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，并研究治疗性 儿童高级别胶质瘤（PHGG）是儿童中枢神经系统中最具侵袭性的肿瘤， 肿瘤和儿童癌症死亡率的最常见原因。PHGG存活率低于5%， 亚型弥漫性中线胶质瘤和20%半球组蛋白3-野生型（H3-wt）PHGG。PHGG高度 侵袭性的并且通常在正常细胞中扩散生长，限制了手术作为治疗选择。辐射 放疗（RT）是短暂有效的，但肿瘤几乎总是复发。尽管进行了数百次临床试验， 化疗在PHGG中显示出明确的生存益处。有效的PHGG治疗至关重要 needed. PHGG可能起源于干细胞样肿瘤起始细胞（PIC）。PHGG肿瘤包括几种 不同的神经胶质细胞来源的细胞类型，比例不同。这种肿瘤的异质性使得理解 PHGG肿瘤生物学和设计疗法。目的1将研究PHGG中每种不同的细胞类型 有助于小鼠模型中的整体肿瘤发生。单细胞RNA-Seq（scRNA-Seq）分析 原位患者来源的PHGG异种移植物（PDX）将用于定义存在的细胞类型并鉴定 差异调节的致癌途径驱动它们的生长。通路表达将被敲除 通过使用稳定的慢病毒转导，用shRNA靶向关键效应基因。对肿瘤生长的影响将 使用存活率、组织学和单细胞RNA-Seq进行评估。 目标2将进行谱系追踪，以确定是否一个单一的PIC细胞类型产生所有的 包括PHGG的增殖细胞类型。将在小鼠PDX模型中进行谱系追踪。单身- 将对PDX肿瘤进行细胞基因组DNA测序。突变特征由单个和 多个核苷酸变异以及拷贝数变异将用于定义每种细胞类型。保守 细胞类型之间的突变模式将用于确定细胞类型之间的层次关系。 一旦确定谱系关系，将在PDX模型中研究对RT的抵抗力。RT是 最一致有效的治疗PHGG，但工作只是暂时细胞再生之前。RT 细胞类型的抗性将基于细胞类型相对于对照的差异存活来确定， RT.将对耐药细胞类型进行药物筛选，以识别放射增敏剂。候选 药物将与RT组合以研究它们在增加RT效应的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.