Marshall Mahoney

Molecular Ultrasound Imaging Using a Targeted Contrast Agent for Assessing Early Tumor Response to Antiangiogenic Therapy

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.

Volumetric Contrast-Enhanced Ultrasound Imaging of Renal Perfusion

To determine whether volumetric contrast‐enhanced ultrasound (US) imaging has the potential to monitor changes in renal perfusion after vascular injury.

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.

Biodistribution of P-selectin targeted microbubbles.

Abstract 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.

A Dual-Reporter, Diagnostic Vector for Prostate Cancer Detection and Tumor Imaging

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-secreted embryonic alkaline phosphatase (SEAP) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels are correlated 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 as a strategy for tumor localization.

Four-dimensional molecular ultrasound imaging of tumor angiogenesis in a preclinical animal model of prostate cancer

This article evaluated volumetric molecular ultrasound (US) imaging with multi-targeted microbubble (MB) contrast agents for detection of angiogenic imaging biomarkers in a preclinical cancer model. Imaging was performed using a modified US system (Ultrasonix Medical Corp) equipped with a 4-dimensional (4D) transducer. Image processing software separated the US signal originating from intravascular MBs that were bound to the overexpressed targets from the freely circulating and unattached contrast agent. Molecular US imaging using targeted MBs was compared to results obtained using non-specific control MBs in the same prostate-cancer bearing mouse. Molecular US signals were significantly higher when imaging the targeted MBs versus imaging of control contrast agent (p <; 0.001). Specifically, it was found that molecular US imaging of the targeted MB contrast agent produced a considerable mean intratumoral enhancement of 19.5 ± 2.2 dB. A significant inverse correlation was found between the molecular US signal and fractional tumor enhancement (ρ = -0.56, p = 0.02). This suggests that as the spatial distribution of the molecular US signal increases, the average intensity of the intratumoral reporter signal decreases. This finding could identify when the neovascularity is inadequate to meet the metabolic demands of the tumor, creating a strong angiogenic response. This high angiogenic response is typically found in aggressive cancers and during early stages of tumor growth. Therefore, molecular US imaging may improve US for the early detection of cancer and help distinguish indolent from aggressive disease.

Molecular ultrasound imaging of tumor response to bevacizumab using a breast cancer animal model

Ultrasound (US) contrast agents, or microbubbles (MBs), have shown immense potential in cancer detection, staging and monitoring drug treatment. Contrast-enhanced US imaging utilizes non-linear oscillations of MBs to improve signal detection from the vasculature. Targeted MBs to over-expressed receptors in a region-of-interest allow enhancement of intratumoral visualization of vasculature. This permits longitudinal studies of angiogenesis development and shows potential in analyzing tumor response to therapy. In this study, breast cancer-bearing mice were analyzed using molecular US imaging for early response to bevacizumab, a vascular disrupting agent. Mice were injected systemically with MBs targeted simultaneously to VEGFR2, p-selectin and αVβ3. Mice underwent molecular US imaging and were analyzed for changes in intratumoral enhancement after delivery of bevacizumab. Individual animal response to therapy was analyzed and established as a 10% or greater reduction in intratumoral enhancement by day 3 post therapy. Intratumoral enhancement was assessed using custom Matlab software. Therapeutic group animals showed a significant response to drug compared to the control group by day 3 (P <; 0.01). Molecular US imaging is a non-invasive, inexpensive approach for analyzing early response to vascular disrupting agents as demonstrated by this study in a breast cancer animal model.

Real-time volumetric contrast-enhanced ultrasound and microbubble destruction-replenishment imaging: Preliminary in vitro and in vivo results

Noninvasive assessment of renal perfusion would help improve treatment and outcome for patients with acute kidney injury. Although there are current methods with the capability of imaging and quantifying renal vascularity perfusion parameters, the nephrotoxic nature of their contrast agents prevents their use on compromised kidneys. Ultrasound (US) contrast agents are both non-toxic and capable of enhancing visualization of vasculature by up to 25 dB. Described in this study are preliminary results for quantifying perfusion parameters in a flow phantom and rat kidney model with volumetric contrast-enhanced US (VCEUS) imaging. Results showed significant correlations between time-to-peak intensity and concentration flow rate in a phantom model (P <; 0.05). Renal perfusion experiments in healthy rats validate VCEUS as a valuable tool for obtaining consistent time-intensity curves and parametric perfusion information. Further study is expected to reveal the advantageous nature of VCEUS for compromised and ischemic kidneys compared to alternative imaging modalities.

Molecular ultrasound imaging using a novel tumor-bearing animal model with variable target receptor expression

Molecular ultrasound (US) imaging of overexpressed endothelial receptors has been shown to increase tumor visualization within a region-of-interest and is of particular interest in cancer research. This study used an in vivo animal model system for evaluating targeted microbubble (MB) contrast agents. 2LMP breast cancer-bearing mice (N = 6) expressed varying intratumoral levels of the target hemagglutinin (HA) receptor through adenoviral (Ad) vector modulation. Animals were administered a “low” and then a “high” Ad vector dose following initial US imaging. Anti-HA antibody-labeled or control MBs were prepared and molecular US imaging was performed 24 hours after Ad vector infection using a SONIX Tablet ultrasound system (Ultrasonix Medical Corp). Custom Matlab programs were developed to evaluate US image intensity from data collected using a MB destruction-replenishment technique. Results showed intratumoral enhancement due to targeted MB accumulation was significantly increased in the “high” Ad vector dosed animals compared to the same animals administered a “low” Ad vector dose (P = 0.001). Control MBs showed no significant differences between imaging days (P = 0.96). Additionally, targeted MBs on average produced a 10.5-fold increase in intratumoral image intensity for the “low” HA receptor group and an 18.8-fold increase in image intensity for the “high” HA receptor group compared to control MBs.

Targeted molecular ultrasound therapy improves chemotherapeutic drug delivery in cancer cells

Microbubble (MB) mediated ultrasound (US) therapy has been shown to non-invasively increase drug uptake through increasing cell membrane permeability and vascular extravasation. Increasing MB-cell interaction through targeted receptors overexpressed in the tumor vasculature promises to increase effectiveness of MB-mediated US therapy. 2LMP breast cancer cells were plated on acoustically transparent tissue culture plates. Using a model system which allows receptor density modulation, molecular US therapy was analyzed for anticancer effects in vitro. Drug (paclitaxel) effectiveness in combination with traditional MB-mediated US therapy or molecular US therapy was analyzed using flow cytometry and ATPlite assays. It was shown that molecular US therapy significantly increases anticancer effects by 25% compared to traditional MB-mediated US therapy (P <; 0.001). This preclinical approach has potential to increase localized delivery through a targeted methodology, thereby potentially decreasing systemically toxicity. This study demonstrates that MBs targeted to cellular receptors are a promising addition to MB-mediated US therapy.