Investigation of tumor stem cell maintenance and cellular hierarchy in pediatric high-grade glioma

项目摘要 该项目的总体目标是确定重要的生物学特征并研究治疗方法 儿童最具侵袭性的中枢神经系统--儿童高级别胶质瘤(PHGG) 肿瘤是儿童癌症死亡的最常见原因。PHGG存活率不到5% 亚型弥漫性中线胶质瘤和20%的半球组蛋白3-野生型(H3-wt)PHGG。PHGG是非常高的 侵袭性的，通常在正常细胞中扩散生长，限制了手术作为一种治疗选择。辐射 治疗(RT)是暂时有效的，但肿瘤几乎总是复发。尽管进行了数百次临床试验，但没有 化疗已经显示出对PHGG患者的明确的生存益处。有效的PHGG疗法至关重要 需要的。 PHGG可能起源于干细胞样的肿瘤起始细胞(PIC)。PHGG肿瘤由几个 不同类型的胶质细胞起源，比例不同。这种肿瘤的异质性使我们很难理解 PHGG肿瘤生物学和设计治疗方法。目标1将研究PHGG中每种不同的细胞类型是如何 对小鼠模型的整体肿瘤形成有贡献。单细胞RNA-Seq(scRNA-Seq)分析 原位患者来源的PHGG异种移植(PDX)将用于定义存在的细胞类型和识别 驱动它们生长的不同调控的致癌途径。途径的表达将被打倒 通过使用稳定的慢病毒转导以shRNA靶向关键效应基因。对肿瘤生长的影响将 通过存活、组织学和单细胞RNA序列进行评估。 AIM 2将执行谱系追踪，以确定单个PIC细胞类型是否产生所有 组成PHGG的增殖细胞类型。血统追踪将在小鼠PDX模型中执行。单人- 将对PDX肿瘤进行细胞基因组DNA测序。突变签名由单个和 多核苷酸变异以及拷贝数变异将被用来定义每种细胞类型。保守的 细胞类型之间的突变模式将被用来确定细胞类型之间的等级关系。 一旦确定了谱系关系，将在PDX模型中研究对RT的抗性。RT是 大多数针对PHGG的治疗一直有效，但仅在细胞再生之前暂时有效。RT 细胞类型的抗性将根据细胞类型与对照组的差异存活来确定 RT.将对耐药细胞类型进行药物筛选，以确定辐射敏感剂。候选人 药物将与RT联合使用，以研究它们在延长RT效应持续时间方面的有效性。

Project Summary The project’s overall goal is to determine important biological characteristics and investigate therapeutic options for pediatric high-grade gliomas (PHGG), the most aggressive of childhood central nervous system tumors and most common cause of childhood cancer mortality. PHGG survival rates are less than 5% for the subtype diffuse midline glioma and 20% for hemispheric histone 3-wild type (H3-wt) PHGG. PHGG are highly invasive and often grow diffusely among normal cells, limiting surgery as a therapeutic option. Radiation therapy (RT) is transiently effective, but the tumors nearly always recur. Despite hundreds of clinical trials, no chemotherapy has shown a definitive survival benefit in PHGG. Effective PHGG therapies are critically needed. PHGG likely originates from stemlike tumor initiating cells (PICs). PHGG tumors comprise several distinct cell types of glial origin, in varying proportions. This tumor heterogeneity complicates understanding PHGG tumor biology and designing therapies. Aim 1 will investigate how each distinct cell type in PHGG contributes to overall tumorigenesis in a mouse model. Single-cell RNA-Seq (scRNA-Seq) analysis of orthotopic patient derived PHGG xenografts (PDX) will be used to define the cell types present and identify differentially regulated oncogenic pathways that drive their growth. Pathway expression will be knocked down by targeting key effector genes with shRNA using stable lentiviral transduction. The effect on tumor growth will be evaluated using survival, histology and single-cell RNA-Seq. Aim 2 will perform lineage tracing to determine whether a single PIC cell type produces all of the proliferating cell types that comprise PHGG. Lineage tracing will be performed in a mouse PDX model. Single- cell genomic DNA sequencing will be performed on PDX tumors. Mutational signatures consisting of single and multiple nucleotide variations as well as copy number variation will be used to define each cell type. Conserved patterns of mutation among cell types will be used to determine the hierarchical relationships among cell types. Once the lineage relationships are worked out, resistance to RT will be studied in the PDX model. RT is the most consistently effective therapy against PHGG but works only temporarily before cells regrow. RT resistance by cell type will be determined based upon differential survival of cell types versus control following RT. Drug screening of resistant cell types to identify radiation sensitizers will be performed. The candidate drugs will be combined with RT to investigate their effectiveness at increasing the duration of the RT effect.