Defining the stem cell origins of brain tumours containing non-neural cells

一些中枢神经系统（CNS）肿瘤的一个显著特征是存在表明神经/胶质和中胚层/间充质（如骨骼/平滑肌、软骨、骨）混合分化的细胞类型。这些“神经中胚层”（NM）肿瘤既影响幼儿也影响成人，它们具有高度侵袭性，预后不良。例如，相当一部分胶质瘤，最常见的成人脑肿瘤，表现出骨、软骨和肌肉分化的间充质特征，其中一些含有明显的肌肉肉瘤成分（胶质瘤）。确定驱动这些肿瘤形成的细胞实体对于设计针对这些恶性肿瘤的有效治疗干预措施至关重要。拟议的博士项目旨在研究NM肿瘤产生于干细胞的假设，这些干细胞类似于多能胚胎前体，即神经中胚层祖细胞（NMPs）或神经嵴（NC）前体。在胚胎早期发育过程中，NMPs产生脊髓神经元以及骨、软骨和骨骼/平滑肌，而NC细胞产生外周神经元、黑素细胞和间充质细胞类型，如平滑肌和软骨。我们推测，纳米肿瘤的发生是由突变驱动的NMP/NC祖细胞在胚胎发生过程中转化为癌症起始细胞，或者是“原位重编程”事件的结果，其中神经/胶质细胞内遗传/表观遗传机制的破坏导致它们转化为NMP/NC样细胞，进而在有利的生态位内触发肿瘤发生。研究计划目前建立脑肿瘤细胞系的常规方法旨在选择神经干细胞（NSC）样细胞，这反映了所有脑肿瘤的起源细胞都是转化的神经祖细胞的假设。因此，黏附胶质瘤干细胞系已经成功建立，使用相同的条件用于神经干细胞的繁殖。然而，这种方法不能用于分离类似于NMPs/NC细胞的双能性NM细胞。我们最近确定了从人多能干细胞（hPSCs）体外诱导NMPs和NC祖细胞的最佳培养条件。成人脑肿瘤将被分离，等量的细胞将在NMP、NC和神经干细胞（NSC）条件下平行培养（阳性对照）。成功的附着、增殖和随后在NMP或NC培养基中传代将标志着在起始肿瘤中存在NM双能干细胞。我们已经获得了有希望的初步数据，表明胶质瘤来源的细胞可以在NMP诱导条件下增殖。通过使用免疫荧光/qPCR和体外分化试验监测NMP和NC标记的表达，将证实所得到的细胞系中存在双能性细胞。将进行RNA-seq分析，将NM肿瘤细胞系的转录组与hpsc衍生的NMPs和NC细胞以及已建立的人类NSC细胞系进行比较，以确定致癌转化的候选驱动因素。通过异种移植/立体定向注射评估NOD-SCID小鼠的致瘤潜力也将进行。如果时间允许，我们还将进行小分子筛选，旨在识别对NM肿瘤细胞系产生细胞毒性/细胞抑制作用的化合物。

A striking feature of some central nervous system (CNS) tumours is the presence of cell types indicative of mixed neural/glial and mesodermal/mesenchymal (e.g. skeletal/smooth muscle, cartilage, bone) differentiation. These "neuromesodermal" (NM) tumours affect both young children and adults and they are highly aggressive being linked to a poor prognosis. For example, a considerable fraction of gliomas, the most common adult brain tumours, display mesenchymal features with elements of bone, cartilage and muscle differentiation and some of them contain a distinct muscular sarcomatous component (gliosarcomas). Defining the cell entity driving the formation of these tumours will be pivotal for the design of effective therapeutic interventions against these malignancies. The proposed PhD project aims to examine the hypothesis that NM tumours arise from stem cells which resemble multipotent embryonic precursors, namely neuromesodermal progenitors (NMPs) or neural crest (NC) precursors. During early embryonic development NMPs give rise to spinal cord neurons as well as bone, cartilage and skeletal/smooth muscle whereas NC cells generate peripheral neurons, melanocytes and mesenchymal cell types such as smooth muscle and cartilage. We speculate that NM tumourigenesis is the result of either a mutation-driven conversion of NMPs/NC progenitors into cancer-initiating cells during embryogenesis or a "reprogramming in situ" event where corruption of the genetic/epigenetic machinery within neural/glial cells results into their conversion into NMP/NC-like cells which in turn trigger tumorigenesis within a favorable niche.Research planCurrent conventional methods for establishing brain tumour cell lines are designed to select for neural stem cell (NSC)-like cells reflecting the hypothesis that the cell-of-origin of all brain tumours is a transformed neural progenitor. Thus adherent glioma stem cell lines have been successfully established using the same conditions as those employed for the propagation of neural stem cells. However, this approach cannot be employed for the isolation of bipotent NM cells resembling NMPs/NC cells. We have recently defined the optimal culture conditions for the induction of NMPs and NC progenitors from human pluripotent stem cells (hPSCs) in vitro. Adult brain tumours will be dissociated and equal numbers of the resulting cells will be cultured in parallel in NMP, NC and neural stem cell (NSC) conditions (positive control). Successful attachment, proliferation and subsequent passaging in NMP or NC culture media would mark the presence of NM bipotent stem cells in the starting tumour. We have already obtained promising preliminary data showing that glioma-derived cells can proliferate in NMP induction conditions. The presence of bipotent cells in the resulting cell lines will be confirmed by monitoring the expression of NMP and NC markers using immunofluorescence/qPCR and in vitro differentiation assays. RNA-seq analysis will be carried out comparing the transcriptomes of NM tumour cell lines to hPSC-derived NMPs and NC cells as well as established human NSC lines to define candidate drivers of oncogenic transformation. Assessment of tumourigenic potential by xenografting/stereotactic injection into NOD-SCID mice will also be performed. If time permits we will also carry out small molecule screens aiming to identify compounds exerting cytotoxic/cytostatic effects on the NM tumour cell lines.