Molecular analysis of physical microenvironmental control of tumor cell invasion

描述(申请人提供)：肿瘤侵袭和转移受到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控。虽然细胞外基质硬度对细胞迁移、黏附和收缩的影响在二维(2D)培养中已经被广泛研究，但事实证明，将这些概念扩展到大多数组织所特有的三维(3D)微环境具有极大的挑战性，因为通常用于改变ECM硬度的操作(例如，基质和交联剂密度的变化)经常同时改变基质孔大小(限制)，这可以产生空间位阻，独立于力学因素调节侵袭速度。为了应对这一挑战，我们开发了一种新型的矩阵平台，该平台基于定义的壁刚度和几何形状的通道的微制造，允许ECM刚度和通道宽度的正交变化。我们已经使用这个平台来表征细胞外基质僵硬和限制对胶质母细胞瘤细胞侵袭的调节，这导致我们发现，由于牵引力的增强，僵硬、狭窄的毛孔最大限度地促进了细胞的侵袭。正如这一发现和其他新发现所证明的那样，这个平台在实验方面提供了两全其美的效果 2D和3D细胞迁移范例，因为它保留了前者的吞吐量、标准化和筛查能力，同时捕获了后者的关键生物物理调节元件。在IMAT R21奖项的支持下，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)开发一种封闭式微流控设备，用于通过定义的几何和刚性通道定向迁移肿瘤细胞；(2)利用该平台筛选小分子文库，以僵硬和约束依赖的方式减缓迁移；以及(3)通过比较蛋白质组学分析，将原代胶质母细胞瘤肿瘤起始细胞的侵袭速度与基因表达联系起来。拟议的研究将解决对能够快速识别药物和基因的平台的未得到满足的需求，这些药物和基因是肿瘤侵袭的物理微环境控制的基础。我们的工作是首次系统地研究细胞外基质硬度和孔大小(限制)在调节肿瘤细胞三维侵袭中的作用，并将高通量分子筛选方法应用于细胞外基质机械生物学中的一个问题。通过将机械生物学、肿瘤干细胞生物学、微流体学和蛋白质组学相结合，我们的工作将创造一种有价值的新发现工具，很可能 为临床肿瘤学打开了新的重大翻译机会。

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.