建模星形细胞瘤发病机制<em>在体外</em>和<em>体内</em>使用星形胶质细胞或条件，遗传工程小鼠神经干细胞
Summary
Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.
Abstract
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Introduction
星形细胞瘤是最常见的原发性脑肿瘤和胶质母细胞瘤（GBM），IV级星形细胞瘤，是最常见的和积极的亚型12-15 1,2个月，中位生存期。弥漫性星形细胞瘤，尤其是大紫荆勋贤，入侵排除手术完全切除，限制了辅助治疗的效果，并不可避免地导致治疗后复发3。患者最初存在要么从头（主）GBM或更低级别星形细胞瘤不可避免地发展到（二级）GBM 4。 GBM是基因组异质性和基因支配三个核心信号通路特征是相互排斥的，共同发生突变：在G1 / S 期 （RB）细胞周期检验点，受体酪氨酸激酶（RTK）和TP53途径5-7。 GBM由四个基因与亚型，类似于大脑不同的细胞类型不同的表达模式，表明GBM亚型是influ原产6,8,9其小区enced。更好的星形细胞瘤模型须在星形细胞瘤的发病机制来定义在特定细胞类型的突变的特定组合的作用。利用这些模型进行更有效的临床药物开发将最终有助于改善患者的预后。当前星形细胞瘤模型包括已建立的人细胞系，来自患者的异种移植物（PDX），遗传修饰的正常人星形细胞和神经干细胞（NSC），以及基因工程化小鼠（GEM）10-14。我们开发了一个替代方案，非生殖细胞创业板（nGEM）模型采用15原发性脑细胞-星形胶质细胞和NSC -从创业板窝藏两侧装接loxP致癌等位基因的不同组合收获。我们的目标是生成星形细胞瘤模型与遗传限定的细胞可以被表型特征在于在体外和在体内和潜在地用于临床药物开发第immune能力的小鼠。
建立的人细胞系是最常用的模型星形细胞瘤的发病机制及在体外和体内的药物反应，它们在技术上是简单的，广泛使用的，并且已经在免疫缺陷小鼠10,11,16-18限定在原位异种移植动力学和致瘤性。其缺点包括不能产生从低级别星形细胞瘤的细胞株，限制研究仅向高级别星形细胞瘤;缺乏定义的起源细胞;的复杂基因组的异常，通常与明显不同的原患者样品的基因组谱的存在;和易感性血清11,17,19-22串行 培养过程中的表型和基因型漂移。在建立人类GBM细胞系个人致癌基因突变的表型结果可以通过多种异常，这实际上是目前，这往往妨碍eluc被屏蔽idation直接基因型 – 表型的影响。
通过病人隔离星形细胞瘤细 胞在免疫缺陷小鼠中，或通过它们的培养中所定义的无血清培养基中之前，原位注射非粘附球状体到免疫缺陷小鼠12,23的脑部皮下通道产生PDX。 PDX更准确地保持人体星形细胞瘤的基因组景观，而是类似建立的人细胞系中，个体的致癌基因突变的表型效果可以由于它们的基因组的复杂性19,24所掩盖。要定义特定的致癌基因突变表型的后果，特别是在应对新的治疗方法，建立了人类细胞系或PDX的面板常常用来建立基因型 – 表型的相关性，显示出普遍性，并尽量减少对细胞的特定影响的可能性。虽然PDX准确地概括星形细胞瘤，INC。的病理特点泸定入侵，建立人类细胞系原位移植瘤一般不21,23,25。另外，正常人星形细胞和NSC已被基因工程化以定义的致癌基因突变，以星形细胞瘤肿瘤的体外和体内 13,14,26建模。这些细胞缺乏建立的人细胞系和PDX的基因组的复杂性，准确地概括人星形细胞瘤组织病理学，但需要异种移植在体内的免疫缺陷的啮齿动物。因为所有的人细胞模型需要免疫缺陷啮齿动物宿主以防止免疫介导的异种移植排斥反应，这些模式不能概括同系系统的天然肿瘤 – 基质相互作用和缺少一个完整的免疫系统，从而限制基质靶向和免疫调节的临床前调查治法10,11。
致癌墓塔预定组合的表型后果创业板证考试期间原位肿瘤发生系统蒸发散体内 。而非条件GEM有所有组织中的突变在整个发展，有条件GEM已经两侧装接loxP的致癌等位基因，使突变的，通过使用细胞类型特异性启动子10,11,15,18限制酶Cre介导的重组到特定的细胞类型定位。有条件的星形细胞瘤GEM已利用一个完整的脑11内阐明在不同的细胞类型中的致癌基因突变的功能角色。 原位 gliomagenesis使用条件GEM的临床前程序是由若干因素，包括1）缺乏体外互相关联，2）难以产生小鼠的大样本与复基因型的原位肿瘤的发展，3）长潜伏期不限，4）和随机肿瘤进展。因为原位肿瘤缺乏对应的体外模型中， 在体外药物测试无法进行瓦特第i个传统的条件创业板模型。在对比其它癌症，脑星形细胞瘤的条件GEM模型由单个致癌基因的突变11很少被诱导。因此，复杂的育种计划必须产生具有多个致癌基因突变的条件GEM。此外，星形细胞瘤起始时具有可变的外显率在这些模型中很长的潜伏期后，而进展到高级别星形细胞瘤一般发生在一个非均匀，随机的方式，最终会引起肿瘤的复杂基因组的风景和快速生长动力学27 28。可变外显率和恶性进展的随机性在有条件的创业板模型要求，个别小鼠经X线影像学筛查的临床前药物试验入组前检测的高级别星形细胞瘤的存在和位置。综合起来，这些限制阻碍了需要公关的产生和创业板条件的大型同伙测试电子临床药物试验。
将RCAS-TVA GEM系统，它利用禽逆转录病毒（RCAS）载体来感染GEM改造以表达病毒受体（TVA）上的特定神经细胞类型中，已被广泛用于模拟星形细胞肿瘤11。相反，创业板的条件，该模型系统能够引进特定细胞类型的多种致癌突变没有复杂的育种计划的要求。然而，它是由可变外显率，为积极分裂的细胞来实现病毒整合的要求，并在随机插入 转基因到宿主基因组29限定。
非种系GEM（nGEM）模型，其中利用细胞从GEM收获，正变得越来越重要，因为它们克服了许多其他模型系统15的限制。发起于星形细胞瘤发病机制的细胞类型和共同发生突变的作用都难以阻止矿用建立的人GBM细胞系或PDX因为它们是从恶性进展的过程中，已经积累了大量的基因突变中未定义的细胞类型，终末期肿瘤衍生的。相比之下，星形细胞瘤各年级可以使用nGEM通过诱导特异性纯化脑细胞类型11,30中定义的基因突变进行建模。因此，特定的基因突变和在细胞和分子表型细胞类型的影响，可以在体外和体内测定。类似建立的人GBM细胞系中，使用nGEM初始体外药物测试可用于优先级药物的体内试验采用相同的细胞。肿瘤在体内然后可通过同种异体移植nGEM细胞原位成的免疫能力的同基因同窝30的大脑来确定。因此，这些原位移植模型允许在体内试验，不仅传统的细胞毒性á次有针对性的治疗，但免疫调节和基质靶向治疗为好。最后，对肿瘤的发生和发展的微环境的作用，可通过使用相同的突变在相同的细胞类型进行比较nGEM和常规GEM模型之间的结果来确定。
我们和其他人已经开发出星形细胞瘤nGEM使用原代细胞-星形胶质细胞，国科会，还是少突胶质前体细胞（OPC） -由创业板30-34收获。后面的星形细胞瘤nGEM发展的理论基础是建立一个模型，以确定在特定的细胞类型的致癌基因突变，可能在免疫感受态的动物可用于体外临床药物试验和体内表型的影响。我们收获的野生表型星形胶质细胞和NSC非Cre表达，维持在> 94％的C57 / BL6背景，两侧装接loxP Rb途径有条件的创业板-皂苷Rb1 的loxP / loxP位 ，或TgGZT121 -和两侧装接loxP RTK / RAS / PI3K通路- NF1 的loxP / loxP位 ， 嘉仕G12D和PTEN 的loxP / loxP位 -基因的不同组合35-39。我们采用编码Cre重组酶的腺病毒载体诱导的基因重组体外 。因为皮质星形细胞收获含有细胞类型的混合物中，我们使用Ad5GFAPCre载体或显性致癌基因的转基因，如TgGZT 121从人GFAP启动子驱动的，以富 集在这些培养物GFAP +星形胶质细胞。我们定义G 1 / S的表型结果（RB），MAPK和PI3K途径的突变在星形胶质细胞和NSC 的体外和体内 。 MAPK和PI3K途径活化的G1 / S期缺陷型星形胶质细胞的分子拟仿人体前神经GBM，一经原位注射，在一个预先定义的位置的均匀生长动力学，短的等待时间形成的肿瘤，和组织病理学人标志人类GBM 30。 体内纵向监测肿瘤的生长有助于治疗反应40通过治疗组群和肿瘤生长的定量分析的归一化的临床前药物测试。我们通过注射荧光素酶表达星形胶质细胞的小鼠的纵向生物发光成像确定肿瘤的生长动力学。因此，星形胶质细胞和NSC从创业板有条件衍生为的星形细胞瘤相关的基因突变功能的后果和临床前药物开发的潜在模型系统定义中的易处理的模型系统。
Protocol
Representative Results
Discussion
最关键的步骤，以确保适当的收获和培养星形胶质细胞的有：1）切除皮质而不采取组织下面的胼胝体，2）以除去脑膜，3），彻底解离的细胞，以及4）以富集GFAP +星形胶质细胞。虽然我们使用机械（摇动）和遗传（遗传重组的限制与显性转化的转基因的Ad5GFAPCre载体或利用（TgGZT 121）根据GFAP启动子控制）的方法来富集GFAP +胶质细胞，其他的技术已被用来净化?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Dulbecco's Modified Eagle Medium (DMEM) (1X) | Invitrogen | 11995065 | DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro |
| Fetal Bovine Serum, Regular | Cellgro | 35-010-CV | |
| Penicillin-Streptomycin | Invitrogen | 15140122 | |
| Sharp-Pointed Dissecting Scissors | Fisher Scientific | 08-395 | |
| Cartilage Thumb Forceps, Curved | Fisher Scientific | 1631 | |
| Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4 | Miltex | 17-301 | |
| Ethanol (200 proof) | Decon Labs | 2710 | |
| Razor Blades | VWR | 55411-050 | |
| Hanks' Balanced Salt Solution (HBSS) (1X), liquid | Invitrogen | 14175-095 | |
| TrypLE Express (1X), Phenol Red | Invitrogen | 12605-010 | Other Trypsin solutions are also suitable for cortical astrocyte havest and culture |
| CULTURE DISH, 60 x 15 mm | Thomas Scientific | 9380H77 | |
| 15 ml tubes | BD Biosciences | 352096 | |
| 50 ml tubes | BD Biosciences | 352070 | |
| Adenovirus stock | Gene Transfer Vector Core, U. Iowa | Ad5CMVCre | Store in 5 µl aliquots at -80 C. Hazardous. Use Bsl2 safetyy precautions |
| Sodium sulfate (Na2SO4) | Sigma-Aldrich | 238597 | |
| Potassium sulfate (K2SO4) | Sigma-Aldrich | 221325 | |
| Magnesium chloride (MgCl2) | Sigma-Aldrich | M8266 | |
| Calcium chloride (CaCl2) | Sigma-Aldrich | 746495 | |
| HEPES potassium salt | Sigma-Aldrich | H0527 | |
| D-(+)- Glucose | Sigma-Aldrich | G8270 | |
| Phenol Red | Sigma-Aldrich | P3532 | |
| Sodium hydroxide (NaOH) | Sigma-Aldrich | S5881 | |
| Papain | Worthington | LS003127 | |
| L-Cysteine-HCl | Sigma-Aldrich | C1276 | |
| Syringe filter (0.22 µm pore size) | Millipore | SLGP033NS | |
| Neurocult proliferation kit, mouse | Stemcell Technologies | 5702 | This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture |
| 0.2% Heparin solution | Stemcell Technologies | 7980 | |
| EGF | Invitrogen | PMG8041 | |
| bFGF | Invitrogen | PHG0261 | |
| Hanks' Balanced Salt Solution (HBSS) (10X) | Invitrogen | 14185-052 | |
| Magnesium sulfate (MgSO4) | Sigma-Aldrich | M7506 | |
| Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | |
| E-64 | Sigma-Aldrich | E3132 | Make 10 mM stock in DMSO, store at -20 °C |
| 6-well plates | Fisher Scientific | 07-200-83 | |
| Cell strainer (40 µm pore size) | Corning | 352340 | |
| Stem cell dissociation solution | Stemcell Technologies | 5707 | Alternatively, use gentle enzyme solutions such as Accutase |
| Methyl cellulose 15 cP | Sigma-Aldrich | M7027 | |
| Dulbecco's Modified Eagle's Media 2X | Millipore | SLM-202-B | For making 5% methyl cellulose solution |
| 1.7mL Snap Cap Microcentrifuge Tube | Corning | 3620 | |
| Hamilton syringe, 250 µl LT no needle | Fisher Scientific | 14-815-92 | |
| PB600-1 Antigen Dispenser | Hamilton | 83700 | |
| Disposable 18 ga needles | Fisher Scientific | NC9015638 | |
| 27 ga 1/2" luer tip needle | Fisher Scientific | 14-826-48 | |
| 2,2,2-Tribromoethanol (Avertin) | Sigma-Aldrich | T48402 | |
| Betadine | Fisher Scientific | NC9386574 | |
| Puralube Opthalmic Ointment | Fisher Scientific | NC9689910 | |
| Model 900 Stereotaxic frame | Kopf Instruments | ||
| VETBOND | Fisher Scientific | NC9259532 | Tissue adhesive |
| Lidocaine | ShopMedVet | RXLIDO-EPI | |
| CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) | Promega | G3580 | |
| Anti-Sox2 | Millipore | AB5603 | |
| Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP) | Dako | Z0334 | |
| IVIS Kinetic | PerkinElmer | For in vivo imaging | |
| D-Luciferin – K+ Salt Bioluminescent Substrate | PerkinElmer | 122796 | For in vivo bioluminescence imaging |
| EdU Imaging Kit | Invitrogen | C10340 | |
| MSCV Luciferase PGK-hygro | Addgene | 18782 |
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