A mu Migration Assay for Temporal Protein Localization

描述(申请人提供)：人脑胶质瘤侵袭邻近的正常脑组织，使手术治疗这种疾病变得不可能。两种不同的小鼠胶质瘤模型显示了两种不同的邻近组织的迁移和侵袭模式：PDGF诱导的少突胶质细胞瘤作为单个细胞侵袭大脑，而RAS驱动的胶质瘤通过沿着血管追踪侵袭。这项翻译的生物工程计划将通过开发和实施一种新的纳米微流控分析来研究这些观察结果，该方法通过实时显示细胞运动和检测信号蛋白质组分的空间和时间活性来检查脑瘤细胞迁移的机制。 我们的项目将开发定制的纳米探针来结合PDGF受体的胞外和胞内结构域，以可视化其在运动性胶质瘤实时迁移过程中的空间分布和时间位置。此外，我们的实验将通过使用我们的微流控迁移分析将可控的一维配体梯度施加到单个细胞上，来确定PDGFR结构域相对于迁移方向的亚细胞定位。 拟议的方法是独特的和高风险的，因为它是第一个开发能够测量由受控的细胞外环境诱导的细胞内蛋白质易位的分析工具。我们建议的方法的成功开发将使未来的研究能够通过细胞内可视化关键蛋白从受体到RAS来研究细胞迁移。这些开创性的结果将阐明肿瘤细胞扩散过程中细胞内蛋白质行为的前所未有的细节。

DESCRIPTION (provided by applicant): Human gliomas aggressively invade the adjacent normal brain tissue to render surgical treatments for this disease impossible. Two different mouse models of glioma demonstrate two distinct patterns of migration and invasion of adjacent tissues: PDGF-induced oligodendrogliomas invade the brain as individual cells, while Ras-driven gliomas invade by tracking along blood vessels. This translational, bioengineering proposal will investigate these observations by developing and implementing a novel, nano-microfluidic assay to examine the mechanisms of brain tumor cell migration via real-time visualization of cell movement, and detection of spatial and temporal activity of signaling protein components. Our project will develop custom nanoprobes to bind, both, the extracellular and intracellular domains of the PDGF receptor in order to visualize its spatial distribution and temporal location during real-time migration in motile glioma. Further, our experiments will determine the subcellular localization of PDGFR domains relative to the direction of migration by using our microfluidic migration assay to impose controllable, one-dimensional ligand gradients onto individual cells. The proposed methodology is unique and high risk because it is the first to develop analytical tools able to measure translocations of intracellular proteins induced by a controlled, extracellular environment. Successful development of our proposed approach will enable future research to investigate cell migration via intracellular visualization of key proteins from receptors to Ras. Such ground-breaking results will elucidate unprecedented details of intracellular protein behavior during tumor cell dispersal.