PMC

Quantitation of CD31, α-SMA, and NG2 expression in control tumors (n = 5; open columns), sunitinib-sensitive tumors (n = 8; striped columns), and sunitinib-resistant tumors (n = 10; solid columns). A, results of double fluorescent staining for CD31 and α-SMA. B, results of double fluorescent staining for CD31 and NG2. Columns, mean; bars, S.D. *, p < 0.05 and **, p < 0.01 compared with the control group by using one-way ANOVA followed by the post hoc Tukey-Kramer multiple comparison test. C and D, representative images for the double fluorescent staining of CD31/NG2 (C) and of CD31/α-SMA (D) in tumor sections from the control, sunitinib-sensitive and sunitinib-resistant groups.

A and B, phosphorylated protein kinases and other kinase substrates protein expression levels in sunitinib-sensitive (A) and sunitinib-resistant tumors (B) compared with control tumors. Up-regulation: a protein expression ratio of the sunitinib-treated tumor to the control tumor being ≥1.5. Down-regulation: a protein expression ratio of the control tumor to the sunitinib-treated tumor being ≥1.5. C and D, network created from up-regulated (C) and down-regulated (D) kinases and other phospho-proteins. Links denote activation (arrowheads) and inhibition (ball-head); dashed arrows represent indirect effects; nodes are color coded by up-regulated (red), down-regulated (blue), and unchanged (light gray); additional nodes that were not measured are color-coded in dark gray. Links are marked based on their database sources, and numbers represent PubMed IDs. eNOS, endothelial nitric-oxide synthase.

Modulated expression levels of ABCG2 and selected phosphorylated and total kinases in sunitinib-sensitive and -resistant tumors. A, Western blot analysis of ABCG2 levels in untreated (n = 4), sunitinib-sensitive (n = 6), and sunitinib-resistant (n = 4) tumors. Anti-β-actin immunoblot is shown as an independent loading control. B, Western blot analysis of phosphorylated and total c-Jun, ERK1/2, GSK3α/β, and PLCγ1 in untreated, sunitinib-sensitive and -resistant tumors. ST^§ indicate sample was not included in the study because of the missing dose on day 30. C, quantitation of Western blotting results using densitometric analysis. The results are plotted as percentage of control. Columns, mean; bars, S.D. Open columns, control group; striped columns, sunitinib-sensitive group; solid columns, sunitinib-resistant group. *, p < 0.05; **, p < 0.01 compared with the control group by using one-way ANOVA followed by the post hoc Tukey-Kramer multiple comparison test. +, p < 0.05; ++, p < 0.01 compared with the sunitinib-sensitive group by using one-way ANOVA followed by the post hoc Tukey-Kramer multiple comparison test.

A and B, unsupervised hierarchical biclustering of the expression patterns of 84 mouse (A) and 84 human (B) angiogenesis associated genes in five control tumors (CT), four sunitinib-sensitive tumors (ST), and seven sunitinib-resistant tumors (RT). The colors represent relative expression levels. Orange represents expression levels greater than the mean for a given gene across all samples; blue represents expression levels less than the mean. Each colored cell in the heat map represents the gene expression value for a probe in a sample. C to F, PCA of the mouse (C, mean values, and E, individual animals) and human (D, mean values, and F, individual animals) angiogenesis-associated gene expression was performed to visualize the overall difference in expression levels across samples and groups. The percentage near each axis of the PCA plots denotes the contribution of the principle component to capturing the overall variability within the data. G and H, expression levels of differentially expressed mouse (G) and human (H) angiogenesis-associated genes exhibiting significant difference between the control, sunitinib-sensitive, and sunitinib-resistant groups (p < 0.05 compared with the control) are visualized as a bi-clustering heat map using the same color scheme and normalization procedure as in A and B.

Differential tumor growth rate in sunitinib-treated tumor-bearing animals. U87MG human glioblastoma xenografts were grown subcutaneously in male NIH Swiss nude mice (nu/nu). Once-daily oral administration of vehicle or 40 mg/kg of sunitinib was initiated 14 days after tumor implantation and continued for 30 days. A, the distribution and median value of the day 30-to-day 0 tumor volume ratio in sunitinib-treated tumor-bearing mice (n = 21). Sunitinib-treated mice with the day 30-to-day 0 tumor volume ratio value being no less than the median value of 4.7 were classified as sunitinib-resistant animals (n = 11), whereas the rest were defined as the sunitinib-sensitive animals (n = 10). Subgroup 1: Tumor samples collected from this group were used for the angiogenesis PCR array, human phosphor-kinase antibody array and immunofluorescence double staining. Subgroup 2: Animals from this group were used in the PK study, and tumor samples collected were used for the immunofluorescence double staining and Western blotting. B, comparison of tumor growth ratio among control, sunitinib-sensitive, and sunitinib-resistant groups. Control animals (n = 5) were sacrificed 16 days after the initiation of the treatment because their tumors reached the allowed maximum size (2000 mm³). ++, p < 0.01 compared with the control group by using one-way ANOVA followed by the post hoc Tukey-Kramer multiple comparison test. **, p < 0.01 compared with the sunitinib-sensitive group by using the Mann-Whitney U test.