Project Summary Cancer continues to affect broad segments of the population, with over 600,000 deaths expected from malignancies this year in the United States.[1] Complete surgical resection, as compared to chemotherapy or radiation alone, offers nearly a nearly tenfold increase in the likelihood of cure for most solid tumors. However, local and systemic recurrence affect 40% of patients undergoing curative-intent resection, due to incomplete disease clearance and positive margins at the index operation.[2] To aid in the completeness of cancer resection—and thus improve patient survival—intraoperative molecular imaging (IMI) has been heralded as an important new tool in surgical oncology. The technology uses fluorescent, tumor-targeted tracers to identify positive margins and preoperatively unrecognized sites of disease. IMI has been applied to thoracic tumors in recent years.[3] However, there is a major unmet need in pulmonary squamous cell carcinoma, which is the second-most common lung cancer subtype and has a particularly low 5 year survival rate at 17.6%.[4] This is a translational proposal with the primary goal of developing and optimizing an optical molecular imaging probe for SCC. We aim to synthesize an EGFR-targeted near infrared (NIR) tracer that is specific to SCC and is biocompatible, using methods established in our lab and by close collaborators. The tracer will first be evaluated in vitro for: 1) receptor binding affinity, 2) nonspecific binding (competitive with excess unlabeled ligand), 3), water solubility, 4) serum protein binding, and 5) photo- and chemical stability. Targeted NIR dye conjugates that perform well in these in vitro analyses will be further evaluated in vivo for: 1) tumor specificity, 2) non- GMP toxicity, 3) biostability, and 4) pharmacokinetics and pharmacodynamics. Following tracer testing, safety and efficacy studies will be performed in a murine model of resection cavity mapping to optimize the timing and dose of the agent. Ultimately, this work will set the stage for human trials of this technology in pulmonary squamous cell carcinoma. We hope that the combined development of innovative contrast agents coupled with advanced intraoperative instrumentation will make a major impact in reducing the local and regional recurrence rates of squamous cell lung cancer after surgery. As a secondary aim, this research will serve as a training platform for the applicant towards competency in tumor biology, tracer design, mammalian cell techniques, and murine experiments, as well as provide the applicant with mentoring towards the goal of becoming an independent investigator.