Engineered fibrillar matrices to study directed cell migration

工程纤维基质用于研究定向细胞迁移

• 批准号: 8256033
• 负责人: Brendon M Baker
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8256033
• 关键词: Adhesions; Architecture; Basement membrane; Behavior; Biocompatible Materials; Blood Vessels; Cancerous; Carcinoma; Cell model; Cells; Cellular Morphology; Cellular biology; Collagen; Collagen Fibril; Disease; Engineering; Environment; Epithelial Cells; Event; Experimental Models; Extracellular Matrix; Fiber; Fibrin; Fibroblasts; Fibrosis; Focal Adhesions; Gel; Glass; Goals; Imaging Techniques; Investigation; Lead; Life; Light; Lymphatic vessel; Malignant Neoplasms; Measures; Mediating; Mediator of activation protein; Melanoma Cell; Microscopy; Molecular; Molecular Biology; Monomeric GTP-Binding Proteins; Movement; Myosin ATPase; Neoplasm Metastasis; Physiological; Population; Primary Neoplasm; Process; Proliferating; Regulation; Role; Secondary to; Signal Pathway; Signal Transduction; Site; Surface; System; Time; Tissues; Traction; Tumor Cell Invasion; Work; body system; cancer cell; cell behavior; cell motility; cellular imaging; directional cell; inhibitor/antagonist; interstitial; killings; migration; mutant; neoplastic cell; novel; novel strategies; polarized cell; prevent; rho; therapeutic target; tumor

DESCRIPTION (provided by applicant): In the progression of a carcinoma, transformed epithelial cells proliferate uncontrollably, eventually breaching through the basement membrane upon which they encounter the fibrillar ECM of the collagen-rich stroma. These cells then migrate through the stroma until they reach blood or lymphatic vessels and intravasate, thereby gaining access to other organ systems. The alignment of collagen fibrils within the tumor stroma is known to promote tumor cell invasion, allowing cells to migrate in a directionally persistent fashion towards neighboring vasculature. The small GTPases Rho and Rac are important players in interactions between the cell and its environment. Of note, Rac signaling is known to be a key mediator of directionally persistent cell migration. Although the specific population of cells responsible for fibrillar reorganization in the stroma is not known, it is likely that cell-generated force via Rho/ROCK driven contractility is a key player in this process. However, much of what is understood about the role of Rac and Rho in cell migration and force transduction has been derived from 2D flat surfaces which fail to recapitulate the 3D fibrillar microenvironment of the tumor stroma. Thus, the focus of the proposed work is to develop a synthetic fibrillar extracellular matrix to study the dynamic function of Rho/Rac signaling in a more physiological context. Importantly, this approach will allow for the independent control of structural features of the fibrillar microenvironment, which currently is not possible using collagen or fibrin gels. The following aims are proposed: Specific Aim 1: Characterize the effect of fibrillar matrix architecture on Rac activity and persistent cell migration. Cell morphology, adhesion, and migratory behavior on fibrillar networks varying in degrees of alignment (from completely isotropic to highly aligned) will be measured with time-lapse microscopy. Rac activity will be quantified and migration will be studied in the presence of Rac inhibitors or constitutively active mutants. Specific Aim 2: Determine if Rho-mediated fibrillar network reorganization is necessary for directional cell migration. Cell clusters will be placed on nonaligned (isotropic) fibrillar networks and fiber reorganization will be examined. Inhibitors of Rho and its downstream effectors will be introduced, as well as constitutively active mutants. Cell migration resulting from Rho-induced alignment will be quantified. Cell migration through fibrillar matrix is relevant to many disease settings, but given the special relevance to fibrosis and metastasis, we will use fibroblasts and melanoma cells as our experimental models for these studies. These investigations will rely heavily on biomaterial engineering, molecular biology, and live- cell imaging approaches in order to understand the interplay between the physical surroundings of the cell, intracellular signaling activities, and resulting cell migratory behavior. The proposed work will not only shed light on signaling mechanisms governing cell migration through extracellular matrix, but will also establish a new approach for devising matrices for the study of fundamental questions in cell biology. PUBLIC HEALTH RELEVANCE: Tumor cell metastasis, the movement of cancerous cells from the primary tumor to secondary sites throughout the body, is the predominant mechanism by which cancer kills. In order to reach blood and lymphatic vessels and gain access to other organ systems, these malignant cells must migrate through fibril- rich interstitial tissues. While tumor cell migration has been widely studied on glass surfaces, considerably less is known about the mechanisms which tumor cells use to migrate through fibrillar tissues. Therefore, the focus of the proposed work is to develop experimental approaches to study cell migration through fibrillar tissues, with the long term goal of identifying key signaling pathways that could become therapeutic targets that prevent metastasis and confine tumor cells to their primary site.

描述(申请人提供)：在癌症的发展过程中，转化的上皮细胞无法控制地增殖，最终突破基底膜，在基底膜上遇到富含胶原的间质的纤维状细胞外基质。然后这些细胞通过基质迁移，直到它们到达血管或淋巴管和血管内，从而获得进入其他器官系统的途径。众所周知，肿瘤间质中胶原纤维的排列可以促进肿瘤细胞的侵袭，使细胞以定向持续的方式向邻近的血管系统迁移。小分子GTP酶Rho和Rac在细胞与环境的相互作用中起着重要作用。值得注意的是，RAC信号被认为是定向持久细胞迁移的关键中介。尽管负责基质纤维重组的特定细胞群尚不清楚，但通过Rho/ROCK驱动的收缩作用产生的细胞力量很可能是这一过程中的关键角色。然而，关于Rac和Rho在细胞迁移和力转导中的作用的了解大多来自2D平面，无法概括肿瘤间质的3D纤维微环境。因此，建议的工作重点是开发一种合成的纤维细胞外基质，以在更生理的背景下研究Rho/Rac信号的动态功能。重要的是，这种方法将允许独立控制纤维微环境的结构特征，这是目前使用胶原蛋白或纤维蛋白凝胶无法实现的。提出了以下目标：特定目标1：表征纤维基质结构对RAC活性和持续细胞迁移的影响。细胞形态、黏附和在不同排列程度(从完全各向同性到高度排列)的纤维网络上的迁移行为将用时间推移显微镜测量。RAC活性将被量化，迁移将在RAC抑制剂或结构性活性突变体存在的情况下进行研究。具体目标2：确定Rho介导的纤维网络重组是否对细胞定向迁移是必要的。细胞团将被放置在非排列(各向同性)的纤维网络上，并将检查纤维重组。将介绍Rho及其下游效应物的抑制剂，以及结构性活性突变体。Rho诱导的比对导致的细胞迁移将被量化。细胞通过纤维基质的迁移与许多疾病相关，但考虑到与纤维化和转移的特殊相关性，我们将使用成纤维细胞和黑色素瘤细胞作为这些研究的实验模型。这些研究将在很大程度上依赖于生物材料工程、分子生物学和活细胞成像方法，以了解细胞物理环境、细胞内信号活动和由此产生的细胞迁移行为之间的相互作用。这项拟议的工作不仅将阐明调控细胞通过细胞外基质迁移的信号机制，而且还将建立一种新的方法来设计用于研究细胞生物学基本问题的矩阵。 公共卫生相关性：肿瘤细胞转移，即癌细胞从原发肿瘤转移到全身次级部位，是癌症致死的主要机制。为了到达血管和淋巴管并进入其他器官系统，这些恶性细胞必须通过富含纤维的间质组织迁移。虽然肿瘤细胞在玻璃表面的迁移已被广泛研究，但对肿瘤细胞在纤维组织中迁移的机制知之甚少。因此，这项拟议工作的重点是开发实验方法来研究细胞在纤维组织中的迁移，长期目标是识别关键的信号通路，这些通路可能成为防止转移并将肿瘤细胞限制在其原发部位的治疗靶点。