Effects of tyrosine kinase inhibitors and CXCR4 antagonist on tumor growth and angiogenesis in rat glioma model: MRI and protein analysis study

Abstract

The aim of the study was to determine the antiangiogenic efficacy of vatalanib, sunitinib, and AMD3100 in an animal model of human glioblastoma (GBM) by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumor protein expression analysis. Orthotopic GBM-bearing animals were randomly assigned either to control group or vatalanib, sunitinib, and AMD3100 treatment groups. Following 2 weeks of drug treatment, tumor growth and vascular parameters were measured using DCE-MRI. Expression of different angiogenic factors in tumor extracts was measured using a membrane-based human antibody array kit. Tumor angiogenesis and invasion were determined by immunohistochemistry. DCE-MRI showed a significant increase in tumor size after vatalanib treatment. AMD3100-treated group showed a significant decrease in a number of vascular parameters determined by DCE-MRI. AMD3100 significantly decreased the expression of different angiogenic factors compared to sunitinib or vatalanib; however, there were no significant changes in vascular density among the groups. Sunitinib-treated animals showed significantly higher migration of the invasive cells, whereas in both vatalanib- and AMD3100-treated animals the invasive cell migration distance was significantly lower compared to that of control. Vatalanib and sunitinib resulted in suboptimal therapeutic effect, but AMD3100 treatment resulted in a significant reduction in tumor growth, permeability, interstitial space volume, and invasion of tumor cells in an animal model of GBM.

Figure 1

Effect of antiangiogenic treatment in U251 tumor determined by MRI. Postcontrast T1WI (A) and T2WI changes (B) after 2 weeks of antiangiogenic treatment with vatalanib, sunitinib, and AMD3100; tumor volumes measured after vehicle and drug treatment (C); T2 maps of U251 tumors following antiangiogenic therapy showed no significant edema or necrosis compared to the vehicle-treated tumor (D). *P < .05. All tumors were in the right hemisphere.

Figure 2

MRI vascular parametric analysis after antiangiogenic therapy. The upper panel shows maps from a representative case showing tumor plasma volume (V_p), vascular forward transfer constant (K^trans), backflow transfer constant (K_b), and interstitial space volume (V_e). The middle and lower panels show quantitative estimations of V_p, K^trans, K_b, and V_e after the drug treatment. Note the significant decreased K^trans, K_b, and V_e in tumors treated with AMD3100. *P < .05. All tumors were in the right hemisphere.

Figure 3

Quantitative analysis of tumor growth factors using protein array kit and ELISA. AMD3100 treatment resulted in a net decrease in the expression of most proangiogenic factors compared to the vehicle treatment. G-CSF expression was significantly increased after treatment with vatalanib compared to the vehicle-treated tumor. Note the significant decrease of VEGFR2 in vatalanib-treated tumors. *P < .05.

Figure 4

Histologic analysis of vascular density. Immunohistochemistry staining with anti-vWF antibody shows tumor neovascularization (upper panel; original magnification, x40) with or without antiangiogenic treatments. Quantitative estimation of MVD by counting vWF-positive areas revealed significant changes in MVD after vatalanib, sunitinib, or AMD3100 treatments compared to the vehicle-treated tumor (lower panel). However, note the dilated and irregular vessels following treatments.

Figure 5

Migration of human marker-positive cells. Representative images of tumor sections were stained with invasion marker MHC-1 (upper panel; original magnification, x10). The drawn lines show the margin of the primary tumor masses and the migrated tumor cells away from the margin. Sunitinib treatment resulted in a significantly increased migration of cells from the main tumor mass (P < .0001). Treatment with vatalanib and AMD3100 resulted in a statistically significant reduction in the tumor cell migration compared to vehicle-treated tumor (P < .0001) (lower panel). *P < .05.

Figure 6

Co-expression of CD44, MMP-2, and vWF in the vicinity of MHC-1-positive cells, following antiangiogenic treatment. Upper panel: Double staining with anti-MHC-1 (green) and anti-MMP-2 (red) antibodies shows correlation between the two markers of tumor invasion. Lower panel: Double staining with anti-vWF (green) and anti-CD44 (red) antibodies showed CD44-positive cells lining the vWF-positive areas, indicating possible incorporation of tumor cells into new vessels (yellow arrow).