Protease Activity in 3D Matrices

描述：细胞迁移和入侵是许多生理过程(如胚胎发育、组织动态平衡、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)蛋白的蛋白分解，以消除物理障碍并调节关键的信号成分。虽然人们普遍认为蛋白质分解在细胞迁移过程中是重要的，但直接检查和量化时空调节和 蛋白水解性基质重塑的协调。我们的目标是开发一种基于材料的荧光报告系统来表征三维环境中的蛋白酶活性，并研究细胞外环境的变化如何在黑色素瘤进展过程中影响这种活性。具体地说，拟议的研究计划旨在：1)开发一个可调的3D培养平台，以研究细胞外微环境如何调节黑色素瘤细胞的蛋白分解活性；2)研究基质细胞如何影响黑色素瘤细胞的蛋白分解和迁移。一系列黑色素瘤细胞株将被培养在含有荧光酶敏感多肽的3D水凝胶中，作为悬挂官能团，以测量三种酶类的蛋白分解：基质金属蛋白酶、组织蛋白和uPA。微环境对蛋白质分解的调节将通过单独或成群培养细胞来研究，以了解同型细胞-细胞相互作用的作用，并通过改变水凝胶的弹性来阐明细胞-基质相互作用对局部和整体蛋白酶活性的作用。最后，使用从健康组织或肿瘤相关成纤维细胞分离的原代成纤维细胞，我们将研究基质细胞共培养对黑色素瘤细胞迁移和蛋白降解的影响。总而言之，这种表征应该会促进对基质重塑和细胞迁移协调的基本理解，提供一种新的方法，允许局部蛋白酶活性的空间表征，并导致对哪些蛋白酶作为更有效的癌症治疗的靶点的新见解。

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.