Screening for human tumor cell supernatants that block the antiangiogenic activity of bortezomib. (A) HUVECs were incubated with either control solution (−) or 10 ng/mL bortezomib (+) in the presence of the respective tumor cell culture supernatant. Cell viability was determined after 24 hours in a WST-1 assay monitoring the mitochondrial succinate reductase activity at 450 nm. Cells in standard culture medium served as control. Among the analyzed cell lines were: myeloma (U266 and OPM-2), breast carcinoma (MCF-7), glioblastoma (U373), colorectal cancer (HRT-18), and prostate carcinoma (PC-3). Bars represent mean ± SEM. *P < .05. (B) Growth and vascularization of human tumor xenografts in the CAM assay. Bortezomib-sensitive glioblastoma (U373) and bortezomib-resistant colorectal cancer (HRT-18) cells with stable overexpression of GFP were grown on the CAM for 2 days. Then xenografts were treated daily with control solution (−) or with 5 ng/mL bortezomib (+). After 3 days, tumors were analyzed under the fluorescence stereomicroscope to visualize GFP-positive tumor cells on the CAM. Bortezomib displayed marked antitumor effects against U373 but not against HRT-18 xenografts on the CAM. Original magnification ×10. Images were acquired using an Olympus SZX10 stereomicroscope (2×/0.2 NA objective) with an Olympus E410 digital camera.