The Y-79 human retinoblastoma cell line and culture medium were obtained from American Type Culture Collection (Manassas, Virginia). The cells, which were grown in a suspension culture in Roswell Park Memorial Institute ([RPMI] Buffalo, New York) medium containing 20% fetal calf serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, were kept at a concentration of 2.5 × 105 cells/mL. To test VEGF expression in normoxic and hypoxic conditions, the cells were incubated at 37°C in an incubator containing either humidified 5% carbon dioxide and 95% air or 5% carbon dioxide and 95% nitrogen. A human umbilical vein endothelial cell (HUVEC) line was obtained from American Type Culture Collection, and the culture medium was purchased from Cambrex BioScience (Walkersville, Inc, Walkersville, Maryland). The cells were plated onto fibronectin (Sigma-Aldrich, Inc, St. Louis, Missouri)–coated cell-culture vessels (Nunc, Roskilde, Denmark) in endothelial basal medium supplemented with 10 ng/mL of human recombinant epidermal growth factor, 1 μg/mL of hydrocortisone, 50 μg/mL of gentamicin, 12 μg/mL of bovine brain extract, and 10% fetal bovine serum. To establish tumor endothelial cells in an in vitro coculture, HUVECs were plated onto 6-well tissue culture plates (5 × 104 cells/well) and were incubated at 37°C in 5% carbon dioxide. After incubation, the HUVECs adhered strongly to the floor of the wells, and the medium was exchanged for a mixed medium of both Y-79 cells and HUVECs, after which 5 × 105 Y-79 cells were seeded into the medium. The cocultured Y-79 cells and HUVECs were treated with 0.01-mg/mL bevacizumab for 3 days. A solution of the drug mixed with the medium was added directly to the cells to ensure a uniform concentration of the drug throughout the well of the tissue culture plate. Untreated cells in the culture medium that were incubated for 3 days served as controls.