Objectives 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. Methods Microbubbles were avidin-biotin linked to antibodies against mouse αVβ-integrin, P-selectin, and vascular endothelial growth factor receptor 2. These receptors are constitutively overexpressed in tumor vasculature. Binding comparisons between targeted microbubble groups were evaluated on mouse SVR angiosarcoma endothelial cells. Levels of the targeted receptors were characterized with flow cytometry. Targeted microbubble groups were administered to human MDA-MB-231 breast cancer tumor-bearing mice (n = 3) followed by contrast-enhanced sonography in a microbubble-sensitive harmonic imaging mode implemented on an ultrasound scanner equipped with a linear array transducer (5 MHz transmit and 10 MHz receive) to evaluate differences in microbubble accumulation in the tumor vasculature. Results In vitro analysis showed a 50% increase (P < .001) in triple-targeted microbubble binding over dual-targeted microbubble groups in mouse SVR cells. Mice bearing MDA-MB-231 tumors showed a 40% increase in tumor image intensity after dosing with triple-targeted microbubbles compared with single- and dual-targeted microbubbles (P = .006). Histologic staining confirmed the presence of αVβ-integrin, P-selectin, and vascular endothelial growth factor receptor 2 in the tumors. Conclusions Microbubble accumulation in the tumor vasculature was improved using a triple-targeted microbubble approach.
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 post scan-converted VCEUS data was achieved at a volume rate of 1 Hz. Following 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. Following administration of an antiangiogenic therapeutic drug (bevacizumab), tumor growth was significantly retarded compared to control tumors (p > 0.03) and reflect 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 MVD counts in control to 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.
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.
Angiogenesis is a critical process for tumor growth and metastatic dissemination. There is tremendous interest in the development of noninvasive methods for imaging tumor angiogenesis, and ultrasound (US) is an emerging platform technology to address this challenge. The introduction of intravascular microbubble contrast agents not only allows real-time visualization of tumor perfusion during an US examination, but they can be functionalized with specific ligands to permit molecular US imaging of angiogenic biomarkers that are overexpressed on the tumor endothelium. In this article, we will review current concepts and developing trends for US imaging of tumor angiogenesis, including relevant preclinical and clinicsal findings.
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 micro-bubbles 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) (R2=0.81) and CD147 (R2=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.
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.
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.
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.
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