Project Summary/Abstract: Breast cancer is the most common cancer in women. For 2021, breastcancer.org projects 281,550 new cases of invasive breast cancer and 49,290 new cases of non-invasive (in situ) breast cancer. 43,600 deaths are also projected. Due to better screening techniques cancers are caught earlier and 75% of patients are candidates for breast conserving surgery (BCS) to remove the cancer. BCS is cosmetically preferable to the alternative (mastectomy) and long-term survival rates are equivalent. Postoperative pathology, the current “gold standard” has significant drawbacks, including the requirement for the patient to return for additional surgery if pathology finds an incomplete resection (i.e., cancer remains in the patient). In addition, current pathology methods only assess about 1/10 of 1% of the entire volume of the removed specimen. A consequence of margin undersampling is that local recurrence occurs in 5-16% of patients with pathologically clean margins, suggesting that one or more regions of tumor had not been sampled during pathological analysis resulting in tumor remaining in the patient. New methods to enhance the quality of BCS resection intraoperatively are being developed. One of the most promising is the use of fluorescent molecular probes to “light up” cancer and guide resection. All current probes, however, require large doses of imaging agent to be administered hours or days before surgery, and require extra labwork, complicating a relatively simple surgical procedure. Moreover, infiltrating cancer cells in tissues surrounding the main mass may not have developed a vasculature, and our work and that of others indicates that it is possible such tumors may not be identified using injected agents, which use such vasculature to reach the diseased tissue. Exploiting increased protease expression at the edge of breast cancers we introduce the novel concept of intraoperative in vivo topical administration of an activatable, microdose, quenched, fluorescent molecular imaging probe to identify cancer that may remain in the surgical cavity after standard- of-care resection. Building on years of preclinical ex-vivo studies, this Phase I project is anchored by a large-animal surgical study to refine and optimize the surgical workflow and technique prior to beginning formal FDA clinical trials. We will synthesize research grade probe, test its performance in PDX rats, complete a surgical optimization project in canines, and submit an IND to FDA. This technology has the potential for an abbreviated regulatory path, offering the potential to reduce re-excisions as well as the false negative rate from pathology undersampling, with a consequent savings in healthcare costs and, enhancement in patient life quality.