Only IFP measurements with stable readings for 3-5 minutes were accepted, and the measurements lasted for 10-20 minutes. Data acquisition was carried out by using LabVIEW software (National Instruments, Austin, TX). Hypoxia, necrosis, and microvessels CD31 was used as a marker for endothelial cells and pimonidazole [1-[(2-hydroxy-3-piperidinyl)-propyl]-2-nitroimidazole] was used as a hypoxia marker. Pimonidazole was dissolved in 0.9% sodium chloride and administered intraperitoneally at a dose of 30 mg/kg. The tumors were resected and fixed in phosphate-buffered 4% paraformaldehyde approximately 4 hours

after the pimonidazole administration. Immunohistochemistry was done by using a peroxidase-based indirect staining method [27]. An anti-pimonidazole rabbit polyclonal antibody

(gift from Prof. J.A. Raleigh, Department of Radiation Oncology, University R788 cell line of North Carolina School of Medicine, Chapel Hill, NC) or an anti-CD31 rabbit polyclonal antibody (Abcam, Cambridge, United Kingdom) was used as primary antibody. Diaminobenzidine was used as chromogen, and hematoxylin was used for counterstaining. Hypoxic fraction was defined as the area fraction showing positive pimonidazole staining (hypoxic fraction = pimonidazole positive area/viable tissue area·100%) and necrotic fraction was defined as the area fraction showing necrotic tissue (necrotic fraction = necrotic tissue area/total area·100%). The area fraction showing GSK-3 phosphorylation positive pimonidazole staining and the area fraction showing necrotic tissue were determined Ureohydrolase by image analysis. Microvascular density was defined as the number

of microvessel profiles per mm2 of viable tumor tissue (microvascular density = number of microvessel profiles/viable tissue area). The number of microvessel profiles was scored manually in immunohisochemical preparations stained with anti-CD31 antibody. Statistical analysis Statistical comparisons of data were carried out by the Student’s t test when the data complied with the conditions of normality and equal variance. Under other conditions, comparisons were done by nonparametric analysis using the Mann-Whitney rank sum test. Probability values of P < 0.05, determined from two-sided tests, were considered significant. The statistical analysis was performed by using the SigmaStat statistical software (SPSS Science, Chicago, IL, USA). Results A-07 tumors were divided into groups with matched tumor sizes to receive sunitinib treatment or no treatment (vehicle). Tumors in both groups grew during the 4-day treatment period (Figure 1). After the treatment, sunitinib-treated tumors did not differ from untreated tumors in size (Figure 1; P > 0.05), indicating that this short-term treatment did not affect tumor growth. Figure 1 Sunitinib treatment did not affect tumor growth. Tumor size before and after 4 days of treatment in mice given vehicle (white colomns) or sunitinib (black columns). Columns, means of 14-15 A-07 tumors, bars SEM.