Pre-clinical studies have established that: 1) proteases of several catalytic-types play causal roles in malignant progression, and 2) the tumor microenvironment (including the tumor site, the composition of the adjacent stroma, infiltration of inflammatory cells, angiogenesis, etc.) affects the expression and activity of proteases and protease inhibitors. Interactions among proteases that result in activation of latent precursor forms or degradation of inhibitors also have been identified. Nonetheless, we do not have a well-delineated picture of which catalytic-types of proteases or individual proteases are appropriate targets for therapies to reduce tumor proteolysis and tumor-associated proteolysis in situ. We propose that studying 'tumor proteolysis' via a confocal assay developed for functional imaging of degradation of quenched-fluorescent (DQ) extracellular matrix proteins by live cells will provide answers. Using this assay, we have demonstrated that stromal cells, in this case fibroblasts alone or in combination with inflammatory macrophages, enhance proteolysis of DQ-collagen IV in live 3D multi-cellular cultures of human colon or breast tumor cells. Cell-based 3D assays have been used to demonstrate that the stroma regulates development of mammary epithelium and its progression of carcinoma. In this proposal, our specific aims are to: 1) optimize cell-based 3D colon and breast tumor models for functional imaging of proteolysis of ECM proteins in 4D (3D + time); 2) use the cell-based 4D models to identify by functional imaging the proteases in live colon and breast tumor cells capable of degrading ECM proteins; 3) use the cell-based 4D models to determine the contribution of live stromal cells to that proteolysis; and, 4) test whether cell-based 4D models can be used as in vitro screens for strategies to reduce the proteolysis of ECM proteins. Our eventual goal is to test alternative strategies for reducing tumor proteolysis, e.g., targeting more than one catalytic-type of protease, targeting stromal as well as tumor proteases, and targeting proteases at times to be defined during progression.