Invasion of glioblastoma multiforme (GBM) cells in organotypic brain slices

器官型脑切片中多形性胶质母细胞瘤 (GBM) 细胞的侵袭

• 批准号: 8596234
• 负责人: Zev Ari Binder
• 金额: $ 4.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2014-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8596234
• 关键词: Adult; Aftercare; Antibodies; Binding; Brain; CD44 gene; Cell Density; Cell Line; Cells; Characteristics; Cilengitide; Clinical; Collagen; Enzyme-Linked Immunosorbent Assay; Excision; Extracellular Structure; Fluorescence; Glioblastoma; Human; Hyaluronic Acid; In Vitro; Individual; Integrins; Invaded; Life; Measurement; Mediating; Mesenchymal; Modeling; Molecular; Movement; Nature; Neurons; Operative Surgical Procedures; Patients; Phase II Clinical Trials; Phase III Clinical Trials; Plastics; Primary Brain Neoplasms; Property; Radiation therapy; Recurrence; Research; Resolution; Role; Slice; Staining method; Stains; Techniques; Time; Traction; Travel; Western Blotting; Work; brain tissue; cell motility; chemotherapy; in vivo; inhibitor/antagonist; knock-down; matrigel; migration; neglect; neoplastic cell; public health relevance; small hairpin RNA; tumor

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most common adult primary brain tumor. The average survival with surgery, radiotherapy, and chemotherapy is 14.6 months and is often marked by tumor recurrence after treatment. Tumor recurrence is caused by tumor cells invading through the normal brain tissue, escaping surgical resection. Current in vitro research in GBM invasion focuses on cell lines grown on plastic and in artificial collagen/matrigel matrices, and in vivo work is often studied using static, fix-and-stain techniques. These approaches either oversimplify the composition of brain tissues or neglect a critical character of GBM cells, i.e. their dynamic invasion. We propose to develop and use a new live-cell platform that will allow us to study the invasion of primary GBM cells at single-cell resolution, in live human brain tissue slices. This dynamic platform will allow us to establish the mode of migration of GBMs during invasion (mesenchymal vs. ameboid), the type of cellular protrusions generated by GBMs to invade, and the role of integrins and CD44 using shRNA-induced knockdown. Additionally, this model provides a mechanistic view of the effect on GBM invasion of cilengitide, a specific ?v?3 integrin inhibitor currently in phase III trials for GBM. Specific Aim 1. To quantify tumor single-cell invasion in brain organotypics, time-lapse fluorescence, differential interference contrast (DIC), and reflection confocal videos will be collected simultaneously to determine baseline movements and invasion characteristics of individual control GBM cells. Measurements include total distance traveled, average cell velocity, and persistence time and distance, as well as protrusion activity and dynamics, intercellular distance (time-dependent tumor cell density in the slice), and cell-induced traction forces on their surrounding microenvironment. Additional information relating tumor cell movement to extracellular structures such as vasculature and neurons will be obtained. Specific Aim 2. To determine the role of integrins and CD44 in the invasion of GBM cells in brain organotypics, we will perform knockdown and inhibition studies. Expression of integrins ?2, ?3, ?5, ?6?1, ?v?3, and ?v?5 and CD44 in GBM tumor cells will be determined using ELISA, PCR, and Western blotting techniques. Then, selective knockdown of expressed integrins ?2, ?3, ?5, ?6?1, ?v?3, and ?v?5, in the GBM tumor cells will be used to quantify changes in the cell invasion. Knocking down one integrin at a time will allow invasion changes to be attributed to the knocked down integrin. Changes in cell motility, protrusion dynamics, microenvironment deformations, and cell density of GBM cells in organotypic brain slices following treatment with cilengitide, a specific ?v?3 integrin inhibitor in phase III trial for GBMs, will be quantified and compared to control cells. The effect on cell motility, protrusion dynamics, microenvironment deformations, and cell density of GBM cells will be determined in the setting of CD44 knockdown. Finally, antibody-mediated inhibition of CD44 binding to hyaluronic acid will be used to determine the effect on cell motility, protrusion dynamics, microenvironment deformations, and cell density of GBM cells in organotypic brain slices.

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是最常见的成人原发脑肿瘤。手术、放疗和化疗的平均生存期为14.6个月，治疗后肿瘤经常复发。肿瘤复发是由于肿瘤细胞侵入正常脑组织，逃避手术切除所致。目前对GBM侵袭的体外研究主要集中在塑料和人造胶原/基质基质上生长的细胞系，而体内工作通常使用静态、固定和染色技术进行研究。这些方法要么过分简化了脑组织的组成，要么忽视了基底膜细胞的一个关键特征，即它们的动态侵袭。我们建议开发和使用一种新的活细胞平台，使我们能够研究原代GBM细胞在单细胞中的侵袭 分辨率，在活的人脑组织切片中。这个动态平台将使我们能够建立 基底膜在侵袭过程中的迁移方式(间充质与变形体)，基底膜侵袭时产生的细胞突起的类型，以及整合素和CD44在shRNA诱导的敲除中的作用。此外，该模型还提供了Cilengiide对GBM侵袭的影响的机制视图，Cilengiide是一种特异性的V3整合素抑制剂，目前正处于GBM的第三阶段试验。具体目的1.为了定量研究肿瘤单细胞在脑器官中的侵袭行为，将同时采集延时荧光、差分干涉对比(DIC)和反射共聚焦视频，以确定单个对照GBM细胞的基线运动和侵袭特征。测量包括总行程、平均细胞速度、持续时间和距离，以及突起活动和动态、细胞间距离(切片中肿瘤细胞密度随时间变化)以及细胞对周围微环境的牵引力。将获得与肿瘤细胞运动与细胞外结构(如血管和神经元)相关的更多信息。具体目的2.为了确定整合素和CD44在脑器质性GBM细胞侵袭中的作用，我们将进行基因敲除和抑制研究。采用酶联免疫吸附试验、聚合酶链式反应和免疫印迹技术检测整合素β2、β3、β5、β6 1、βV 3、βV 5和CD44在GBM肿瘤细胞中的表达。然后，选择性地敲除GBM肿瘤细胞中表达的整合素？2、？3、？5、？6？1、？V？3和？V？5，以量化细胞侵袭的变化。一次敲除一个整合素将允许侵袭性的变化归因于被击倒的整合素。器官型脑片中的GBM细胞在用cilengitide治疗后，细胞运动性、突起动力学、微环境变形和细胞密度的变化将被量化，cilengiide是一种特异性的？v？3整合素抑制剂，用于GBM的III期试验，并 与对照细胞相比。CD44基因敲除对GBM细胞的运动性、突起动力学、微环境变形和细胞密度的影响将在CD44基因敲除的背景下确定。最后，抗体介导的CD44与透明质酸结合的抑制将被用来确定对器官型脑片中GBM细胞的细胞运动性、突起动力学、微环境变形和细胞密度的影响。