Glycocalyx and NCOR2-Notch-Mediated Stemness in Glioblastoma Aggressiveness

描述（由申请人提供）：由于放疗和化疗的相对无效以及弥漫性侵入健康脑组织，高级别多形性胶质母细胞瘤（GBM）患者的平均生存时间为6-12个月。虽然任何GBM亚型的确切起源“顶细胞”仍然不确定，但有合理的共识认为，最具侵略性的GBM来自干细胞，或至少来自多能细胞。迄今为止，绝大多数GBM研究都集中在GBM细胞独特的遗传和生化信号成分上，很少或根本没有关注生物物理特征。本文提出的工作旨在表征GBM细胞的细胞内在生物物理特征，使其具有攻击性。这项跨学科工作的成功完成有可能提供一个新的范例，以澄清增强生存，侵袭性和治疗抗性的GBM的基础。目的：长期目标是揭示更有效的GBM治疗模式，延长患者生存期，并通过将物理科学家的概念与基础和临床癌症生物学研究相结合来提高GBM患者的生活质量，以探索细胞内在力量在GBM侵袭中的作用。具体而言，由于GBM的侵袭性归因于其干细胞样特性，因此该提案测试了干细胞性和抗死亡行为是通过改变的糖萼（细胞相关碳水化合物层）引起的独特细胞内在机械表型介导的想法，并询问了负责其上调的分子机制，假设其通过NCoR 2和Notch起作用。方法概述：拟议的工作测试Notch和NCoR 2信号传导之间的功能联系，并阐明它们的联合作用是否协同驱动高水平糖蛋白在细胞表面的沉积，这反过来又可以通过生物物理手段驱动整合素和Notch激活，以改变存活、侵袭和治疗抗性。牵引力、扫描角干涉、TIRF、二次谐波发生、旋转圆盘和激光共聚焦显微镜等技术将用于表征WT人GBM细胞以及具有修饰的NCoR 2或Notch的细胞的生物物理特性。为了探究在体外维持这些干细胞样特性的机制，将使用GBM细胞的基因工程和生物材料工程策略。将采用人GBM的原位异种移植小鼠模型来测试体内靶向NCoR 2-Notch-糖萼回路的有效性。

DESCRIPTION (provided by applicant): Patients with high grade glioblastoma multiforme (GBM) have a mean survival time of 6-12 months due to the relative ineffectiveness of radio and chemotherapy and diffuse invasion into healthy brain tissue. Although the exact "apex cell" of origin for any GBM subtype remains uncertain, there is reasonable consensus that the most aggressive GBMs arise from stem cells, or at the very least from pluripotent cells. Overwhelming majority of GBM research to date has focused on GBM cell's unique genetic and biochemical signaling components with little or no focus on the biophysical features. Work proposed here aims to characterize the cell-intrinsic biophysical features of GBM cells that render them aggressive. Successful completion of this interdisciplinary work has the potential to offer a new paradigm with which to clarify the basis for enhanced survival, invasiveness, and treatment resistance of GBMs. Objectives: The long term goal of proposed work is to reveal more effective GBM treatment modalities, extend patient survival, and improve the quality of life for GBM patients by integrating physical scientist's concepts with the basic and clinical cancer biology research in order to explore the role of cell-intrinsic force in GBM aggression. Specifically, since aggressive nature of GBMs has been attributed to their stem-like properties, the proposal tests the idea that stemness and death-resistant behavior is mediated though a unique cell-intrinsic mechano-phenotype due to altered glycocalyx, the cell-associated carbohydrate layer, and interrogates the molecular mechanism responsible for its upregulation, which is hypothesized to act through NCoR2 and Notch. Overview of Approach: Proposed work tests a functional link between Notch and NCoR2 signaling and elucidates whether their combined action synergistically drives deposition of high levels glycoproteins at the cell surface which can in turn drive integrin and notch activation by biophysical means to modify survival, invasion, and treatment resistance. Techniques such as traction force, scanning angle interference, TIRF, second harmonic generation, spinning disc, and laser confocal microscopies will be used to characterize the biophysical properties of WT human GBM cells as well those with modified NCoR2 or Notch. To interrogate the mechanism sustaining these stem-like properties in vitro, genetic engineering of GBM cells and biomaterial engineering strategies will be used. Orthotopic xenograft mouse models of human GBMs will be employed to test the effectiveness of targeting NCoR2-Notch-glycocalyx circuitry in vivo.