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

Each year more than half a million people are diagnosed as having head and neck squamous cell carcinoma (HNSCC) world-wide.1 Beyond visual inspection and palpation, the clinical tools with which a surgeon may assess head and neck cancer are often found lacking in improving patient outcomes. The result is an increasing reliance on computed tomography and magnetic resonance imaging studies that still may not discover small foci of meta-static disease. Furthermore, these modalities do not permit cancer-specific detection techniques. Ultrasound (US) is a cost-effective imaging tool that is already used to broadly assess head and neck cancers and to aid in fine-needle biopsy. Ultrasonography offers immediate feedback that could greatly aid in surgical decision making, but poor resolution often renders it unsuitable as a stand-alone diagnostic test.2 Recently, microbubble (MB) US contrast agents have been shown to dramatically improve US resolution through imaging of tumor microvasculature.3 Micro-bubbles have been approved by the US Food and Drug Administration for use in cardiology, and owing to the superficial location of many head and neck lesions, will likely play an important role in the clinical detection of head and neck disease. Ultrasonographic contrast agents are gas-filled MBs that are administered intravenously and act as intravascular tracers during US imaging. Contrast MBs are typically about one-hundredth of a millimeter in size and will enter tumor micro-vasculature relatively unimpeded.4 The MB shell can be made of protein, polymer, lipid, or carbohydrate surfactant, and to oppose in vivo Bernoulli forces from blood vessels, a poorly soluble biologically inert perfluorinated gas is frequently used as the particle core.5 Microbubble contrast agents offer excellent detection sensitivities, can be imaged routinely with most current US systems,6 and are currently being studied as potential diagnostic agents in thyroid cancer.7 However, so far they have suffered from poor sensitivity and specificity in differentiating benign from malignant lesions. To further improve tumor resolution using MBs, we hypothesize that monoclonal antibodies can be linked to the MB shell via protein conjugation for molecular targeting of ligands specific to HNSCC. Epidermal growth factor receptor (EGFR) and extracellular matrix metalloproteinase inducer (CD147) are both known to be overexpressed by epithelial tumor cells and contribute to tumor progression. Up-regulated expression and subsequent activation of EGFR is known to be associated with tumor proliferation,8 and CD147 is a membrane glycoprotein greatly enriched on the surface of cancer cells that stimulates neighboring stromal cells to increase the synthesis of matrix metalloproteases necessary for metastatic spread.9 Though these cell surface proteins are predominantly expressed on tumor cell membranes, there is evidence that tumor-derived endothelial cells express these ligands as well,10 making them superb objectives for a targeted MB contrast agent. Subsequently, we hypothesize that MBs conjugated to antibodies against these cell surface ligands may improve US detection of HNSCC. Targeted contrast-enhanced US is an emerging imaging modality that has yet to be explored in head and neck cancer. Herein, we report on the feasibility of US imaging of head and neck cancer with targeted contrast agents both in vitro and in vivo. Because of the widespread clinical availability and relative ease of use, targeted contrast-enhanced US may have a growing role in the clinical assessment of head and neck cancer.