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: 10240477
• 负责人: Guohao Dai
• 金额: $ 53.41万
• 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/10240477
• 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; Prognosis; Proliferating; Radiation; Radiation therapy; Recording of previous events; Recurrence; Regulation; Relapse; Reporter; Resistance; Resolution; SCID Mice; Specific qualifier value; Speed; 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; public health relevance; radiation resistance; real-time images; stem cell population; stem cells; stem-like cell; stressor; 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.

概括 胶质母细胞瘤（GBM）是最常见和最恶性的脑肿瘤类型，由于 尽管有治疗，但复发。肿瘤细胞休眠（静止）是肿瘤复发的主要根源，如 常规疗法主要针对增殖细胞。此外，静态细胞具有特权 特殊壁ches中干细胞的亚群，可以重新唤醒肿瘤的产卵肿瘤 传播。由于难以追踪，剖析肿瘤休眠因素仍然具有挑战性 这个人口。我们已经开发了可耐二霉素诱导的组蛋白2b-GFP静止报告基因。在脉冲中 - 追逐范式，静止的肿瘤细胞保留H2B-GFP标签，而增殖细胞顺序稀释GFP。 我们在颅内GBM移植中的试点研究表明，静态细胞优先驻留在近距离 接近脉管系统。这表明周围的壁re可以促进神经胶质瘤干细胞（GSC） 休眠。为了检验这一假设，我们采用多学科的方法来开发生物工程的血管 GBM类器官模型。患者衍生的神经胶质瘤干细胞（GSC）用于产生GBM器官，该器官 然后将其嵌入具有灌注的多尺度血管网络中。我们的飞行员数据显示出渗透性 GSC沿脉管系统的生长，概括了GBM的关键临床特征。在AIM 1中，我们将分析 H2B-GFPHIGH与我们的器官模型中的H2B-GFPLOW种群，以定义周围壁细分市场之间的联系 和GSC休眠。基于我们的飞行员转录组分析，显示了矩阵修饰符的上调 休眠细胞中的基因，我们将测试顶级候选者，以确定其在促进GSC休眠方面的功能。 可以使用相同的GSC线在SCID小鼠中进行平行的体内移植实验，以验证 我们模型的仿生本质。在AIM 2中，我们将利用血管模型的独特功能 这允许调节灌注速度和氧张力，以检验代谢应激源的假设 例如缺氧会促进GSC休眠。为了揭示GBM细胞的代谢异质性，我们将部署 双重记者同时检测缺氧（RFP）和静止（H2B-GFP），以解决是否可以解决 血管周期壁ches赋予休眠GSC的低氧微环境。我们还将在我们的模型中测试 通过缺氧和交叉链接DNA激活的新型pro-proug（TH-302）的功效，以靶向低氧 休眠GSC。我们的初步研究表明，GSC静止与 辐射（XRT）电阻。在AIM 3中，我们将测试血管周期和缺氧是否进一步增强了XRT- 静态GSC的阻力。从机械上讲，我们将确定DNA修复途径是否被激活 休眠GSC中缺氧。最后，我们将与XRT一起测试缺氧激活药物的潜在协同作用 在杀死缺氧状态下休眠的GBM细胞时。总而言之，我们的建议介绍了一种新颖的3D血管GBM模型 通过灌注，逐步审问治疗因素，增强了GSC休眠。我们的仿生模型 提供了一个强大的平台，用于测试基因功能和新型药物以靶向GBM休眠状态。