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神经/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)肿瘤影响儿童和成人，它们具有很强的侵袭性，与预后不良有关。例如，相当一部分的胶质瘤是最常见的成人脑肿瘤，表现为具有骨、软骨和肌肉分化成分的间质特征，其中一些含有明显的肌肉肉瘤成分(胶质肉瘤)。确定驱动这些肿瘤形成的细胞实体将是设计针对这些恶性肿瘤的有效治疗干预措施的关键。拟议的PHD项目旨在检验NM肿瘤起源于类似于多能胚胎前体的干细胞的假设，即神经中胚层前体(NMPs)或神经脊(NC)前体。在胚胎早期发育过程中，NMPs分化为脊髓神经元、骨、软骨和骨骼/平滑肌，而NC细胞分化为外周神经元、黑素细胞和间充质细胞，如平滑肌和软骨。我们推测，NM肿瘤的发生要么是胚胎发育过程中NMPs/NC前体细胞突变驱动转化为致癌细胞的结果，要么是神经/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，从而在有利的生态位内触发肿瘤发生的结果。研究计划目前建立脑肿瘤细胞系的传统方法被设计为选择神经干细胞(NSC)样细胞，反映了所有脑肿瘤的起源细胞是转化的神经前体细胞的假设。因此，使用与神经干细胞增殖相同的条件，成功地建立了粘附性胶质瘤干细胞系。然而，这种方法不能用于分离类似于NMPs/NC细胞的双能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，并在NMP、NC和神经干细胞(NSC)条件下平行培养相同数量的细胞(阳性对照)。在NMP或NC培养液中成功的附着、增殖和随后的传代将标志着NM双能干细胞在起始肿瘤中的存在。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，将证实在所得到的细胞系中存在双能细胞。将进行RNA-SEQ分析，将NM肿瘤细胞系与hPSC来源的NMP和NC细胞以及已建立的人NSC系进行转录比较，以确定致癌转化的候选驱动因素。还将通过异种移植/立体定向注射到NOD-SCID小鼠来评估肿瘤生成潜力。如果时间允许，我们还将进行小分子筛选，以识别对NM肿瘤细胞株具有细胞毒性/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.