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Protease Activity in 3D Matrices

3D 矩阵中的蛋白酶活性

基本信息

批准号：
8684387
负责人：
KRISTI S. ANSETH
金额：
$ 18.37万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8684387
关键词：
Address Affect Atherosclerosis Biological Assay Biological Models Blood Vessels Cathepsins Cell Communication Cell Culture Techniques Cell Line Cell physiology Cells Cellular biology Cleaved cell Coculture Techniques Collaborations Complex Cultured Cells Dimensions Disease Elasticity Embryonic Development Encapsulated Engineering Environment Enzymes Ethylene Glycols Exposure to Extracellular Matrix Extracellular Matrix Proteins Fibroblasts Goals Growth Homeostasis Hydrogels Image In Situ Knowledge Lead Letters Libraries Life Malignant Neoplasms Matrix Metalloproteinases Measurement Measures Mechanics Mediating Melanoma Cell Metastatic Melanoma Metastatic to Methods Microscopy Monitor Neoplasm Metastasis Peptide Hydrolases Peptides Physiological Processes Primary Neoplasm Proteolysis Radial Regulation Regulation of Proteolysis Reporter Research Role Secondary to Signal Transduction Site Specialist Staging Stromal Cells System Techniques Technology Time Tissues Urokinase Work Wound Healing base cancer therapy cell motility cell type ethylene glycol experience extracellular functional group in vitro Model in vivo insight melanoma migration neoplastic cell new growth protein aminoacid sequence public health relevance scaffold spatiotemporal time use tool tumor tumor progression

项目摘要

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.

描述：细胞迁移和侵袭是许多生理过程（例如胚胎发生、组织稳态、伤口愈合）和疾病状态（例如癌症、动脉粥样硬化）中的关键步骤。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) 蛋白的蛋白水解，以消除物理障碍并调节关键信号传导成分。虽然人们普遍认为蛋白水解在细胞迁移过程中很重要，但很难直接检查和量化时空调节和蛋白水解基质重塑的协调。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。具体而言，拟议的研究计划旨在：1) 开发可调节的 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水解活性；2) 检查基质细胞如何影响黑色素瘤细胞的蛋白水解和迁移。一系列黑色素瘤细胞系将在 3D 水凝胶中培养，其中荧光酶敏感肽作为悬垂功能基团并入，以测量三种酶类的蛋白水解：基质金属蛋白酶、组织蛋白酶和 uPA。将通过单个或成簇培养细胞来研究微环境对蛋白水解的调节，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水解的影响。总的来说，这种表征应该促进对基质重塑和细胞迁移协调的基本理解，提供一种允许对局部蛋白酶活性进行空间表征的新方法，并导致对哪些蛋白酶针对更有效的癌症治疗有新的见解。