Fig. 4.

Role of exosomes in hypoxia-dependent cross-talk between malignant cells and vascular cells. (A) Mouse aortas were incubated in the absence (Ctrl) or presence of exosomes (Exo) (25 µg/mL) derived from normoxic (N) or hypoxic (H) GBM cells for 24 h and then embedded in Matrigel overlaid with medium supplemented with 2% mouse serum ± Exo. Shown are representative photomicrographs of microvessels at day 7. (B) Quantitative analysis of microvessel number (Left) and length (Right) presented as the mean ± SD, n = 6 aortas per group. *P < 0.05. (C) HUVECs were cultured for 24 h in the absence (Ctrl) or presence of Exo (10 µg/mL) and then grown on Matrigel for 20 h. Shown are representative photomicrographs of EC tubes from the different treatment groups. (Scale bar: 500 µm.) (D) Quantitative analysis of tubes/microscopic field presented as the mean ± SD, n = 6 per group. *P < 0.05. (E) HUVECs were cultured in the presence of varying concentrations of Exo derived from N or H GBM cells for a total period of 72 h and assessed for proliferation by [³H]-thymidine incorporation. (F) HUVECs were cultured in the presence of varying concentrations of Exo derived from N or H GBM cells for 48 h and assessed for cell death by 7-AAD staining. (G) Hypoxia potentiates autocrine stimulation by Exo. GBM cells were assessed for transwell migration over a period of 6 h in the absence (Ctrl) or presence of Exo (50 µg/mL) derived from of N or H GBM cells. (E–G) Data are presented as fold of untreated, control cells and are the mean ± SD, n ≥ 6 per group. *P < 0.05. (H) Primary HBVPs were assessed for transwell migration over a period of 6 h in serum-free medium (Ctrl) or in the different media as indicated. EC preconditioning with H GBM cell Exo significantly increased paracrine stimulation of pericyte migration (compare EC CM and EC + Exo CM). (I) Pericytes were analyzed for proliferative activity with the different treatments, as indicated. EC Exo preconditioning significantly increased pericyte proliferation, and the activity was in the soluble, vesicle-free CM fraction (compare EC + Exo CM and EC + Exo CM Sol). (J and K) Similar experiment as in H and I, respectively, with GBM cells; EC Exo preconditioning significantly increased paracrine stimulation of GBM cell migration (J) and proliferation (K). (H–K) Data are presented as fold of untreated, control cells, and are the mean ± SD, n ≥ 6 per group. *P < 0.05.