Background: Local recurrence is a common mode of failure for patients with cancer. Intra-operative detection of microscopic residual disease in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce the rate of re-excision, and tailor adjuvant therapy. Previously, we utilized a primary mouse model of soft tissue sarcoma (STS) to develop a novel imaging system to detect cancer (Cancer. 2012). Because cathepsin proteases are preferentially expressed in tumors compared to normal tissue, we used cathepsin-activatable fluorescent imaging agents with a novel imaging device for intra-operative assessment of residual cancer within tumor beds of mice. We demonstrated that residual fluorescence detected in the tumor bed by the imaging system correlates with local recurrence and that image guided surgery improves outcomes for mice with positive residual fluorescence. LUM015 is a pegylated cathepsin-activated imaging agent containing a Cy5 fluorophore attached to a quencher by a polypeptide linker. Upon cleavage of the linker by cathepsin proteases, the quencher is released, allowing fluorescence to be detected. A phase I clinical trial is open at Duke to test the safety of LUM015 (NCT01626066). Methods: This open-label nonrandomized trial compares up to four dose cohorts of LUM015 (cohorts -1 to 3: 0.25, 0.5, 1 and 1.5 mg/kg) in order to determine a safe and recommended phase II dose of LUM015 that labels tumors in human patients. Subjects with STS or breast cancer receive LUM015 by peripheral intravenous injection 2-72 hours prior to scheduled surgical resection. Safety evaluations prior to and during the 24-hour period after injection consist of vital signs, ECG, PFTs, blood and urine studies and documentation of any adverse pharmacological activity (APA). This evaluation is repeated at 2, 7 and 14 days after study agent delivery and an end of study APA assessment occurs 30-35 days after surgery. Pharmacokinetic parameters are also determined. Correlative studies include quantitative imaging as well as histological and biochemical analyses of the resected tissues. To date, cohorts 1 and 2 (n=9 patients) have been enrolled without APA. Enrollment in cohort 3 began in January 2014. Clinical trial information: NCT01626066.