3D/4D Functional Imaging of Tumor-Associated Proteolysis: Impact of Microenvironment

Abstract Proteases play causal roles in many aspects of the aggressive phenotype of tumors, yet many of the implicated proteases originate from tumor-associated cells or from responses of tumor cells to interactions with other cells. Therefore, to obtain a comprehensive view of tumor proteases, we need to be able to assess proteolysis in tumors that are interacting with their microenvironment. As this is difficult to do in vivo , we have developed functional live-cell optical imaging assays and 3D and 4D (i.e., 3D over time) coculture models. We present here a description of the probes used to measure proteolysis and protease activities, the methods used for imaging and analysis of proteolysis and the 3D and 4D models used in our laboratory. Of course, all assays have limitations; however, we suggest that the techniques discussed here will, with attention to their limitations, be useful as a screen for drugs to target the invasive phenotype of tumors.