Glycocalyx and NCOR2-Notch-Mediated Stemness in Glioblastoma Aggressiveness

描述(申请人提供)：高级别多形性胶质母细胞瘤(GBM)患者平均生存期为6-12个月，这是由于放化疗相对无效和弥漫性侵犯健康脑组织所致。尽管任何GBM亚型的确切起源仍不确定，但已有合理的共识认为，最具侵袭性的GBM来自干细胞，或至少来自多能细胞。到目前为止，绝大多数的GBM研究集中在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其生物物理特征。这里提出的工作旨在描述使基底膜细胞具有侵袭性的细胞固有的生物物理特征。这项跨学科工作的成功完成有可能提供一种新的范式，用来阐明提高基底膜的存活率、侵袭性和治疗耐药性的基础。目的：拟议工作的长期目标是通过将物理学家的概念与基础和临床癌症生物学研究相结合，揭示更有效的基底膜治疗方式，延长患者的生存时间，改善基底膜患者的生活质量，以探索细胞内在力量在基底膜侵袭中的作用。具体地说，由于GBM的侵略性被归因于它们的干细胞样特性，该提案测试了这样的想法，即茎和抗死亡行为是通过细胞相关碳水化合物层糖萼的变化引起的独特的细胞内在机械表型来调节的，并询问了其上调的分子机制，该机制被假设通过NCoR2和Notch发挥作用。方法概述：拟议的工作测试了Notch和NCoR2信号之间的功能联系，并阐明了它们的联合作用是否协同推动高水平糖蛋白在细胞表面沉积，进而通过生物物理手段驱动整合素和Notch的激活，以改善生存、侵袭和治疗耐药性。将使用牵引力、扫描角干涉、TIRF、二次谐波产生、旋转圆盘和激光共聚焦显微镜等技术来表征WT人GBM细胞以及修饰NCoR2或Notch的细胞的生物物理特性。为了在体外探究维持这些干细胞特性的机制，将使用GBM细胞的基因工程和生物材料工程策略。将使用人GBM的原位异种移植小鼠模型在体内测试靶向NCoR2-Notch-Glycoalyx电路的有效性。

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.