人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用
Summary
目前的胶质母细胞瘤（GBM）的离体模型并没有针对人肿瘤浸润的生理学相关研究进行优化。在这里，我们提出了新鲜人类GBM组织的器官切片培养物的产生和维持方案。提供延时显微术和定量细胞迁移分析技术的描述。
Abstract
尽管手术，化学治疗和放射治疗，胶质母细胞瘤（GBM）仍然具有极差的临床预后。进入周围脑实质的肿瘤侵袭代表着持久的治疗挑战。为了开发GBM的抗迁移疗法，为控制实验提供生理学相关背景的模型系统至关重要。在这里，我们提出了一种用于在手术切除期间获得的人类GBM组织产生切片培养物的方案。这些培养物允许离体实验，而不通过动物异种移植物或单细胞培养物传代。此外，我们描述了使用延时激光扫描共聚焦显微镜与细胞跟踪，以定量研究肿瘤细胞的迁移行为和与治疗相关的反应。手术组织采集后90分钟内可重复产生切片。逆转录病毒介导的荧光细胞随后在培养的两周内完成蜂鸣，共聚焦成像和肿瘤细胞迁移分析。我们已经成功地使用这些切片培养物来发现人类GBM中与迁移行为增加相关的遗传因素。此外，我们已经验证了模型检测针对抗迁移疗法的患者特异性变异的能力。展望未来，人类GBM切片培养是快速离体评估治疗剂对肿瘤敏感性的有吸引力的平台，以推进个性化神经肿瘤治疗。
Introduction
胶质母细胞瘤（GBM）的实验室研究受到缺乏忠实地重现人类疾病所需病理特征（即肿瘤细胞迁移和侵袭）的模型的阻碍。 2D和3D 的体外侵袭测定的比较研究，以及3D啮齿动物切片培养物模型已经发现在这两个上下文机理上不同的细胞迁移的程序，从2D系统发现的译可能限制与人类疾病1，2，3。这里描述的器官型肿瘤切片培养和成像范例允许研究从手术切除获得的离体人肿瘤组织的切片内的肿瘤细胞迁移。因此，手术切除的肿瘤组织的切片培养物与延时共聚焦显微镜结合提供了研究本地的肿瘤细胞迁移的平台微环境无组织溶解或培养传代。
有一种使用由人类肿瘤异种移植物，逆转录病毒诱导的肿瘤和细胞产生的叠加来研究肿瘤侵袭1，2，3，4，5 GBM的啮齿动物脑切片培养物模型的大量文献。最近，几个小组已经直接从人GBM组织6，7，8，9，10中描述的器官切片培养物的产生。然而，关于切片技术和培养基的公开方案之间存在明显差异。此外，使用器官切片培养物已经集中在包括细胞信号变化的静态实验终点ng，增殖和死亡。本文所述的方案通过采用时间推移激光扫描共聚焦显微镜的动态肿瘤细胞行为的时间分辨观察来扩展先前的切片培养范例。的帧间11和瘤内12最近发现，在人GBM 13遗传变异强调联这种异质性与肿瘤细胞的行为和其对肿瘤对治疗的反应的影响的重要性。在这里，我们报告了使用来自人类癌症组织的直接切片培养物的简化和可重复的方案，以近似实时地观察肿瘤细胞迁移。
Protocol
在开始收集患者组织样本之前，必须根据批准的机构评估委员会（IRB）方案从每个患者获得知情同意书。该协议的作者收到了科罗拉多大学医院和Inova Fairfax医院批准的IRB协议下所述的工作的同意。从这些切片培养物收集的数据不用于指导患者护理决定。 预切片准备在计划肿瘤切除和组织收集前（或在2周内利用以前产生的培养基）前一天准备“组织加工”培养基和“?…
Representative Results
我们的小组成功地从50多位接受GBM初始切除术的患者中获得了切片培养。这种切片生成，培养，逆转录病毒标记，成像和迁移分析协议已经精简到可重复的工作流程中（ 图1 ）。重要的是，这些器官型GBM切片与整个培养物中的起始肿瘤组织一致，包括在培养物中维持长达15天的病理标志物和小胶质细胞（ 图2 ）。此外，我们利用该系统对微环?…
Discussion
来自人类癌症组织的有机切片培养物为临床前的翻译实验提供了有吸引力和利用率不足的平台。了解肿瘤细胞在天然肿瘤微环境中的迁移，增殖和细胞死亡的人群行为缺乏。重要的是，在细胞行为水平上以动态，时间分辨的方式研究肿瘤对治疗的反应可以揭示治疗抵抗的新机制。人类肿瘤切片培养提供人类疾病过程与当前离体和体内建模技术之间的联系19 。最近，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢Lee Niswander博士和Rada Massarwa博士对这里描述的切片培养共聚焦成像方案的技术专长和贡献。进一步感谢Kalen Dionne博士提供了关于优化脑肿瘤组织切片和培养参数的专业知识。
Materials
| DMEM High Glucose | Invitrogen (Gibco) | 11960-044 | |
| Neurobasal-A Medium, minus phenol red | Invitrogen (Gibco) | 12349-015 | |
| B-27 Supplement (50X), serum free | Invitrogen (Gibco) | 17504-044 | |
| Penicillin-Streptomycin (10,000 U/mL) | Invitrogen (Gibco) | 15140-122 | |
| GlutaMAX Supplement | Invitrogen (Gibco) | 35050-061 | |
| L-Glutamine (200 mM) | Invitrogen (Gibco) | 25030-081 | |
| HEPES (1 M) | Invitrogen (Gibco) | 15630-080 | |
| Nystatin Suspension | Sigma-Aldrich | N1638-20ML | 10,000 unit/mL in DPBS, aseptically processed, BioReagent, suitable for cell culture |
| UltraPure Low Melting Point Agarose | Invitrogen (Gibco) | 16520-050 | Melts at 65.5 C, Remains fluid at 37 C, and sets rapidly below 25 C. |
| Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 647 Conjugate | Thermo Fisher (Molecular Probes) | I32450 | Used in media to label Microglia/Macrophages |
| pRetroX-IRES-ZsGreen1 Vector | Clonetech | 632520 | |
| Retro-X Concentrator | Clonetech | 31455 | Binding resin for non-ultracentrifugation concentration of viral supernatants |
| pVSG-G Vector | Clonetech | 631530 | part of the Retro-X Universal Retroviral Expression System |
| GP2-293 Viral packaging cells | Clonetech | 631530 | part of the Retro-X Universal Retroviral Expression System |
| Cyanoacrylate Glue (Super Glue) | Sigma-Aldrich | Z105899 | Medium-viscosity |
| Equipment | |||
| Peel-A-Way Embedding Mold (Square – S22) | Polysciences, Inc. | 18646A-1 | Molds for tumor sample embedding |
| Stainless Steel Micro Spatulas | Fisher Scientific | S50823 | Bend instrument 45 degrees at the neck of the spoon blade |
| Curved Fisherbrand Dissecting Fine-Pointed Forceps | Fisher Scientific | 08-875 | |
| Single Edge Razor Blade (American Safety Razors) | Fisher Scientific | 17-989-001 | Blade edge is 0.009" thick. Crimped blunt-edge cover is removed before loading onto vibratome. |
| Leica VT1000 S Vibratome | Leica Biosystems | VT1000 S | |
| Hydrophilic PTFE cell culture insert | EMD Millipore | PICM0RG50 | 30 mm, hydrophilic PTFE, 0.4 µm pore size |
| 35 mm Glass Bottom Dishes | MatTek | P35G-1.5-20-C Sleeve | 20mm glass diameter. Coverslip glass thickness 1.5 |
| LSM 510 Confocal Micoscope | Zeiss | LSM 510 | 10x Air Objective (c-Apochromat NA 0.45) |
| PECON Stagetop Incubator | PeCON Germany | (Discontinued) | Incubator PM 2000 RBT is a comprable product designed for use with Zeiss Microscopes. |
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Citer Cet Article
Parker, J. J., Lizarraga, M., Waziri, A., Foshay, K. M. A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs. J. Vis. Exp. (125), e53557, doi:10.3791/53557 (2017).