Pathomimetic Avatars for Live‐Cell Imaging of Tumor Proteolysis and Drug Screening

Abstract

To define protease‐related druggable pathways involved in malignant progression of cancer, our laboratory has pioneered novel techniques for functional live‐cell imaging of protease activity, initially concentrating on pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by breast cells (benign and cancerous) in 3‐D cultures over time (4D). In order to recapitulate the cellular composition and architecture of breast tissue, we include other tumor‐associated cells (e.g., fibroblasts, myoepithelial cells, lymphatic endothelial cells). We also model non‐cellular aspects of the tumor microenvironment, e.g., an acidic pericellular pH. Use of these pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for drug screening, we have designed culture chambers with multiple wells that are either individual or connected by a bridge to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. Covers contain integrated gas exchange ports and sensors to monitor oxygen levels, pH and temperature over the extended time periods in culture and to insure maintenance of such experimental conditions as hypoxia and/or a pericellular acidic pH. We predict that the pathomimetic avatars will accelerate identification of druggable pathways, screening of drug and natural product libraries and the entry of validated drugs or natural products into clinical trials.Support or Funding InformationSupported in part by National Institute of Health R21 CA 131990 (BFS) and the President's Research Enhancement Fund of Wayne State University (BFS and YX). Live‐cell imaging was performed in the Microscopy, Imaging and Cytometry Resources Core of Wayne State University, which was supported in part by National Institutes of Health P30 CA22453 to the Karmanos Cancer Institute and by the Perinatology Research Branch of the National Institute of Child Health and Human Development.