Reshu Saini

A Triple-Targeted Ultrasound Contrast Agent Provides Improved Localization to Tumor Vasculature

Actively targeting ultrasound contrast agents to tumor vasculature improves contrast‐enhanced sonography of tumor angiogenesis. This report summarizes an evaluation of multitargeted microbubbles, comparing single‐, dual‐, and triple‐targeted motifs.

Quantitative mapping of tumor vascularity using volumetric contrast-enhanced ultrasound

Objective:The goal of this research project was to develop a volumetric strategy for real-time monitoring and characterization of tumor blood flow using microbubble contrast agents and ultrasound (US) imaging. Materials and Methods:Volumetric contrast-enhanced US (VCEUS) imaging was implemented on a SONIX RP US system (Ultrasonix Medical Corp, Richmond, BC) equipped with a broadband 4DL14–5/38 probe. Using a microbubble-sensitive harmonic imaging mode (transducer transmits at 5 MHz and receives at 10 MHz), acquisition of postscan-converted VCEUS data was achieved at a volume rate of 1 Hz. After microbubble infusion, custom data processing software was used to derive microbubble time-intensity curve-specific parameters, namely, blood volume (IPK), transit time (T1/2PK), flow rate (SPK), and tumor perfusion (AUC). Results:Using a preclinical breast cancer animal model, it is shown that millimeter-sized deviations in transducer positioning can have profound implications on US-based blood flow estimators, with errors ranging from 6.4% to 40.3% and dependent on both degree of misalignment (offset) and particular blood flow estimator. These errors indicate that VCEUS imaging should be considered in tumor analyses, because they incorporate the entire mass and not just a representative planar cross-section. After administration of an antiangiogenic therapeutic drug (bevacizumab), tumor growth was significantly retarded compared with control tumors (P > 0.03) and reflects observed changes in VCEUS-based blood flow measurements. Analysis of immunohistologic data revealed no differences in intratumoral necrosis levels (P = 0.70), but a significant difference was found when comparing microvessel density counts in control with therapy group tumors (P = 0.05). Conclusions:VCEUS imaging was shown to be a promising modality for monitoring changes in tumor blood flow. Preliminary experimental results are encouraging, and this imaging modality may prove clinically feasible for detecting and monitoring the early antitumor effects in response to cancer drug therapy.

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 to Assess Early Response to Apoptosis-Inducing Anti-Death Receptor 5 Antibody Therapy in a Breast Cancer Animal Model

The objective of this study was to determine whether volumetric contrast‐enhanced ultrasound (US) imaging could detect early tumor response to anti–death receptor 5 antibody (TRA‐8) therapy alone or in combination with chemotherapy in a preclinical triple‐negative breast cancer animal model.

Molecular Targeting of Ultrasonographic Contrast Agent for Detection of Head and Neck Squamous Cell Carcinoma

OBJECTIVE To investigate the feasibility of ultrasonographic (US) imaging of head and neck cancer with targeted contrast agents both in vitro and in vivo. We hypothesize that conjugation of microbubble contrast agent to tumor-specific antibodies may improve US detection of head and neck squamous cell carcinoma (HNSCC). DESIGN Preclinical blinded assessment of anti-EGFR and anti-CD147 microbubble contrast agents for US imaging of HNSCC. SETTING Animal study. SUBJECTS Immunodeficient mice. INTERVENTION Injection of targeted microbubbles. MAIN OUTCOME MEASURE Microbubble uptake in tumors as detected by US. RESULTS In vitro assessment of anti-epidermal growth factor receptor (EGFR) and anti-CD147-targeted microbubbles in 6 head and neck cancer cell lines yielded a 6-fold improvement over normal dermal fibroblasts (P < .001). Binding of targeted agents had a positive correlation to both epidermal growth factor receptor (EGFR) (R(2) = 0.81) and CD147 (R(2) = 0.72) expression among all cell lines. In vivo imaging of flank tumors in nude mice (N = 8) yielded enhanced resolution of anti-EGFR-and anti-CD147-targeted microbubble agents over IgG control (P < .001), while dual-targeted contrast agents offered enhanced imaging over single-targeted contrast agents (P = .02 and P = .05, respectively). In a blinded in vivo assessment, targeted contrast agents increased intratumoral enhancement of flank tumors over controls. Targeted US contrast agents to both EGFR and CD147 were 100% sensitive and 87% specific in the detection of flank tumors. CONCLUSION This preclinical study demonstrates feasibility of using molecular US to target HNSCC for contrast-enhanced imaging of HNSCC tumor in vivo.

Systemic delivery of a breast cancer-detecting adenovirus using targeted microbubbles

One of the major limitations of cancer gene therapy using recombinant human adenovirus (Ad) is rapid Ad inactivation from systemic delivery. To eliminate this, biotin-coated ultrasound contrast agents, or microbubbles (MBs), were streptavidin-coupled with biotinylated antibodies to three distinct tumor vasculature-associated receptors (αVβ3 integrin, P-selectin and vascular endothelial growth factor receptor-2) for systemic targeting of a previously generated vector Ad5/3-Id1-SEAP-Id1-mCherry. This cancer-specific, dual-reporter vector was loaded in the targeted MBs and confirmed by confocal microscopy. MB loading capacity was estimated by functional assays as 4.72±0.2 plaque forming unit (PFU) per MB. Non-loaded (free) Ad particles were effectively inactivated by treatment with human complement. The Ad-loaded, targeted-MBs were injected systemically in mice bearing MDA-MB-231 tumors (Grp 1) and compared with two control groups: Ad-loaded, non-targeted MBs (Grp 2) and free Ad (Grp 3) administered under the same conditions. Two days after administration the blood levels of secreted embryonic alkaline phosphatase (SEAP) reporter in Grp 1 mice (16.1 ng ml–1±2.5) were significantly higher (P<0.05) than those in Grp 2 (9.75 ng ml–1±1.5) or Grp 3 (4.26 ng ml–1±2.5) animals. The targeted Ad delivery was also confirmed by fluorescence imaging. Thus, Ad delivery by targeted MBs holds potential as a safe and effective system for systemic Ad delivery for the purpose of cancer screening.

MODEL SYSTEM USING CONTROLLED RECEPTOR EXPRESSION FOR EVALUATING TARGETED ULTRASOUND CONTRAST AGENTS

This report details a model system for evaluating targeted ultrasound (US) contrast agents using adenoviral (Ad) vectors to regulate target receptor expression. Receptor density in vitro was modulated in breast cancer cells by varying the multiplicity of infection (MOI) from 0 to 100. Target receptors were induced using a green fluorescent protein (GFP) reporter Ad vector for gene transfer and expression of the hemagglutinin (HA) tag. These reporter genes were under the control of the ubiquitous cytomegalovirus (CMV) promoter. Subsequently, receptor expression and anti-HA antibody (Ab) binding was examined with flow cytometry. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or isotype control Ab to the surface of biotin coated MBs via a streptavidin bridge. Targeted MBs were incubated with Ad infected 2LMP cells to evaluate in vitro MB binding. Experimental results found GFP expression to be directly correlated with Ad MOI (r² = 0.96). Increasing the Ad MOI produced a corresponding increase in binding and accumulation of anti-HA Ab on the cell surface (p < 0.01). However, no difference was found between Cy5-labeled anti-HA Ab exposed cell groups at an MOI of 0 (p > 0.29). Additionally, no difference was found between the isotype control Ab group (p > 0.44) indicating minimal nonspecific binding. No difference was found between cell groups incubated with isotype-targeted MBs (p > 0.42) regardless of receptor density. However, cells exposed to HA-targeted MBs showed increased levels of cell binding proportional to induced receptor expression levels (p < 0.02).

Optical fluorescent imaging to monitor temporal effects of microbubble-mediated ultrasound therapy

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p <; 0.05) using the small molecule, but not in the large molecule group (p >; 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p <; 0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors.

Ultrasound imaging of whole tumor response to drug therapy

Use of volumetric contrast-enhanced ultrasound (US) imaging of cancer-bearing mice allows description of whole tumor perfusion and monitoring response to drug treatment. Thirty mice were implanted with breast cancer cells. Following sufficient tumor growth, mice were sorted into four treatment groups: Abraxane, TRA-8, Abraxane + TRA-8, or control. Mice were administered drug treatment on days 0, 3, 7, 10, 14, and 17. Whole tumor contrast-enhanced US imaging was performed on days 0, 1, 3, and 7 before drug dosing. At termination, tumors were excised and immunohistologic analyses were performed. Therapeutic efficacy was detected within 7 days after drug dosing with volumetric contrast-enhanced US imaging. The most significant reduction in tumor perfusion as compared to control mice measurements was observed in the TRA-8 + Abraxane dosed animals (P = 0.001). The reduction in perfusion observed in the TRA-8 + Abraxane group coincided with a reduction in tumor size during the same time period. Survival curves illustrate that combination TRA-8 + Abraxane improves drug efficacy compared to monotherapy. Overall, preliminary results demonstrate that volumetric contrast-enhanced US imaging of changes in whole tumor perfusion levels allows early monitoring of cancer response to drug therapy.

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.