1 tumor-bearing mice received control, metformin (225 mg/kg), imatinib (60 mg/kg) for the combined treatment. Fluorescent signaling of series thin-layer scanning were reconstructed for 3D observation of VSMCs on vessels. VSMCs No. is at the bottom of the panel and indicated as mean ± SEM (n = 8). (b) Quantification of ratio of α-SMA⁺ area/CD31⁺ area (upper), CD31⁺ area per μm² (middle) and percentage of α-SMA⁺ VSMCs associated with vessels (bottom) in 4 T1 cancers (n = 8). (c) Representative images showing decreased vascular leakage in shRNA-PDGF-B- than shRNA-Con-transfected 4T1 tumors. Fitc-dextran (green) was injected through tail vein 10 mins before sacrifice. Tumor sections was counterstained with anti-CD31 antibody (Red). White arrows indicate the dextran leaking outside the vessel wall. Bars: 100 μm. (d) Representative images showing more lectin-perfused CD31⁺ vessels in shRNA-PDGF-B- than shRNA-Con-transfected 4T1 tumors (n = 8). Red: perfused lectin; Green: CD31⁺ vessels. White arrows indicate CD31⁺ vessels with lectin perfusion. Percentage of Lectin⁺/CD31⁺ vessels (of CD31⁺ vessels) is indicated as mean ± SEM (at the bottom of the panel). (e) H&E staining of sections of 4T1 cancers (Left) and quantification of necrotic and hemorrhagic areas (Right; n = 8). shRNA-PDGF-B or shRNA-Con-transfected 4T1 tumor-bearing mice received low dose CTX (20 mg/kg•day). “N” indicates “necrosis”; black arrows indicate tumor hemorrhage. (f) Decreased primary tumor lung metastasis in mice bearing shRNA-PDGF-B 4T1 cancer cells than that in mice bearing shRNA-Con 4T1 cells. (Upper) H&E staining for 4T1 tumor sections; (Lower) quantification of 4T1 metastatic index (metastatic nodules per gram of primary tumor; n = 8). Red asterisk indicates lung metastasis nodule. Magnification: 200Х. (g) Schematic diagram of metformin-induced vascular remodeling in metastatic breast cancers. Metastatic breast cancers are angiogenic with hypoperfusion and vascular immaturity, which contribute to vascular leakage, chemoresistance, hypoxia and distant metastasis. By decreasing PDGF-B of those metastatic breast cancers, metformin inhibits angiogenesis and improved the vascular maturity and functionality, therefore improving the chemosensitivity and reducing the distant metastasis