Combination of low doses of NPI-0052 plus bortezomib inhibits neovascularization and proteasomal activities in vivo in xenografted MM tumors. (A) Top panel shows blood vessels in tumor sections from untreated or NPI-0052 (0.025 mg/kg) plus bortezomib (0.25 mg/kg)–treated mice, as identified by staining with a marker for angiogenesis factor VIII. Photograph is representative of similar observations in 3 different mice receiving same treatment. Bottom panel shows the quantification of blood vessels in tumor sections from mice treated with indicated doses of NPI-0052, bortezomib, or combined NPI-0052 plus or minus bortezomib. Blood vessels were enumerated in nonnecrotic areas of each section using light microscopy (magnification, 40×/0.75 NA oil). Error bars represent standard deviation. Arrows: Arrow in left hand panel indicates blood vessel. Arrow in right hand panel shows immunostained tumor section of mice receiving indicated doses of agents. (B) VEGFR1 expression in tumor sections from untreated or NPI-0052 (0.025 mg/kg) plus bortezomib (0.25 mg/kg)–treated mice as identified by IHC staining with VEGFR1 Abs. Photograph is representative of similar observations in 2 different mice receiving same treatment. (C) NPI-0052 plus bortezomib triggers synergistic inhibition of CT-L, C-L, and T-L proteasomal activities in vivo in xenografted MM tumors. For the analysis of proteasome activities in tumors, mice were treated with NPI-0052 (intravenously), bortezomib (intravenously), or NPI-0052 plus bortezomib (as in panel A) and killed after 90 minutes of drug administration; tumors were then examined for CT-L, C-L, and T-L proteasome activity. The data represent percentage of inhibition compared with vehicle control–treated animals from 2 independent experiments with similar results. A CI less than 1 indicates synergy.