Designing technologies to visualize protease activity in cancer models

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PROJECT SUMMARY Cancer is a collection of diseases that are caused by the uncontrolled proliferation of cells. It is the second leading cause of death in the United States of America and approximately 40% of Americans will get diagnosed with cancer at some point in their life. Proteases, which are enzymes that cleave peptide bonds, play vital roles in many steps in cancer progression, including migration and metastasis. Drugs that target protease activity have entered clinical trials, however they were all unsuccessful. New anti-protease therapies are being developed with improved enzyme specificity to reduce unwanted side-effects. Thus increasing our understanding of protease activity in the tumor microenvironment is needed to bring these promising drugs to the clinic. The objective of this project is to develop biomolecular conjugates which can visualize protease activity within model tissues in order to understand how cancerous cells modulate the activity of other local cells. These conjugates combine nucleic acids and peptide such that a protease cleaving the peptide activates the nucleic acid for binding. This will be used quantify protease activity around individual cell types. Since nucleic acid hybridization is sequence-specific, multiple protease can be studied simultaneously within a single hydrogel. The conjugates will be coupled to the hydrogel using “click” chemistry. This enables the molecules to be added during cell culture in order to study protease activity at specified time points. These conjugates have been designed so that they can be made on automated synthesizers, and can be easily incorporated into most biomaterials platforms. This research plan includes two specific aims that were designed to develop protease-responsive conjugates, incorporate them into hydrogels, and visualize spatiotemporal protease activity in a model tissue. The first aim will develop nucleic acid-peptide conjugates which are able to bind fluorophore-labelled complementary strand only after they been “activated” by proteases. The second aim will incorporate these conjugates into hydrogels for simultaneous spatiotemporal visualization proteases in a hydrogels containing both cancerous and noncancerous cells in order to better understand metastatic processes. We hypothesize that cancer cells cultured with non-cancerous cells will have altered protease activity. This approach is powerful because it can be easily adapted by other labs, can be used for many proteases and incorporated into most biomaterial systems. Since proteases catalyze the cleavage of a peptide bond, they are especially useful for making stimuli-responsive therapies. Thus this research can help researchers across disciplines develop more effective biomedical interventions.