Use of ultrasound contrast agent microbubbles in preclinical research: recommendations for small animal imaging.

Ultrasound contrast imaging techniques represent a real opportunity to improve efficiency in the preclinical drug discovery and development process. Ultrasound contrast agents (UCAs) combined with specific ultrasound contrast detection modes provide real-time, high spatial resolution of both organ and lesion blood perfusion, the so-called dynamic contrast-enhanced ultrasound imaging. With the advent of targeted UCA, ultrasound molecular imaging is gaining momentum in molecular imaging, particularly because of the simultaneous real-time anatomical and functional/molecular imaging capabilities. In preclinical research, contrast-enhanced ultrasound imaging, with either nontargeted or targeted UCA, is a fast-growing imaging modality that has not yet been standardized compared with other imaging modalities. Contrast-enhanced ultrasound imaging is an operator-dependent imaging modality, requiring adherence to rigorous step-by-step protocols. In this article, which is intended for advanced, hands-on researchers, we report key factors that can lead to variability in preclinical results and recommend some preventive methods to limit or cancel their effect on the final results. Standardized procedures are a prerequisite for acceptance of new contrast-enhanced ultrasound imaging methods to eliminate factors that could distort results, improve the reproducibility between different centers and studies, and, therefore, allow translation to clinical application.

Effects of acoustic radiation force on the binding efficiency of BR55, a VEGFR2-specific ultrasound contrast agent.

This work describes an in vivo study analyzing the effect of acoustic radiation force (ARF) on the binding of BR55 VEGFR2-specific contrast-agent microbubbles in a model of prostatic adenocarcinoma in rat. A commercial ultrasound system was modified by implementing high duty-cycle 3.5-MHz center frequency ARF bursts in a scanning configuration. This enabled comparing the effects of ARF on binding in tumor and healthy tissue effectively in the same field of view. Bubble binding was established by measuring late-phase enhancement in amplitude modulation (AM) contrast-specific imaging mode (4 MHz, 150 kPa) 10 min after agent injection when the unbound bubbles were cleared from the circulation. Optimal experimental conditions, such as agent concentration (0.4 × 10(8)-1.6 × 10(8) bubbles/kg), acoustic pressure amplitude (26-51 kPa) and duty-cycle (20%-95%) of the ARF bursts, were evaluated in their ability to enhance binding in tumor without significantly increasing binding in healthy tissue. Using the optimal conditions (38 kPa peak-negative pressure, 95% duty cycle), ARF-assisted binding of BR55 improved significantly in tumor (by a factor of 7) at a lower agent dose compared with binding without ARF, and it had an insignificant effect on binding in healthy tissue. Thus, the high binding specificity of BR55 microbubbles for targeting VEGFR2 present at sites of active angiogenesis was confirmed by this study. Therefore, it is believed that based on the results obtained in this work, ultrasound molecular imaging using target-specific contrast-agent microbubbles should preferably be performed in combination with ARF.

Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55.

OBJECTIVES: To evaluate BR55, a new VEGFR2-specific ultrasound contrast agent, for imaging prostate tumors in an orthotopic model in the rat. MATERIALS AND METHODS: Rat prostate adenocarcinoma were established by injection of G Dunning R-3327 tumor cells in one lobe of the prostate of Copenhagen rats. Imaging experiments were performed with BR55, SonoVue, and streptavidin-functionalized microbubbles coupled with an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody using a clinical ultrasound scanner. Contrast enhancement in the tumor and healthy prostate was followed over time by intermittent imaging at low acoustic power. Signal quantification and statistical analysis were performed in the tumor and healthy tissue to compare the behavior of the 3 contrast agents. Immunohistochemistry was performed on the prostate and tumor specimen to determine the expression of VEGFR2. RESULTS: Comparable contrast enhancement was observed in tumors at peak intensity for BR55 and SonoVue. Then, once unbound microbubbles had cleared from the circulation, a strong enhancement of the tumor was obtained with BR55, whereas no significant microbubble accumulation was detected in the healthy prostate tissue. SonoVue microbubbles were rapidly eliminated, and no significant binding was observed in the tumor. The tumor to prostate ratio calculated after signal quantification was about 20 for the 3 doses of BR55 tested. The enhancement obtained with BR55 in the tumor was not significantly different from the one observed with antibody-coupled streptavidin microbubbles. Intense staining for VEGFR2 was detected in the tumor vessels by immunohistochemistry. CONCLUSIONS: This study showed that BR55 binding to prostate tumors resulted in a strong enhancement of the lesions as early as a few minutes after contrast injection, whereas minimal nonspecific accumulation occurred in the healthy part of the gland. BR55, like SonoVue, provide information on tissue perfusion during the early vascular phase, but BR55 binding to the tumoral endothelium allows to gain additional information by highlighting the sites of active angiogenesis. The late phase enhancement of the tumor should be particularly valuable for prostate cancer detection and for biopsy guidance.