The purpose of the project is to develop and test, in preclinical models, a sensitive, specific imaging peptide probe to distinguish GBM tissue from healthy tissue. The novel imaging probe targets a cell-surface marker, matrix metalloproteinase14 (MMP-14) that is expressed to a much greater degree in GBM cells than in healthy cells. Upon binding to MMP-14, a dual modality fluorescent tag is activated. This tag is detectable by positron emission tomography (PET) for pre-operative tumor assessment, and by near infrared fluorescence (NIRF) imaging for real-time surgical guidance in distinguishing tumor cells from healthy cells. Results from the project indicate that GBM cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptide probes. Resected mouse brains with patient derived xenograft (PDX) GBM tumors showed tumor-to-background NIRF ratios of 7.6-11.1 at 4 h after i.v. injection of the peptides. PET images showed localization of activity in orthotopic PDX tumors after i.v. injection of radiolabeled peptide probes; uptake of the radiolabeled probes in tumors was significantly reduced (p<0.05) by blocking with the non-labeled peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors.