SUMMARY Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the United States for which surgery remains a critical treatment option. The appropriate extent of resection for NSCLC is highly dependent on intra-operative decision-making. Specifically, the diagnosis of NSCLC is often determined intra-operatively by a limited resection of a suspicious pulmonary nodule, and intraoperative diagnosis is required before proceeding to a larger, cancer-specific resection during the same procedure. Further, intraoperative evaluation of resection margins is required in NSCLC surgeries to assure complete cancer removal before leaving the operating room (OR). Thus, the ability to accurately diagnose NSCLC and determine surgical margins in the OR is critical in the care for patients with NSCLC. However, the current technology used for intraoperative evaluation of NSCLC diagnosis and margins is frozen section analysis, a century-old technique that is labor-intensive and prone to error. Frozen section analysis also extends operative time, subjecting patients to increased risks of surgical complications. New technologies that can provide surgeons with the ability to rapidly and accurately diagnose NSCLC in the OR and evaluate surgical margins with high accuracy are critically needed to improve surgical care and outcomes for patients. We have reported the development of an innovative technology, the MasSpec Pen (MSPen), for rapid and non-destructive diagnosis of cancer tissues. The MSPen deploys a single droplet of water to gently extract molecules from tissues, which are then analyzed by mass spectrometry (MS) and statistical classifiers to provide diagnosis in seconds. We have demonstrated that the MSPen can accurately diagnose NSCLC using banked tissues with 97% accuracy. We have also shown that the MSPen system can be translated to the OR for use by surgeons for rapid in vivo and ex vivo tissue analysis. Through a collaborative partnership between academic scientists and surgeons at Baylor College of Medicine and the R&D team at MSPen Technologies Inc., we now propose to refine and validate the MSPen for intraoperative diagnosis and surgical margin evaluation in NSCLC. Our objective is to optimize and translate new MSPen technology for NSCLC detection, test its functionality, and validate its performance in the OR setting, thus delivering a potentially improved platform for intraoperative lung tissue analysis to surgeons - the end users. Our specific aims are: Aim 1. Optimize the MSPen Technology for intraoperative use in NSCLC operations. We will develop a next-gen MSPen platform for use in open and robotic NSCLC surgeries; Aim 2. Validate the MSPen for NSCLC diagnosis. We will conduct a prospective observational study allowing thoracic surgeons to validate the technology for NSCLC diagnosis in the OR; Aim 3. Validate the MSPen for intraoperative surgical margin evaluation in NSCLC. We will validate the MSPen technology for surgic...