应用肺转移法研究骨肉瘤转移性肺定植的实用思考
Summary
本文的目的是为肺转移试验 (美洲狮) 的协议提供详细的描述。该模型允许研究人员使用 widefield 荧光或共聚焦激光扫描显微镜研究肺组织中的转移性骨肉瘤 (OS) 细胞生长。
Abstract
肺转移试验 (彪马) 是一种体外肺外植体和封闭细胞培养系统, 允许研究人员通过荧光显微镜研究骨肉瘤 (OS) 的肺定植生物学. 本文详细描述了该协议, 并讨论了使用 widefield 或共聚焦荧光显微镜平台获取转移生长图像数据的示例。美洲狮模型的灵活性使研究人员不仅可以研究在肺部微环境中的 OS 细胞的生长, 而且还可以评估随着时间推移抗转移治疗的效果。共焦显微镜允许前所未有的高分辨率成像的 OS 细胞相互作用的肺实质。此外, 当美洲狮模型与荧光染料或荧光蛋白基因记者相结合时, 研究人员可以对转移的 OS 细胞的肺微环境、细胞和亚型结构、基因功能和启动子活动进行分析。美洲狮模型提供了一个新的工具, 骨肉瘤研究人员发现新的转移生物学和评估活动的新型抗转移, 靶向治疗。
Introduction
改善儿童转移性骨肉瘤 (OS) 患者的预后仍然是一个关键的未满足的临床需要1。这突出了发展新的分子靶向治疗的重要性。常规化疗靶向肿瘤细胞增殖没有证明是有效的治疗转移性疾病, 因此, 新的战略必须针对转移过程本身2。本文讨论了一种相对较新型的体外肺转移模型、门多萨和同事 3开发的肺转移试验 (彪马) 的实际应用, 为发现新的操作系统中肺转移进展的分子驱动程序4,5。然而, 在继续之前, 应该谨慎地简单地接触几种目前的转移模型, 以及美洲狮模型如何比传统的体外检测提供了一些优势。
大多数用于研究转移的实验模型包括体外和体内系统, 它概括了转移级联的特定步骤或几个步骤。这些步骤包括: 1) 肿瘤细胞从原发肿瘤转移到附近的血管 (血液或淋巴) 和转运内的中转, 3) 在第二部位的拘捕, 4) 在次生部位的渗出和存活, 5) 形成淋巴结和 6) 成长为血管化转移 (图 1)。体外模型的转移可以包括2维 (2D) 迁移和3维 (3D) 胶入侵检测, 并在其他地方详细审查6。对于体内模型, 两种常用的模型系统包括: 1)自发性转移模型是将肿瘤细胞原位注射到特定组织类型中, 形成局部肿瘤, 自发地脱落转移细胞。到遥远的地点;2)实验性转移模型是将肿瘤细胞注入靶器官上游血管的地方。例如, 肿瘤细胞的尾静脉注射导致发展肺转移瘤5,7,8。其他实验性转移模型包括将肿瘤细胞注入脾脏或肠系膜静脉, 导致肝转移的发展9,10。韦尔奇11详细讨论了这些体内模型的实际注意事项。另一个用于研究小儿肉瘤转移的体内模型是肾肾包膜下肿瘤植入模型, 它导致局部肿瘤形成和自发转移到肺部12, 13.更技术上苛刻的技术, 如活体 videomicroscopy 可以直接可视化, 实时, 转移癌细胞与转移部位 (即肺或肝脏) 的微血管的相互作用, 如麦克唐纳14和 Entenberg15, 或胚膜中的癌细胞渗出, 如 Kim 16所述。
美洲狮模型是一个前体,肺组织外植体, 封闭培养系统, 其中荧光肿瘤细胞的生长可以通过荧光显微镜在一个月内纵向观察 (见图 2A)。该模型概括肺定植的初始阶段 (步骤3至 5) 在转移级联。美洲狮模型优于传统的体外模型的一些主要优势是: 1) 它提供了一个机会, 纵向测量转移癌细胞生长在3D 微环境中, 保留许多特点的肺部微环境在体内 3;2) 美洲狮允许研究人员评估在3D 肺微环境下, 候选基因或药物治疗的击倒是否具有抗转移活性;3) 美洲狮模型具有多种类型的荧光显微平台 (图 2B) 的灵活性, 如 widefield 荧光显微镜或激光扫描共焦显微术, 其中的示例显示在图 2C & D中,分别.本文将讨论如何使用美洲狮模型获得的纵向成像数据的转移生长的增强绿色荧光蛋白 (eGFP) 表达, 人高和低转移性骨肉瘤细胞 (MNNG 和居屋细胞, 分别) 使用低放大 widefield 荧光。用共聚焦激光扫描显微镜对美洲狮模型中的线粒体进行标记的荧光染料的成像实例, 并对其进行了研究。
Protocol
Representative Results
Discussion
以下技术文章描述了美洲狮模型在研究 OS 中肺定植的一些实用方面。在《议定书》中, 研究人员应格外注意的一些关键步骤包括以下几个方面:
a) 气管插管。气管在解剖周围的肌肉和结缔组织时容易受损。此外, 导管针可以很容易地通过气管。在插入套管时要密切注意针的斜角如何进入气管。
b) 刺穿或损害肺部的表面。肺可以很容易刺穿或损坏的解剖剪刀…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢 Arnulfo 门多萨博士, 他提供了美洲狮技术的训练。 此外, 我们还要承认 Drs. 卡拉姆·昌德·沃赫拉肯纳, 苏珊. 加菲尔德 (NIH) 和 Sam 加亚尔多·阿帕里西奥 (BC 癌症机构) 在本研究过程中提供他们的显微镜使用。这项研究是支持 (部分) 由国家卫生研究院的内部研究计划, 癌症研究中心, 儿科肿瘤科。M.M.L. 得到美国国立卫生研究院 (15335 奖) 的支持, 目前在转移研究中得到了琼帕克奖学金的支持。P.H.S. 得到不列颠哥伦比亚省癌症基金会的支持。
Materials
| Table 2 | |||
| Cell culture reagents for A-media, B-media, and complete media | |||
| MNNG-HOS | ATCC | CRL-1547 | highly metastatic OS cell line |
| HOS | ATCC | CRL-1543 | poorly metastatic OS cell line |
| MG63.3 | Amy LeBlanc Laboratory (NCI) | N/A | highly metastatic OS cell line |
| MG63 | ATCC | CRL-1427 | poorly metastatic OS cell line |
| 10X M199 media | Thermofisher | 11825015 | Base media for A-media and B-media |
| Distilled Water (sterilized) | Thermofisher | 15230-147 | Component of A-media & B-media |
| 7.5% sodium bicarbonate solution | Thermofisher | 25080094 | Component of A-media & B-media |
| Hydrocortizone | Sigma-Alrich | H6909 | Component of A-media & B-media |
| Retinol acetate-water soluable | Sigma-Alrich | R0635-5MG | Component of A-media & B-media |
| Penicillin/Streptomycin 10X concentrated (10000 U/ml) solution | Thermofisher | 15140122 | Component of A-media & B-media, complete media. |
| Bovine insulin solution (10mg/ml) | Sigma-Alrich | I0516-5ML | Component of A-media & B-media |
| DMEM, high glucose | Thermofisher | 11965092 | Base media of Complete Media |
| L-Glutamine (200 mM) | Thermofisher | 25030081 | Component of Complete Media |
| Fetal Bovine Serum | Thermofisher | 16000044 | Component of Complete Media |
| Dulbecco’s Phosphate Buffered Saline | Thermofisher | 14190144 | Used in cell culture. |
| Hank’s Buffered Salts Solution, no calcium, no magnesium, no phenol red | Thermofisher | 14175095 | Used to resuspend cell pellet prior to injection |
| Trypsin-EDTA (0.25%), phenol red | Thermofisher | 25200114 | Used in cell culture. |
| DAR4M | Enzo | ALX-620-069-M001 | Used to label lung parenchyma. |
| Name | Company | Catalog Number | Comments |
| Table 3 | |||
| Materials for PuMA | |||
| Zeiss 710 Confocal LSM | Zeiss | N/A | Upright LSM confocal microscope |
| Zeiss 780 Confocal LSM | Zeiss | N/A | Inverted LSM confocal microscope |
| SCID mice | Charles River | N/A | NOD.CB17-Prkdcscid/NcrCrl, female, age 6-8 weeks |
| GelFoam | Harvard Apparatus | 59-9863 | Used as a support for lung tissue sections. |
| SeaPlaque Agarose | Lonza | 50100 | Used during insufflation of the lung. |
| 1 ml syringe with 27 gauge needle | Fisherscientific | 14-826-87 | Used for tail vein injection. |
| 10 ml syringe | BD | 309604 | Used for insufflation of the lung. |
| 20 gauge catheter | Terumo | SR-OX2032CA | Used during insufflation of the lung. |
| Abbott IV extension set (30", Sterile) | Medisca | 8342 | Used during insufflation of the lung. |
| Alcohol swabs | BD | 326895 | For wiping tail vein before injection |
| Sterile surgical gloves | Fisherscientific | Varies with size | Asceptic handing of mouse lungs |
| 30 cm ruler | Staples | Used for insufflation of the lung. | |
| Support stand for ruler | Pipette.com | HS29022A | Used for insufflation of the lung. |
| 35 mm glass-bottomed culture dish | Ibidi | 81158 | Used during imaging of lung slices |
| Absorbent Underpads with Waterproof Moisture Barrier | VWR | 56617-014 | Used to line the sterile work area in the biological hood. |
| Catgut Plain Absorbable Suture | Braun | N/A | Used to tie off cannulated trachea. |
| Name | Company | Catalog Number | Comments |
| Table 4 | |||
| Surgical instruments for PuMA | |||
| Micro Dissecting Scissors 3.5" Straight Sharp/Sharp | Roboz | RS-5910 | For cutting lung sections |
| 4” (10 cm) Long Serrated Straight Extra Delicate 0.5mm Tip | Roboz | RS-5132 | For manipulating/holding lung sections. |
| 4” (10 cm) Long Serrated Slight Curve 0.8mm Tip | Roboz | RS5135 | For manipulating/holding lung sections. |
| Thumb Dressing Forceps; Serrated; Delicate; 4.5" Length; 1.3 mm Tip Width | Roboz | RS-8120 | For general dissection. |
| Thumb Dressing Forceps 4.5" Serrated 2.2 mm Tip Width | Roboz | RS-8100 | For general dissection. |
| Extra Fine Micro Dissecting Scissors 3.5" Straight Sharp/Sharp, 20mm blade | Roboz | RS-5880 | For general dissection. |
| Knapp Scissors; Straight; Sharp-Blunt; 27mm Blade Length; 4" Overall Length | Roboz | RS-5960 | For general dissection. |
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