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Molecular Imaging of Vascular Endothelial Growth Factor Receptor 2 Expression Using Targeted Contrast-Enhanced High-Frequency Ultrasonography

Department of Radiology and Radiological Sciences (A.L., A.C.F., J.C.G.), Department of Radiation Oncology (J.H., D.E.H.), and Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism (M.B.), Vanderbilt University Medical Center, Nashville, Tennessee USA; and Vanderbilt University Institute of Imaging Science, Nashville, Tennessee USA (A.L., J.C.G.).

Address correspondence to Andrej Lyshchik, MD, PhD, Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, CCC-1118 MCN, 1161 21st Ave, Nashville, TN 37232-2675, USA. E-mail: andrej.lyshchik{at}vanderbilt.edu

Objective. The aim of our study was to investigate the use of targeted contrast-enhanced high-frequency ultrasonography for molecular imaging of vascular endothelial growth factor receptor 2 (VEGFR2) expression on tumor vascular endothelium in murine models of breast cancer. Methods. Highly invasive metastatic (4T1) and nonmetatstatic (67NR) breast cancer cells were implanted in athymic nude mice. Tumors were examined in vivo with targeted contrast-enhanced high-frequency ultrasonography using a scanner with a 40-MHz probe. Randomized boluses of ultrasound contrast agents (UCAs) conjugated with an anti-VEGFR2 monoclonal antibody or an isotype control antibody (immunoglobulin G) were injected into the animals. Sonograms were analyzed by calculation of the normalized video intensity amplitudes caused by backscatter of the bound UCA. After ultrasonography, the tumor samples were harvested for analysis of VEGFR2 expression by immunoblotting and immunocytochemistry. Results. The mean video intensity amplitude caused by backscatter of the retained VEGFR2-targeted UCA was significantly higher than that of the control UCA (mean ± SD: 4T1 tumors, 15 ± 3.5 versus 7 ± 1.6 dB; P < .01; 67NR tumors, 50 ± 12.3 versus 12 ± 2.6 dB; P < .01). There was a significant difference in VEGFR2-targeted UCA retention between 4T1 and 67NR tumors (normalized video intensity amplitudes, 15 ± 3.5 and 50 ± 12.3 dB, respectively; P < .001), and this correlated well with relative VEGFR2 expression in the two tumor types. Conclusions. Targeted contrast-enhanced high-frequency ultrasonography may enable in vivo molecular imaging of VEGFR2 expression on the tumor vascular endothelium and may be used for noninvasive longitudinal evaluation of tumor angiogenesis in preclinical studies.

Key Words: breast cancer • molecular ultrasonography • vascular endothelial growth factor receptor 2

Abbreviations: IgG, immunoglobulin G • TBST, Tris-buffered saline–Tween 20 • UCA, ultrasound contrast agent • VEGFR2, vascular endothelial growth factor receptor 2

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