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 αvβ3-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.

Objective Volumetric contrast-enhanced ultrasound (VCEUS) imaging has the potential to monitor changes in renal perfusion following vascular injury. Methods VCEUS utilizes a series of planar image acquisitions, capturing the non-linear second harmonic signal from microbubble (MB) contrast agents flowing in the vasculature. Tissue perfusion parameters (peak intensity, IPK; time-to-peak intensity, TPK; wash-in rate, WIR; area under 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 kidney. For the flow phantom studies, either the concentration of MB contrast agent 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) and the contrast agent concentration was varied (5, 10, and 20 µL/L). Animal studies were performed using either healthy rats or those that underwent renal ischemia-reperfusion injury. A series of renal studies were performed using healthy rats (N = 4) while the angle of the transducer was varied for each VCEUS image acquisition (reference or 0°, 45°, and 90°) to assess if 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 MB concentrations injected into the flow system and the IPK, WIR, and AUC perfusion measures (R2 > 0.56, P < 0.005). Further, there was a linear relationship between changes in volume flow rate and the TPK, WIR, and AUC metrics (R2 > 0.77, P < 0.005). No significant difference was found between the transducer angle during data acquisition and any of the derived renal perfusion measures (P > 0.60). After induction of renal ischemia-reperfusion injury in a rat animal model (N = 4), VCEUS imaging of the injured kidney revealed an initial reduction in renal perfusion when compared to control animals followed by a progressive recovery of vascular function. Conclusions Preliminary results are encouraging and VCEUS-based renal perfusion imaging may prove clinically feasible for detecting and monitoring acute kidney injury.

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.

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-99m using the HYNIC method. Tc-99m 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-99m 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 ± .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.

Detection of prostate-specific antigen (PSA) as a screening strategy for prostate cancer is limited by the inability of the PSA test to differentiate between malignant cancer and benign hyperplasia. Here, we report the use of a cancer-specific promoter, inhibition of differentiation-1 (Id1), to drive a dual-reporter system (Ad5/3-Id1-SEAP-Id1-mCherry) designed for detection of prostate cancer using a blood-based reporter SEAP (secreted embryonic alkaline phosphatase) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels correlate with increased Gleason grade and disease progression. To evaluate the performance of the dual-reporter system, a prostate cell panel with varying aggressive phenotypes was tested. Following infection with the Ad5/3-Id1-SEAP-Id1-mCherry vector, expression of the SEAP and mCherry reporters was shown to increase with increasing levels of cellular Id1. No correlation was observed between Id1 and PSA. To evaluate in vivo performance, flank tumors were grown in athymic male mice using three prostate cancer cell lines. Following intra-tumoral injection of the vector, tumors formed by cells with high Id1 had the greatest reporter expression. Interestingly, tumors with the lowest levels of Id1 and reporter expression produced the greatest amounts of PSA. These data support the use of Ad5/3-Id1-SEAP-Id1-mCherry as a predictor of prostate cancer malignancy and a strategy for tumor localization.