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.