Engineered fibrillar matrices to study directed cell migration

描述（由申请人提供）：在癌的进展中，转化上皮细胞不受控制地增殖，最终冲破基底膜，在基底膜上遇到富含胶原基质的纤维状ECM。然后，这些细胞通过基质迁移，直到到达血液或淋巴管并进入血管内，从而进入其他器官系统。已知肿瘤基质内胶原原纤维的排列促进肿瘤细胞的侵袭，允许细胞以定向持续的方式向邻近的脉管系统迁移。小的gtp酶Rho和Rac在细胞与其环境的相互作用中起着重要的作用。值得注意的是，已知Rac信号是定向持续细胞迁移的关键介质。虽然在基质中负责原纤维重组的特定细胞群尚不清楚，但通过Rho/ROCK驱动的细胞产生的力很可能在这一过程中起关键作用。然而，我们对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，这些平面无法再现肿瘤基质的3D纤维微环境。因此，本研究的重点是开发一种合成纤维细胞外基质，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，目前使用胶原蛋白或纤维蛋白凝胶是不可能的。具体目标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、粘附和纤维网络上不同程度排列（从完全各向同性到高度排列）的迁移行为将用延时显微镜测量。Rac活性将被量化，迁移将在Rac抑制剂或本构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定向细胞迁移是必要的。细胞团将被放置在不连续的（各向同性）纤维网络和纤维重组将被检查。将介绍Rho抑制剂及其下游效应物，以及本构活性突变体。由rho诱导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，但鉴于其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，以了解细胞的物理环境、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。这项工作不仅将揭示细胞通过细胞外基质迁移的信号机制，而且还将为设计用于研究细胞生物学基本问题的基质建立新的方法。

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.