Gain of VEGF-B function in VEGF-A–null tumors inhibits tumor growth by altering angiogenesis and vascular functions in mouse tumors. (A) Expression levels of VEGF-B protein in VEGF-A–null 528ras fibrosarcoma tumor cells. (n = 3 samples per group). (B) Tumor growth curves of vector- and VEGF-B-528ras fibrosarcoma tumors (n = 20 mice per group). (C, Left) Tumor vasculature of vector- and VEGF-B-528ras tumors. Arrowheads point to vascular branches, and arrows indicate perivascular cell coverage. (Scale bar, 50 μm.) (Right) Vessel numbers, coverage of perivascular cells, branch points, and diameters of tumor vessels were quantified (n = 24 random fields per group). (D, Left) Blood perfusion (2,000-kDa dextran) and vascular permeability (70-kDa dextran) of vector- and VEGF-B-528ras tumors. Arrowheads indicate perfused or leaked dextran signals. (Scale bar, 50 μm.) (Right) Quantification of blood perfusion and leakiness (n = 24 random fields per group). (E, Left) FACS measurement of F4/80⁺ and CD206⁺ macrophages of vector- and VEGF-B-528ras tumors. (Right) Quantification is presented as the percentage of positive signals versus the total gated events (n = 12 samples per group). (F, Left) FACS analysis of CTCs in mice bearing vector- and VEGF-B-528ras tumors. (Right) Quantification of CTCs is presented as the percentage of positive signals versus the total gated events (n = 12 samples per group). (G, Left) Pulmonary metastasis in SCID mice bearing vector- and VEGF-B-528ras tumors. Arrowheads indicate metastatic nodules. Dashed lines encircle metastatic nodules. [Scale bar, 5 mm (Top); 100 μm (Middle and Bottom).] (Right) The bar chart shows the percentage of mice with visible lung metastases (n = 12 animals per group). (H, Top and Middle) Dissemination of DiI-labeled mouse vector- (Top) and VEGF-528ras (Middle) tumor cells in zebrafish. Dashed lines encircle primary tumor sites. Arrowheads point to the disseminated tumor cells. [Scale bar, 500 μm (Top); 200 μm (Middle).] (Bottom) The bar charts show the quantification of disseminated tumor cells in the whole body of zebrafish embryos (n = 10 zebrafish embryos per group). All error bars represent SEM. All P values were analyzed according to Student’s t test. BF, bright field; ns, not significant.

VEGF-B promotes cancer metastasis in a zebrafish model. (A) Dissemination of DiI-labeled human nontransfected WT, scrambled-shRNA-, and VEGFB-shRNA- MDA-MB-435 melanoma cells in zebrafish. Dashed lines encircle primary tumor sites. Arrowheads indicate the disseminated tumor cells. [Scale bar, 500 μm (Upper); 200 μm (Lower).] (B) Quantification of disseminated WT, scrambled-shRNA-, and VEGFB-shRNA-MDA-MB-435 melanoma cells in the whole body of zebrafish embryos (n = 10 zebrafish embryos per group). (C) Dissemination of DiI-labeled human vector- and VEGF-B-UACC-62 melanoma cells in zebrafish. Dashed lines encircle primary tumor sites. Arrowheads indicate the disseminated tumor cells. [Scale bar, 500 μm (Upper); 200 μm (Lower).] (D) Quantification of disseminated vector- and VEGF-B-UACC-62 melanoma cells in the whole body of zebrafish embryos (n = 10 zebrafish embryos per group). (E) Dissemination of DiI-labeled vector- and VEGF-B-T241 tumor cells in zebrafish. Dashed lines encircle primary tumor sites. Arrowheads indicate the disseminated tumor cells. [Scale bar, 500 μm (Upper); 200 μm (Lower).] (F) Quantification of disseminated vector- and VEGF-B-T241 tumor cells in the whole body of zebrafish embryos (n = 10 zebrafish embryos per group). All error bars represent SEM. All P values were analyzed according to Student’s t test. ns, not significant.