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

摘要 胶质母细胞瘤(GBM)是最常见和恶性的脑肿瘤类型，由于 尽管接受了治疗，但复发了。肿瘤细胞休眠(静止)是肿瘤复发的主要根源，AS 传统疗法主要针对增殖细胞。此外，静止的牢房拥有特权 干细胞亚群在特殊的小生境中，可以被重新唤醒以再次滋生肿瘤- 传播。由于追踪困难，剖析肿瘤休眠的决定因素仍然具有挑战性 这群人。我们开发了一种多西环素诱导的组蛋白2B-GFP静息报告。在脉搏中- Chase范式中，静止的肿瘤细胞保留了H2B-GFP标记，而增殖的细胞则依次稀释了GFP。 我们在颅内GBM移植中的初步研究表明，静止的细胞优先居住在近距离。 接近血管系统的。这表明血管周围的壁龛可能促进胶质瘤干细胞(GSC)的生长。 休眠。为了验证这一假设，我们采取了多学科的方法来开发生物工程血管。 GBM类器官模型。患者来源的胶质瘤干细胞(GSC)被用来产生GBM有机类化合物， 然后嵌入一个多尺度的血管网络中进行灌流。我们的试点数据显示 GSC沿着血管系统生长，概括了GBM的一个关键临床特征。在目标1中，我们将分析 我们的器官模型中H_2B-GFP高种群与H_2B-GFP低种群的对比，以确定血管周围生态位之间的联系 和GSC休眠。根据我们的飞行员转录组图谱显示基质修饰物上调 对于休眠细胞中的基因，我们将测试排名靠前的候选基因，以确定它们在促进GSC休眠中的功能。 将使用相同的GSC系在SCID小鼠身上进行平行的体内移植实验，以验证 我们的模型的仿生本质。在目标2中，我们将利用血管模型的独特功能 允许调节灌流速度和氧分压来测试代谢应激源的假设 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 双报告同时检测缺氧(RFP)和静止(H2B-GFP)以解决是否 血管周围的微环境为休眠的GSC提供了一个低氧的微环境。我们还将在我们的模型中测试 低氧和DNA交联激活的新型前药(TH-302)对低氧靶向作用的研究 休眠的GSC。我们的初步研究显示了令人信服的证据表明GSC静止和 耐辐射(XRT)。在目标3中，我们将测试血管周围生态位和缺氧是否进一步增强XRT- 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 休眠状态下GSC的低氧。最后，我们将测试缺氧激活药物与xrt的潜在协同作用。 在低氧状态下杀死休眠的GBM细胞。综上所述，我们的方案引入了一种新的3D血管GBM模型 通过灌流逐步询问增强GSC休眠的控制因素。我们的仿生模型 为检测基因功能和靶向GBM休眠的新药提供了强大的平台。

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.