Deconstructing Directional Cell Motility in Metastasis thru Substratum nanopatter

这项建议的目标是开发抗转移治疗药物。为了实现这一目标，我们 提出一种新的、多学科的方法，将纳米技术与分子和细胞生物学相结合。 其基本原理是解构作为侵袭性基础的定向细胞运动机制 控制单元格的几何形状。该方法的前提是对单元几何的控制将允许我们引出 典型的结构和功能细胞表型，具有明确的运动周期阶段的特征。 基本相同的“设计单元”阵列将为探测复杂的方向提供一个独特的平台 并建立高含量、可翻译的筛选程序。 我们将首先开发纳米技术来控制几何形状，从而控制运动机械 细胞。将开发与活细胞成像和电子显微镜兼容的技术。 新技术将包括湿冲压(WET)，以产生透明的粘接岛，受控 蚀刻(GET)从微图案生成纳米图案，并行阵列浸渍笔光刻(DPN) 直写纳米功能。基于这项技术，我们将生成图案化的基板，包括 被卫星纳米斑点包围的圆形、三角形、泪滴和多边形解构方向 动力机制。我们将集中在焦点粘连(FA)-微管(MT)相互作用和焦点粘连 周转；细胞极化和片脂突起；丝状体形成和MT相互作用；以及 膜蛋白水解酶的募集。基因沉默与基因芯片高含量筛选相结合 相同的“设计者”细胞将导致识别对定向至关重要的分子靶点和途径 能动性。这些靶点和途径将作为筛选识别小分子的基础 转移抑制因子。

The goal of this proposal is the development of anti-metastasis therapeutics. To achieve this goal, we propose a novel, multidisciplinary approach that combines nano-technology with molecular and cell biology. The rationale is to deconstruct the directional cell motility mechanisms that underlie invasiveness through control of cell geometry. The premise of the approach is that control of cell geometry will allow us to elicit stereotypical structural and functional cell phenotypes characteristic of defined phases of the motility cycle. Arrays of essentially identical "designer cells" will provide a unique platform for probing complex directional motility mechanisms and for establishing high-content, translational screening procedures. We will first develop the nano-technology to control the geometry and, therefore, the motility machinery of cells. Technologies will be developed that are compatible with live cell imaging and electron microscopy. Novel technologies will include wet stamping (WETS) to create transparent adhesive islands, controlled etching (GET) to generate nano-patterns from micro-patterns and parallel array dip pen lithography (DPN) to direct write nano-features. Based on this technology, we will then generate patterned substrata, including circles, triangles, tear-drops and polygons surrounded by satellite nano-spots to deconstruct directional motility mechanisms. We will focus on focal adhesion (FA)-microtubule (MT) interaction and focal adhesion turnover; cell polarization and lamellipodial protrusion; filopodial formation and MT interaction; and membrane protease recruitment. Gene silencing in combination with high-content screening of arrays of identical, "designer" cells will lead to identification of molecular targets and pathways crucial for directional motility. These targets and pathways will then serve as the basis for screens to identify small molecule inhibitors of metastasis.