Molecular events that differentiate cancerous tissues from normal, healthy tissues may be used as targets of pharmacologic interventions and diagnostic imaging. Protease activity is particularly suited for detection as the activity of the protein, not just the expression. For example, members of the matrix metalloproteinase family, which include collagenases and gelatinases, have higher activity in aggressive, metastatic cancers than benign tumors. Thus, methods for measuring protease activity in living systems would provide diagnostic and prognostic information. .Optical imaging is an excellent method for low cost, high-throughput preclinical imaging using safe and stable contrast agents. Rather than radioactive contrast agents, optical imaging uses fluorescent compounds that can be visualized throughout the body for days to weeks after injection with no evident toxicity. Multiple agents, injected separately or in the form of activatable agents can be detected throughout the body for days to weeks after injection. In the proposed work, fluorescent contrast agents will be investigated that target cancer-specific cell-surface receptors and extracellular enzymes. .Cancer therapy induces a negative effect on tumor biology with variable magnitude. This effect is generally measured by the change in tumor size and the prolongation of patient survival. These changes can take weeks to months to observe using the available imaging capabilities. Efforts are under way to shorten this delay by evaluating biomarkers such as changes in cellularity, microvascular density using MRI and metabolism using nuclear imaging. We will investigate the use of optical molecular imaging agents for detection of therapy-induced changes in cancer at the molecular level. Protease-activatable fluorescent molecular probes and optical imaging techniques will be used to measure the expression of cancer-specific enzymes before and after therapy. The goal is to develop quantitative metrics to predict the long-term response of cancer based on early changes in molecular expression and activity.