Tumor Angiogenic Marker Expression Levels during Tumor Growth: Longitudinal Assessment with Molecularly Targeted Microbubbles and US Imaging

Abstract

Purpose:To evaluate the use of molecularly targeted microbubbles (MBs) and ultrasonography (US) in the noninvasive assessment of the level of expression of three angiogenic markers, a v b 3 integrin, endoglin, and vascular endothelial growth factor receptor (VEGFR) 2, on tumor vascular endothelial cells in vivo during tumor growth. Materials and Methods:All procedures using laboratory animals were approved by the Institutional Administrative Panel on Laboratory Animal Care. Binding specifi city of three types of targeted MBs (MB Integrin , MB Endoglin , MB VEGFR2 ) was tested in cell culture under fl ow shear stress conditions. In vivo targeted contrast material-enhanced US imaging signal using the three MB types was measured at three tumor stages (small, medium, large) in three subcutaneous cancer xenografts (breast, ovarian, pancreatic cancer) in mice ( n = 54). In vivo US imaging signal was correlated with ex vivo angiogenic marker expression. Signifi cant differences were evaluated by using the Student t , analysis of variance, Wilcoxon, and Tukey Honest Signifi cant Difference tests. Results:Cell attachment of all three MB types was signifi cantly ( P = .016) higher compared with control MBs, and this attachment could be signifi cantly ( P = .026) decreased by blocking antibodies. Angiogenic marker-expressing cells bound signifi cantly ( P = .003) more targeted MBs than negative control cells, and MB attachment signifi cantly ( P , .001) correlated with marker expression levels on cells ( r = 0.87). In early stage breast and ovarian cancers, in vivo targeted contrastenhanced US demonstrated signifi cantly ( P Յ .04) higher endoglin expression than both a v b 3 integrin and VEGFR2 expression, whereas in early stage pancreatic cancer, marker expressions were not signifi cantly different ( P Ն .07). There was good correlation ( r Ն 0.63; P Յ .05) between in vivo targeted contrast-enhanced US imaging signals using the three MB types and ex vivo immunoblotting results regarding expression levels of the three angiogenic markers. Immunofl uorescence confi rmed expression of a v b 3 integrin, endoglin, and VEGFR2 on tumor vascular endothelial cells. Conclusion:Targeted contrast-enhanced US imaging allows noninvasive in vivo assessment of the expression levels of a v b 3 integrin, endoglin, and VEGFR2, which vary during tumor growth in subcutaneous cancer xenografts.

Squamous Cell Carcinoma Xenografts: Use of VEGFR2-targeted Microbubbles for Combined Functional and Molecular US to Monitor Antiangiogenic Therapy Effects

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Noninvasive monitoring of early antiangiogenic therapy response in human nasopharyngeal carcinoma xenograft model using MRI with RGD-conjugated ultrasmall superparamagnetic iron oxide nanoparticles

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