Fig. 4

Stimulatory effects of cancer cell-derived sEVs on endothelial cells and tumor growth depend on VEGF. a–c HUVEC were stimulated with equivalent amounts of sEVs of VEGF^+/+ cancer cells, sEVs of VEGF^−/− cancer cells or rVEGF₁₆₅, and then assayed for VEGFR2 phosphorylation (a) and tube formation (b, c). In b, mean ± SD of n = 4 independent experiments. In c, representative images of tube formation. Scale bar = 100 μm. d–f Nude mice were inoculated i.p. with ES2 VEGF^−/− cells that stably expressed GFP. At 7 days thereafter when tumors were palpable, mice were randomized into groups (n = 6 mice per group) and then administered equivalent amounts of sEVs of ES2 VEGF^+/+ cells or sEVs of ES2 VEGF^−/− cells, three times a week for 2 weeks. Negative and positive control groups of tumor-bearing mice were administered saline and rVEGF₁₆₅, respectively. In d, representative images of GFP-expressing tumors in the abdominal cavity viewed under a fluorescence stereomicroscope. Arrows indicate tumors on the omentum. Scale bar = 10 mm. In e, immunofluorescence staining of CD31 (red) in sections of omental tumors (Ome T) adjacent to the pancreas (Panc). Scale bar = 100 μm. In f, amount of i.p. tumor burden, numbers of intratumoral CD31⁺ cells, and volume of ascites in each mouse in each of the groups. I.p. tumor burden is expressed as % of area of GFP fluorescence in the abdominal cavity. Numbers of CD31⁺ cells were scored in five random 100× fields per omental tumor section and an average score was determined for each mouse. Error bars in f represent SD. ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni’s corrections in b and f. Source data used for graphs in b and f can be found in Supplementary Data 4