Molecular analysis of physical microenvironmental control of tumor cell invasion

描述（由申请人提供）：肿瘤侵袭和转移受肿瘤细胞和细胞外基质（ECM）之间的生物物理相互作用的强烈调节。虽然已经在二维（2D）培养中广泛研究了ECM刚度对细胞迁移、粘附和收缩性的影响，但是考虑到通常用于改变ECM刚度的操作（例如，基质和交联密度的变化）经常同时改变基质孔径（限制），这可以产生独立于力学调节侵入速度的空间位阻。为了解决这一挑战，我们已经开发了一种新的矩阵平台的基础上定义的壁刚度和几何形状，允许ECM刚度和通道宽度的正交变化的通道的微细加工。我们已经使用这个平台来表征ECM刚度和限制对胶质母细胞瘤细胞侵袭的调节，这使我们发现，由于牵引力的增强极化，刚性窄孔使细胞侵袭最大化。正如这个和其他新的发现所证明的那样，这个平台在实验方面提供了两全其美的方法。 2D和3D细胞迁移范例，因为它保留了前者的通量，标准化和筛选能力，同时捕获后者的关键生物物理调节元件。在IMAT R21奖的支持下，我们现在建议将该平台开发为用于高通量分子筛选和分析的微流控技术。我们将围绕三个具体目标组织我们的研究：（1）开发一种封闭的微流体装置，用于肿瘤细胞通过限定几何形状和刚度的通道定向迁移;（2）使用该平台筛选小分子文库，以刚度和限制依赖的方式减缓迁移;（3）通过比较蛋白质组学分析，探讨胶质母细胞瘤原发细胞侵袭速度与基因表达的关系。拟议的研究将解决对能够快速识别肿瘤侵袭的物理微环境控制基础药物和基因的平台的未满足需求。我们是第一个系统的努力，研究ECM刚度和孔径（限制）在调节肿瘤细胞的三维侵袭的作用，并应用高通量分子筛选方法在细胞ECM机械生物学的问题之一。通过整合机械生物学、肿瘤干细胞生物学、微流体学和蛋白质组学，我们的工作将创造一种有价值的新发现工具， 为临床肿瘤学开辟了重要的新的转化机会。

DESCRIPTION (provided by applicant): Tumor invasion and metastasis are strongly regulated by biophysical interactions between tumor cells and the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility has been extensively studied in two-dimensional (2D) culture, extension of these concepts to three- dimensional (3D) microenvironments characteristic of most tissues has proven extremely challenging given that manipulations normally used to vary ECM stiffness (e.g., variation of matrix and crosslink density) often concurrently alter matrix pore size (confinement), which can create steric barriers that regulate invasion speed independently of mechanics. To address this challenge, we have developed a novel matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows orthogonal variation of ECM stiffness and channel width. We have used this platform to characterize the regulation of glioblastoma cell invasion by ECM stiffness and confinement, which has led us to discover that stiff, narrow pores maximize cell invasion as a consequence of enhanced polarization of traction forces. As evidenced by this and other novel findings, this platform offers the best of both worlds with respect to experimental 2D and 3D cell migration paradigms, in that it retains the throughput, standardization, and screening power of the former while capturing key biophysical regulatory elements of the latter. With the support of this IMAT R21 award, we now propose to develop this platform as a microfluidic technology for high-throughput molecular screening and analysis. We will organize our research around three specific aims: (1) To develop an enclosed microfluidic device for the directed migration of tumor cells through channels of defined geometry and stiffness; (2) To use the platform to screen small molecule libraries for agents that slow migration in a stiffness- and confinement-dependent fashion; and (3) To relate invasion speed to gene expression in primary glioblastoma tumor initiating cells through comparative proteomic analysis. The proposed studies will address an unmet need for platforms capable of rapidly identifying drugs and genes that underlie physical microenvironmental control of tumor invasion. Ours is one of the first systematic efforts to study the roles of ECM stiffness and pore size (confinement) in regulating tumor cell invasion in 3D and to apply high-throughput molecular screening approaches to a problem in cell-ECM mechanobiology. By integrating mechanobiology, tumor stem cell biology, microfluidics, and proteomics, our work will create a valuable new discovery tool that is likely to open significant new translational opportunities for clinical oncology.