Volumetric contrast-enhanced ultrasound imaging of renal perfusion.

OBJECTIVES: To determine whether volumetric contrast-enhanced ultrasound (US) imaging has the potential to monitor changes in renal perfusion after vascular injury. METHODS: Volumetric contrast-enhanced US uses a series of planar image acquisitions, capturing the nonlinear second harmonic signal from microbubble contrast agents flowing in the vasculature. Tissue perfusion parameters (peak intensity [IPK], time to peak intensity [TPK], wash-in rate [WIR], and area under the curve [AUC]) were derived from time-intensity curve data collected during in vitro flow phantom studies and in vivo animal studies of healthy and injured kidneys. For the flow phantom studies, either the contrast agent concentration was held constant (10 µL/L) with varying volumetric flow rates (10, 20, and 30 mL/min), or the flow rate was held constant (30 mL/min) with varying contrast agent concentrations (5, 10, and 20 µL/L). Animal studies used healthy rats or those that underwent renal ischemia-reperfusion injury. Renal studies were performed with healthy rats while the transducer angle was varied for each volumetric contrast-enhanced US image acquisition (reference or 0°, 45°, and 90°) to determine whether repeated renal perfusion measures were isotropic and independent of transducer position. Blood serum biomarkers and immunohistology were used to confirm acute kidney injury. RESULTS: Flow phantom results revealed a linear relationship between microbubble concentrations injected into the flow system and the IPK, WIR, and AUC (R(2) > 0.56; P < .005). Furthermore, there was a linear relationship between volume flow rate changes and the TPK, WIR, and AUC (R(2) > 0.77; P < .005). No significant difference was found between the transducer angle during data acquisition and any of the perfusion measures (P > .60). After induction of renal ischemia-reperfusion injury in the rat animal model (n = 4), volumetric contrast-enhanced US imaging of the injured kidney revealed an initial reduction in renal perfusion compared to control animals, followed by progressive recovery of vascular function. CONCLUSIONS: Volumetric contrast-enhanced US-based renal perfusion imaging may prove clinically feasible for detecting and monitoring acute kidney injury.

Biodistribution of P-selectin targeted microbubbles.

PURPOSE: To evaluate binding of P-selectin targeted microbubbles (MB) in tumor vasculature; a whole-body imaging and biodistribution study was performed in a tumor bearing mouse model. METHODS: Antibodies were radiolabeled with Tc-99 m using the HYNIC method. Tc-99 m labeled anti-P-selectin antibodies were avidin-bound to lipid-shelled, perfluorocarbon gas-filled MB and intravenously injected into mice bearing MDA-MB-231 breast tumors. Whole-body biodistribution was performed at 5 min (n = 12) and 60 min (n = 4) using a gamma counter. Tc-99 m-labeled IgG bound IgG-control-MB group (n = 12 at 5 min; n = 4 at 60 min), Tc-99 m-labeled IgG-control-Ab group (n = 5 at 5 min; n = 3 at 60 min) and Tc-99 m-labeled anti P-selectin-Ab group (n = 5 at 5 min; n = 3 at 60 min) were also evaluated. Planar gamma camera imaging was also performed at each time point. RESULTS: Targeted-MB retention in tumor (60 min: 1.8 ± 0.3% ID/g) was significantly greater (p = 0.01) than targeted-MB levels in adjacent skeletal muscle at both time points (5 min: 0.7 ± 0.2% ID/g; 60 min: 0.2 ± 0.1% ID/g) while there was no significant difference (p = 0.17) between muscle and tumor retention for the IgG-control-MB group at 5 min. CONCLUSIONS: P-selectin targeted MBs were significantly higher in tumor tissue, as compared with adjacent skeletal tissue or tumor retention of IgG-control-MB.

Enhancement of adenovirus delivery after ultrasound-stimulated therapy in a cancer model.

Improving the efficiency of adenovirus (Ad) delivery to target tissues has the potential to advance the translation of cancer gene therapy. Ultrasound (US)-stimulated therapy uses microbubbles (MBs) exposed to low-intensity US energy to improve localized delivery. We hypothesize that US-stimulated gene therapy can improve Ad infection in a primary prostate tumor through enhanced tumor uptake and retention of the Ad vector. In vitro studies were performed to analyze the degree of Ad infectivity after application of US-stimulated gene therapy. A luciferase-based Ad on a ubiquitous cytomegalovirus (CMV) promoter (Ad5/3-CMV-Luc) was used in an animal model of prostate cancer (bilateral tumor growth) to evaluate Ad transduction efficiency after US-stimulated therapy. Bioluminescence imaging was employed for in vivo analysis to quantify Ad infection within the tumor. In vitro studies revealed no difference in Ad transduction between groups receiving US-stimulated therapy using high, low or sham US intensity exposures at various multiplicities of infection (MOIs) (p = 0.80). In vivo results indicated that tumors receiving US-stimulated therapy after intra-tumoral injection of Ad5/3-CMV-Luc (1 × 10(6) plaque-forming units) exhibited a 95.1% enhancement in tumor delivery compared with control tumors receiving sham US (p = 0.03). US-stimulated therapy has significant potential to immediately affect Ad-based cancer gene therapy by improving virus bioavailability in target tissues.

An animal model allowing controlled receptor expression for molecular ultrasound imaging.

Reported in this study is an animal model system for evaluating targeted ultrasound (US) contrast agents binding using adenoviral (Ad) vectors to modulate cellular receptor expression. An Ad vector encoding an extracellular hemagglutinin (HA) epitope tag and a green fluorescent protein (GFP) reporter was used to regulate receptor expression. A low and high receptor density (in breast cancer tumor bearing mice) was achieved by varying the Ad dose with a low plaque forming unit (PFU) on day 1 and high PFU on day 2 of experimentation. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or IgG isotype control antibodies to the MB surface with biotin-streptavidin linkage. Targeted and control MBs were administered on both days of experimentation and contrast-enhanced US (CEUS) was performed on each mouse using MB flash destruction technique. Signal intensities from MBs retained within tumor vasculature were analyzed through a custom Matlab program. Results showed intratumoral enhancement attributable to targeted MB accumulation was significantly increased from the low Ad vector dosing and the high Ad vector dosing (p = 0.001). Control MBs showed no significant differences between day 1 and day 2 imaging (p = 0.96). Additionally, targeted MBs showed a 10.5-fold increase in intratumoral image intensity on day 1 and an 18.8-fold increase in image intensity on day 2 compared with their control MB counterparts.

Molecular ultrasound imaging using a targeted contrast agent for assessing early tumor response to antiangiogenic therapy.

OBJECTIVES: Contrast-enhanced ultrasound (US) and targeted microbubbles have been shown to be advantageous for angiogenesis evaluation and disease staging in cancer. This study explored molecular US imaging of a multitargeted microbubble for assessing the early tumor response to antiangiogenic therapy. METHODS: Target receptor expression of 2LMP breast cancer cells was quantified by flow cytometric analysis and characterization established with antibodies against mouse α(V)ß3- integrin, P-selectin, and vascular endothelial growth factor receptor 2. Tumor-bearing mice (n = 15 per group) underwent contrast-enhanced US imaging of multitargeted microbubbles. Microbubble accumulation was calculated by destruction-replenishment techniques and time-intensity curve analysis. On day 0, mice underwent baseline imaging. Next, therapy group mice were injected with a 0.2-mg dose of bevacizumab, and controls received matched saline injections. Imaging was repeated on days 1 and 3. After imaging was completed on day 3, the mice were euthanized and tumors excised. Histologic analysis of microvessel density and intratumoral necrosis was completed on tumor sections. RESULTS: On day 3 after bevacizumab dosing, a 71.8% change in tumor vasculature was shown between the therapy and control groups (P = .01). The therapy group had a 15.4% decrease in tumor vascularity, whereas the control group had a 56.4% increase. CONCLUSIONS: Molecular US imaging of angiogenic markers can detect the early tumor response to drug therapy.