Detection and monitoring of tumor boundaries are vital for the effective treatment of cancer and for improving our understanding of cancer progression. Recent studies by SRI International's (SRI's) collaborators have identified an enzyme, tissue transglutaminase (tTG) that demarcates the border between normal and injured/malignant tissue. SRI hypothesizes that the activity of this enzyme can be exploited to outline tumors using optical detection. To test this hypothesis, SRI proposes an R21 project with the following specific aims: (1) Modify optical instrumentation for in vitro assays and in vivo imaging using optically labeled tTG substrates. We will modify a fluorescence microscope and in vivo imaging system design to use appropriate excitation and emission wavelengths for the labeled substrates. (2) Synthesize and evaluate plasma protein-based tTG substrates with optical labels. Substrates acted on by tTG will be conjugated with cyanine dyes suitable for near-infrared in vivo imaging. Labeled substrate will be evaluated in an in vitro assay on an endothelial cell line. (3) Test activity on labeled substrates in vivo using infrared imaging. This imaging will be performed on a wound-healing model, a substrate clearance model, and a tumor model in rats. In the longer term, we plan to use labeled tTG substrates to answer basic questions regarding tumor progression and to develop a methodology for clinical demarcation of tumor boundaries suitable for use as an intraoperative diagnostic during biopsy or tumor removal. The technique would also be of value for imaging thrombi.