Effect of members of the miR-17-92 cluster on paracrine activity of tumor cells in vitro and tumor angiogenesis in vivo. (A-B) HUVECs or LLC1 tumor cells were transfected with the respective precursors. Medium was changed to Dulbecco modified Eagle medium with 0.05% bovine serum albumin after 1 day. Conditioned medium was collected at day 2, and 10 times concentrates were transferred to collagen-embedded spheroids with nontransfected HUVECs as illustrated in panel A. Quantification of spheroid sprout length was performed after incubation of the spheroids with the conditioned medium for 24 hours; n = 4. (C-D) LLC1 tumor cells were subcutaneously injected in mice. Antagomir-17/20 (8 mg/kg body weight) was intravenously injected once as indicated. Tumor size was measured daily (C), and tumor volume and weight were detected in explanted tumors (D) at day 13; n = 7 for Antagomir-Co and n = 6 for Antagomir-17. (E) Tumor angiogenesis was detected in sections stained with the endothelial marker endomucin. A secondary antibody conjugated to Alexa Fluor 555 was used. The number of vessels was counted manually; n = 7 for Antagomir-Co and n = 6 for Antagomir-17. (F) Perfused vessels were detected by intravenous infusion of fluorescein isothiocyanate-conjugated lectin and were quantified by automatic measurement of the pixel region in each section; n = 7 for Antagomir-Co and n = 6 for Antagomir-17. Scale bars represent 20 μm.