Protease Activity in 3D Matrices

3D 矩阵中的蛋白酶活性

• 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
• 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

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：细胞迁移和侵入是许多生理过程中的关键步骤（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）。这些运动相关细胞功能的关键组分是细胞外基质（ECM）蛋白的蛋白水解，以去除物理屏障以及调节关键信号传导组分。虽然公认蛋白水解在细胞迁移期间是重要的，但难以直接检查和量化时空调节， 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，以表征蛋白酶在三维环境中的活性，并研究在黑色素瘤进展过程中细胞外环境的变化如何影响这种活性。具体而言，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水解活性，2）研究基质细胞如何影响黑色素瘤细胞的蛋白水解和迁移。黑色素瘤细胞系的进展将在3D水凝胶中培养，其中掺入荧光酶敏感肽作为侧官能团，以测量三种酶类别的蛋白水解：基质金属蛋白酶、组织蛋白酶和uPA。通过单独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全局蛋白酶活性的作用。最后，利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，这种表征应该推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，并导致对蛋白酶靶向更有效的癌症治疗的新见解。