Matching panels of in vivo and in vitro model system of pediatric brain tumors

项目摘要 脑瘤是儿童癌症相关死亡的主要原因。对相互作用的理解较差 肿瘤细胞，尤其是肿瘤干细胞(CSCs)与正常脑组织之间起到了一定的作用。由于 获得肿瘤组织的困难，只有有限的生物学上准确和匹配的 体外(肿瘤有机物)和体内(PDOX)模型系统。正常的脑组织更难获得，使 发育正常的大脑器官几乎是不可能的。为了克服这些障碍，我们建议利用我们的 作为来源来源的现有(n=127)和未来已建立的患者来源异种移植(PDOX)模型 以形成相匹配的肿瘤器官。这些PDOX模型复制了原始患者的生物学 它代表了新发现的各种恶性脑肿瘤的分子亚型。 使用24个PDOX模型，我们建立了13个(54.1%)肿瘤有机体。认识到正常的稀缺性 脑组织，我们建议使用尸检组织。事实上，我们有冷冻保存的正常可存活细胞 取自不同年龄儿童的大脑、小脑、脑干和脑室下区细胞，并显示 身体解剖的脑细胞可以在体外增殖形成神经球。因此，我们假设1) 尸检的正常脑组织和PDOX模型可以提供急需的和生物学上准确的细胞 对于正常和肿瘤类器官的发育；2)这些类器官的生长依赖于 生长因子的位置和环境受不同类型细胞之间的动态相互作用的调节。 放射治疗是儿童脑肿瘤最重要的治疗方案之一。自基因突变以来 组蛋白修饰基因在儿童脑肿瘤中很常见，我们的第三个假设是这种表观遗传学 改变影响对辐射的反应和抵抗力，并影响有机物和PDOX的靶向成功 肿瘤将导致新的联合疗法。我们提出了三个具体目标。目标1：发育正常大脑 所有年龄段的正常大脑、小脑、脑干和脑室下区的有机化合物 并从PDOX模型中建立肿瘤有机化合物并确定其位置和发育阶段 依赖的生态位因子组合。目标2：了解肿瘤之间的动态相互作用 CSCs和非干细胞肿瘤)细胞和正常脑细胞。我们将使用共培养试验来确定利基 决定CSC和正常脑细胞的细胞多样性和存活率的因子组合，并执行全局 多组学分析和单细胞RNAseq阐明分子机制，随后在体内 在PDOX模型中验证我们的发现。目的3：确定组蛋白甲基化在调节CSC中的作用 恶性脑肿瘤在受到辐射干扰时的存活和自我更新。我们将使用正常组织和肿瘤组织 有机化合物以确定先前存在的组蛋白甲基化状态如何影响其存活率并确定关键 耐辐射甲基化(S)，通过将辐射与一组告密性小分子探针相结合 在有机体和PDOX模型中，选择性地针对肿瘤表观遗传调控回路中的不同节点。

Project Summary Brain tumors are the leading cause of cancer-related death in children. Poor understanding of the interactions between tumor cells, especially cancer stem cells (CSCs), and normal brain tissues have played a role. Due to the difficulties of obtaining tumor tissues, there is a limited availability of biologically accurate and matched in vitro (tumor organoid) and in vivo (PDOX) model systems. Normal brain tissue is more difficult to obtain, making it nearly impossible to develop normal brain organoids. To overcome these barriers, we propose to utilize our existing (n=127) and future established patient derived orthotopic xenograft (PDOX) models as the source supply of tumor cells to develop matching tumor organoids. These PDOX models replicate the biology of original patient tumors and represent a wide spectrum of the newly discovered molecular subtypes of malignant brain tumors. Using 24 PDOX models, we have established 13 (54.1%) tumor organoids. Recognizing the rarity of normal brain tissues, we propose to use autopsied tissues. Indeed, we have cryopreserved viable cells of normal cerebrum, cerebellum, brain stem, and sub-ventricular zone cells from children of different ages and showed that the autopsied cerebral cells can proliferate in vitro to form neurospheres. We, therefore, hypothesize that 1) autopsied normal brain tissues and PDOX models can provide critically needed and biologically accurate cells for the development of normal and tumor organoid, and 2) the growth of these organoid are dependent on the growth factor milieu of their location and modulated by the dynamic interactions between different types of cells. Radiation therapy is one of the most important treatment regimens for pediatric brain tumors. Since mutation of histone modification genes are frequent in pediatric brain tumors, our 3rd hypothesis is that such epigenetic changes affect responses and resistance toward radiation, and successful targeting in organoid and PDOX tumors will lead to new combination therapies. We propose three Specific Aims. Aim 1: Develop normal brain organoids from autopsied normal cerebrum, cerebellum, brain stem and subventricle zone from all age groups and establish tumoral organoids from PDOX models and identify their location- and developmental-stage dependent niche factor combinations, respectively. Aim 2: Understand the dynamic interactions between tumor (CSCs and non-stem tumor) cells and normal brain cells. We will use co-culture assays to identify the niche factor combinations that dictate cellular diversity and survival of CSC and normal brain cells, and perform global multi-omics analysis and single cell RNAseq to elucidate the molecular mechanisms, followed by in vivo validation of our findings in PDOX models. Aim 3: Determine the role of histone methylation in regulating CSC survival and self-renewal of malignant brain tumors when perturbed by radiation. We will use normal and tumoral organoids to determine how pre-existing histone methylation status affect their survival and to identify key radiation-resistant methylation(s) by combining radiation with an informer set of small-molecule probes that selectively target distinct nodes in tumor epigenetic regulatory circuity in organoids and in PDOX models.