Protease Activity in 3D Matrices

描述：细胞迁移和侵袭是许多生理过程（如胚胎发生、组织稳态、伤口愈合）和疾病状态（如癌症、动脉粥样硬化）的关键步骤。这些与运动相关的细胞功能的一个关键组成部分是细胞外基质（ECM）蛋白的蛋白质水解，以消除物理障碍并调节关键信号成分。虽然人们普遍认为蛋白质水解在细胞迁移过程中很重要，但很难直接检测和量化其时空调节和作用

DESCRIPTION: Cell migration and invasion are critical steps in many physiological processes (e.g., embryogenesis, tissue homeostasis, wound healing) and disease states (e.g., cancer, atherosclerosis). A key component to these motility related cellular functions is proteolysis of extracellular matrix (ECM) proteins to remove physical barriers as well as modulate key signaling components. While it is well accepted that proteolysis is important during cellular migration, it has been difficult to directly examine and quantify the spatiotemporal regulation and coordination of proteolytic matrix remodeling. We aim to develop a materials based fluorescent reporter system to characterize protease activity in three- dimensional environments and study how changes in the extracellular environment influence this activity during melanoma progression. Specifically, the proposed research plans aims to: 1) Develop a tunable 3D culture platform to investigate how extracellular microenvironment regulates melanoma cell proteolytic activity and 2) Examine how stromal cells influence melanoma cell proteolysis and migration. A progression of melanoma cell lines will be cultured in 3D hydrogels with fluorescent enzyme sensitive peptides incorporated as pendant functional groups to measure proteolysis of three enzyme classes: matrix metalloproteinases, cathepsins, and uPA. Regulation of proteolysis by the microenvironment will be investigated by culturing cells singly or in clusters to understand the role of homotypic cell-cell interactions and by varying the elasticity of the hydrogel to elucidate the role of cell-matrix interactions on local and global protease activity. Finally, usin primary fibroblasts isolated from healthy tissue or tumor associated fibroblasts, we will investigate the effect of stromal cell co- culture on melanoma cell migration and proteolysis. Collectively, this characterization should advance the basic understanding of the coordination of matrix remodeling and cell migration, provide a new method that allows spatial characterization of local protease activity, and lead to new insights into which proteases to target for more effective cancer therapies.