The EGFR is highly expressed on the surface of OSC-19-luc2-cGFP cells. B. 0.34 and 2.72 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hours after administration of the EGFR-specific nanobody. After injection of 75 g 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly recognized intraoperatively using a recently developed fluorescent EGFR targeting nanobody. Translation of this approach to the medical center would potentially improve the rate of radical surgical resections. Keywords:image-guided surgery, near-infrared fluorescence, oral squamous cell carcinoma, nanobody, Epidermal Growth Factor Receptor == INTRODUCTION == In oncology, a range of noninvasive imaging modalities, including X-ray, ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI), enable early detection, staging, and treatment evaluation of malignancy. However, in most cases surgeons still discriminate healthy tissue from cancerous tissue by means of visual inspection and palpation during surgery. Given the fact that adequate tumor free margins are of paramount importance for patient prognosis and end result, and that irradical resections still frequently occur, novel imaging modalities are needed. Despite the main objective of achieving macroscopic clearance of 1 1 cm in the surgical management of oropharyngeal or oral squamous cell carcinoma (OSCC), the presence of tumor positive margins has been reported in 16% of patients.1Numerous reports have indicated that involved margins imply deteriorated prognoses.2On the other hand, applying wider surgical margins will result in functional impairment in most cases.3,4Therefore, clearer delineation of the tumor during surgery may improve the quantity of radical resections, thus increasing patients survival rates while maintaining postoperative functionality. Near-infrared (NIR) fluorescence imaging is usually a novel imaging technique that provides the doctor with real-time visualization of tumors during surgery.57In the NIR region (650 800 nm), less absorption Tilfrinib of light by tissue components allows much deeper penetration of light. Furthermore, lower fluorescence from endogenous Tilfrinib fluorophores decreases the nonspecific background transmission. As the human eye is not sensitive to NIR fluorescent light, a specific NIR fluorescence imaging system is needed to visualize the fluorescence transmission. At the same time, since NIR light is usually invisible to the human eye, it will not alter the surgical field.8 One of the main challenges in intraoperative fluorescence imaging lies in the development, validation and clinical introduction of a tumor specific agent. Being widely overexpressed in OSCC, the epidermal growth factor receptor (EGFR) serves as an interesting target for intraoperative fluorescence imaging.9,10The EGFR is a transmembrane glycoprotein that is involved in DNA synthesis and cell proliferation. Overexpression contributes to Tilfrinib oncogenesis by proliferation, dedifferentiation, inhibition of apoptosis, invasiveness and lack of adhesion dependence.11Furthermore, EGFR overexpression is often associated with a poor prognosis for patients with OSCC.12 For tumor targeting using molecular imaging techniques, antibodies are promising as they can be raised specifically against practically any molecular target. Nevertheless, due to their large hydrodynamic diameter, intact antibodies accumulate in the liver. Moreover, long half-life in the bloodstream and slow blood clearance via the liver results in high contrast images only several days after injection. A very appealing alternative is the use of nanobodies.13Nanobodies are the smallest functional antigen-binding fragments Tilfrinib derived from naturally-occurring heavy-chain only antibodies.14They show very specific binding to their targets and their size of approximately 15 kDa ensures efficient distribution and tissue penetration, as well as rapid clearance from the body.1517 This study assesses the feasibility of intraoperative fluorescence delineation of orthotopic OSCC and microscopic lymph node metastases, using an anti-EGFR nanobody and a clinically available fluorescence camera system. For this, the IL6R anti-EGFR nanobody 7D12 was conjugated to the NIR fluorophore IRDye800CW.18, as previously described by Oliveira et al.17. Tumor accumulation and specificity of 7D12-800CW was compared to the unfavorable control nanobody R2-800CW and to 800CW alone. == MATERIAL and METHODS == == Cell lines.