Dissect governing factors for tumor stem cell dormancy in biomimetic vascular/GBM model

剖析仿生血管/GBM模型中肿瘤干细胞休眠的控制因素

• 批准号: 9751425
• 负责人: Guohao Dai
• 金额: $ 62.47万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751425
• 关键词: 3-Dimensional; Address; Arteries; Biomedical Engineering; Biomimetics; Blood Vessels; Brain Neoplasms; Cell Line; Cell Proliferation; Cells; Cerebrovascular system; Clinical; DNA; DNA Damage; DNA Repair Gene; DNA Repair Pathway; Data; Doxycycline; Drug Screening; Engineering; Environment; Exhibits; Extracellular Matrix; Gene Expression Profiling; Gene-Modified; Genes; Genetic Engineering; Glioblastoma; Glioma; Goals; Growth; Heterogeneity; Hypoxia; Individual; Infiltrative Growth; Kinetics; Label; Link; Location; Malignant - descriptor; Malignant neoplasm of brain; Metabolic; Modeling; Modification; Molecular; Mus; Nature; Operative Surgical Procedures; Organoids; Oxygen; Patients; Perfusion; Pharmaceutical Preparations; Physiologic pulse; Physiological; Pilot Projects; Plant Roots; Population; Positioning Attribute; Prodrugs; Proliferating; Radiation; Radiation therapy; Recording of previous events; Recurrence; Regulation; Relapse; Reporter; Resistance; Resolution; SCID Mice; Specific qualifier value; Speed; Stem cells; Sum; Target Populations; Testing; Time; Transplantation; Tumor Stem Cells; Up-Regulation; Veins; Xenograft procedure; base; bioprinting; cancer stem cell; conventional therapy; crosslink; enhancing factor; experimental study; gene function; in vivo; innovation; interdisciplinary approach; neoplastic cell; novel; novel therapeutics; nutrition; outcome forecast; public health relevance; radiation resistance; real-time images; stem cell population; stem-like cell; stressor; study population; synergism; targeted treatment; transcriptome; tumor; tumor microenvironment

SUMMARY Glioblastoma (GBM), the most common and malignant type of brain tumor, has a dismal prognosis due to recurrence despite treatment. Tumor cell dormancy (quiescence) is a major root for tumor relapse, as conventional therapies target mostly proliferating cells. Moreover, quiescent cells harbor a privileged subpopulation of stem-like cells in special niches, which can be reawakened to spawn tumor re- propagation. Dissecting determining factors for tumor dormancy remains challenging due to difficulty to track this population. We have developed a doxycycline-inducible Histone2B-GFP quiescence reporter. In pulse- chase paradigms, quiescent tumor cells retain H2B-GFP label, while proliferative cells sequentially dilute GFP. Our pilot studies in intracranial GBM transplants showed that quiescent cells preferentially reside in close proximity to vasculature. This suggests that perivascular niche may promote glioma stem cell (GSC) dormancy. To test this hypothesis, we take a multidisciplinary approach to develop a bioengineered vascular GBM organoid model. Patient-derived glioma stem cells (GSC) are used to generate GBM organoids, which are then embedded in a multi-scale vascular network with perfusion. Our pilot data demonstrated infiltrative growth of GSC along vasculature, recapitulating a key clinical feature of GBM. In Aim 1, we will analyze the H2B-GFPhigh vs. H2B-GFPlow populations in our organoid model to define the link between perivascular niches and GSC dormancy. Based on our pilot transcriptome profiling that showed upregulation of matrix modifier genes in dormant cells, we will test top candidates to determine their function in promoting GSC dormancy. Parallel in vivo transplant experiments will be carried out in SCID mice using identical GSC lines to verify the biomimetic nature of our model. In Aim 2, we will take advantage of the unique features of our vascular model that allow regulation of perfusion speed and oxygen tension to test the hypothesis that a metabolic stressor such as hypoxia promotes GSC dormancy. To reveal metabolic heterogeneity of GBM cells, we will deploy dual reporters to simultaneously detect hypoxia (RFP) and quiescence (H2B-GFP) to address whether perivascular niches confer a hypoxic microenvironment to dormant GSC. We will also test in our model the efficacy of a novel pro-drug (TH-302), which is activated by hypoxia and crosslinks DNA, to target hypoxic dormant GSC. Our preliminary study showed compelling evidence of the link between GSC quiescence and radiation (XRT) resistance. In Aim 3, we will test if perivascular niche and hypoxia further enhance XRT- resistance of quiescent GSC. Mechanistically, we will determine if DNA repair pathways are activated by hypoxia in dormant GSC. Lastly, we will test potential synergy of the hypoxia-activated drug together with XRT in killing dormant GBM cells in hypoxic state. In sum, our proposal introduces a novel 3D vascular GBM model with perfusion to step-wise interrogate governing factors that enhance GSC dormancy. Our biomimetic model provides a powerful platform for testing gene function and novel drugs to target GBM dormancy.

总结 胶质母细胞瘤（GBM）是最常见的恶性脑肿瘤类型，其预后很差， 尽管治疗复发。肿瘤细胞休眠（静止）是肿瘤复发的主要根源， 常规疗法主要靶向增殖细胞。此外，静止的细胞拥有一个特权， 在特殊的壁龛中，干细胞样细胞亚群可以被重新唤醒以产生肿瘤， 传播由于难以追踪，因此解剖肿瘤休眠的决定因素仍然具有挑战性 这个人口。我们已经开发了一种强力霉素诱导的组蛋白2B-GFP静止报告基因。在脉搏上- 在chase范例中，静止的肿瘤细胞保留H2 B-GFP标记，而增殖细胞依次稀释GFP。 我们在颅内GBM移植中的初步研究表明，静止细胞优先驻留在近 接近脉管系统。这表明血管周围小生境可能促进胶质瘤干细胞（GSC） 休眠为了验证这一假设，我们采取多学科的方法来开发生物工程血管， GBM类器官模型。患者来源的神经胶质瘤干细胞（GSC）用于产生GBM类器官， 然后嵌入到多尺度血管网络中进行灌注。我们的飞行员数据显示 GSC沿着脉管系统生长，概括了GBM的关键临床特征。在目标1中，我们将分析 我们的类器官模型中的H2 B-GFP高与H2 B-GFP低群体，以定义血管周围小生境之间的联系 和GSC休眠基于我们的试验性转录组分析，显示基质修饰剂的上调 在休眠细胞中的基因，我们将测试最佳候选人，以确定他们的功能，促进GSC休眠。 将使用相同的GSC系在SCID小鼠中进行平行的体内移植实验，以验证GSC的表达。 我们模型的仿生性质。在目标2中，我们将利用血管模型的独特功能 可以调节灌注速度和氧分压，以检验代谢应激源 如低氧促进GSC休眠。为了揭示GBM细胞的代谢异质性，我们将部署 双报告基因，同时检测缺氧（RFP）和静止（H2 B-GFP），以解决是否 血管周围的小生境为休眠的GSC提供了缺氧的微环境。我们还将在模型中测试 缺氧激活交联DNA的新型前体药物TH-302靶向缺氧的疗效 休眠的GSC我们的初步研究显示了令人信服的证据，证明GSC静止和 抗辐射（XRT）。在目标3中，我们将测试血管周围小生境和缺氧是否进一步增强XRT-1。 静态GSC的电阻。从机制上讲，我们将确定DNA修复途径是否被激活， 休眠GSC缺氧。最后，我们将测试缺氧激活药物与XRT的潜在协同作用 在缺氧状态下杀死休眠的GBM细胞。总之，我们的建议介绍了一种新的三维血管GBM模型 通过灌注逐步询问增强GSC休眠的控制因素。我们的仿生模型 为测试基因功能和靶向GBM休眠的新药提供了强大的平台。