Immunohistochemistry was done by using a peroxidase based indirect staining method. An anti pimonidazole rabbit polyclonal antibody, an anti CD31 rabbit polyclonal antibody, an anti collagen IV rabbit polyclonal antibody, Nilotinib or an anti SMA rabbit polyclonal antibody was used as primary antibody. Diaminobenzidine was used as chromogen, and hematoxylin was used for counterstaining. Hypoxic area fractions were determined by image analysis. Number of microvessel profiles per mm2 of tumor tissue was scored manually and used as parameter for micro vascular density. Statistical analysis Statistical comparisons of data were carried out by the Students t test when the data complied with the condi tions of normality and equal variance. Under other con ditions, comparisons were done by nonparametric analysis using the Mann Whitney rank sum test.
The Kolmogorov Smirnov method was used to test for nor mality, and the Levenes test was used to test for equal variance. Probability values of P 0. 05, determined from two sided tests, were considered significant. The statis tical analysis was performed by using the SigmaStat stat istical software. Results Short term sunitinib treatment did not affect tumor growth Mice were divided in groups with matched tumor size to receive sunitinib treatment or no treatment. Sunitinib treatment was started 6 or 12 days after tumor initiation. At these time points, defined as day 0, A 07 GFP and R 18 GFP tumors were of similar size, and had developed vascular net works. During the short treatment period, kg day sunitinib, or R 18 GFP tumors were treated with 40 mg kg day sunitinib.
Sunitinib treatment reduced vessel density To investigate effects of sunitinib treatment on vascular morphology, mice treated with sunitinib or vehicle were submitted to intravital microscopy. Sunitinib treatment reduced vessel density in both A 07 GFP and R 18 GFP tumors. This is shown qualitatively in Figure 3 which shows representative intravital microscopy images of an untreated A 07 GFP tumor, an A 07 GFP tumor treated with sunitinib, an untreated R 18 GFP tumor, and an R 18 GFP tumor treated with sunitinib. To quantify these qualitative observations, vascular masks were produced and vessel densities and interstitial distances were calcu lated. Sunitinib treated tumors had significantly lower vessel densities and significantly higher interstitial dis tances than untreated tumors .
The sunitinib induced reduction in vessel densities was more pronounced for A 07 GFP tumors than for R 18 GFP tumors. For sunitinib treated A 07 GFP tumors, the density of small vessels decreased more than the density of large vessels, implying that sunitinib treat ment selectively pruned small vessels. For sunitinib treated R 18 GFP tumors, the densities of small and large vessels were similarly reduced.