A triple-targeted ultrasound contrast agent provides improved localization to tumor vasculature

The emergence of combination antiangiogenic cancer therapy has led to an increased demand for noninvasive modalities to monitor and evaluate tumor vasculature for determining an early treatment response. Recently, contrast-enhanced sonography was validated as a powerful tool for monitoring antiangiogenic therapy in cancer.1,2 In addition, clinical trials are ongoing to determine the importance of contrast-enhanced sonography for evaluating tumor vasculature changes during presurgical therapy.3 The evolution of contrast-enhanced sonography has led to actively targeting microbubbles to tumor vasculature to provide improved visualization of angiogenesis and vessel assessment.4 Microbubbles are gas-filled colloidal particles surrounded by a flexible outer core composed of surfactant, polymer, or lipid molecules. Originally designed to improve ultrasound visualization during cardiac shunt evaluation,5 current microbubbles are stable in the circulation with a systemic half-life of several minutes. Their small size (1–10 μm) permits pulmonary passage, yet they are large enough to resist extravasation.6 During ultrasound exposure, microbubbles expand and contract in rapid oscillation and generate a nonlinear backscattered signal that can be isolated to improve contrast relative to surrounding soft tissue. These strategies ultimately improve vasculature visualization, making microbubbles useful ultrasound contrast agents. To improve contrast-enhanced visualization of circulation and angiogenesis, targeted microbubbles have been generated that actively bind molecular markers expressed on the vessel lumen. These microbubbles are conjugated to proteins through either covalent linkage or avidin-biotin interactions.7 Although the use of avidin-biotin linkage is limited to preclinical conditions because of avidin immunogenicity, the application remains useful for investigators to explore the wide range of receptors available to improve targeting. Microbubbles conjugated with receptor-targeting ligands bind to receptors expressed on the vessel lumen in the tissue of interest. The accumulation of microbubbles through vessel wall interaction ultimately improves contrast-enhanced sonography and the visualization of angiogenesis.6 For specific targeting of microbubbles to the tumor vasculature, overexpressed receptors involved in survival mechanisms such as angiogenesis and adhesion are commonly used. Endothelial receptors involved in angiogenesis are routinely shown to be overexpressed in cancer and contribute to overall cancer progression.8-11 Vascular endothelial growth factor (VEGF) is a chemical ligand produced by cells undergoing hypoxia. It generates neovascularization by forming a chemotactic gradient for the recruitment of bone marrow-derived endothelial cells and through binding of vascular endothelial growth factor receptor 2 (VEGFR2) on preexisting endothelial cells, stimulating growth and proliferation.12 Vascular endothelial growth factor receptor 2 mediates all cellular responses to VEGF, and targeting overexpression of the receptor has led to successful strategies in antiangiogenic therapy.13-15 The targeting of microbubbles to this receptor through antibody-microbubble coupling has been shown to improve contrast-enhanced sonography.16,17 Another commonly overexpressed surface molecule in tumor endothelial cells is the cell adhesion molecule P-selectin,15 which is expressed on stimulated endothelial cells and activated platelets. It participates in the recruitment of leukocytes to areas of inflammation, which are common in the tumor vasculature.18,19 In addition, the presence of P-selectin permits the adhesion of platelets and cancer cells to the tumor endothelium. Recruitment of activated platelets has been shown to induce localized production of VEGF, thereby stimulating angiogenesis and overall tumor enrichment.20 Techniques in contrast-enhanced sonography have used the expression of P-selectin in echocardiography and atherosclerotic plaque detection with P-selectin–targeted microbubbles.21,22 The overexpression of P-selectin in the tumor vasculature by stimulated endothelial cells makes it a viable target for intravascular microbubble binding. Integrins mediate attachment and interactions between cells and their surrounding tissues, which include neighboring cells and the extracellular matrix.23 αVβ3-Integrin is commonly overexpressed in the vasculature of breast cancer.24 The αVβ3-Integrins are involved in leukocyte recruitment, tumor progression, and angiogenesis and have been targeted in many cancer therapies.25-27 Targeting by antibody- or binding peptide-labeled microbubbles has been shown to improve contrast-enhanced sonography of the tumor vasculature.17,28 Improvements in contrast-enhanced sonography have been driven in part by the need for sensitive detection and characterization of vascular abnormalities. First-generation targeted microbubbles were single-targeted microbubbles to enhance visualization of inflammation and tumor angiogenesis.7,29 Soon thereafter, dual-targeted microbubbles emerged as superior agents for targeted contrast-enhanced sonography. For tumor vasculature imaging, dual combinations of VEGFR2 and αVβ3-integrin,17 P-selectin and vascular cell adhesion molecule 1,21 and intercellular adhesion molecule 1 and sialyl Lewisx30 were all shown to improve visualization over their single-targeted counterparts. Triple-targeted microbubbles may further improve contrast-enhanced sonography. In this report, microbubbles targeted to the commonly overexpressed vasculature receptors αVβ3-integrin, P-selectin, and VEGFR2 were used in a triple-targeted strategy to enhance microbubble binding and overall visualization of tumor vasculature.