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Medicina

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

Published: June 28, 2018
doi:
10.3791/56650
, ,
1Department of Pathology,Yale University School of Medicine, 2Department of Medical Oncology,Yale University School of Medicine

Summary

本文介绍一种通过对气道损伤进行预适应来显著提高肺癌细胞原位植入的方法。这种方法也可以应用于研究肺微环境中的间质相互作用, 转移传播, 肺癌的共同发病, 并更有效地产生病人的移植。

Abstract

肺癌是一种致命的治疗顽固性疾病, 是生物异构的。为了了解和有效地治疗胸恶性肿瘤的完整临床谱, 需要额外的动物模型来重述不同的人肺癌亚型和分期。同种异体或异种移植模型是多种多样的, 可以利用小鼠或人类的恶性细胞,在体内癌变能力的量化。然而, 以前所描述的肺癌细胞植入的方法已经在非生理部位进行, 如小鼠的侧面, 由于细胞移植到肺部的效率低下。在本研究中, 我们描述了一种通过对小鼠肺纤维化诱发博莱霉素进行预调节来增强原位肺癌细胞植入的方法。作为一种概念验证实验, 我们将这种方法应用于从老鼠或人源获得的肺腺癌亚型的嫁接肿瘤细胞, 并将其转化为不同品系的小鼠。我们证明, 在肿瘤细胞注射前, 与博莱霉素的呼吸道损伤增加了肿瘤细胞从0-17% 到71-100% 的植入。显著地, 该方法通过不同模型和小鼠菌株增强肺肿瘤发生率和后续生长。此外, 嫁接肺癌细胞从肺部传播到相关的远端器官。因此, 我们提供了一个协议, 可用于建立和维持新的肺癌原位模型, 限制数量的细胞或 biospecimen, 并定量评估肺癌细胞在生理相关设置的癌变能力.

Introduction

肺癌是全球1癌症相关死亡的主要原因。肺癌患者最终从转移到远端器官, 主要是中枢神经系统, 肝脏, 肾上腺, 骨骼2,3,4。胸恶性肿瘤历来被归类为小细胞肺癌或非小细胞肺癌 (NSCLC)5。NSCLC 是最常见的恶性肿瘤, 可分为不同的组织学亚型, 包括肺腺癌 (LUAD) 和肺鳞状细胞癌 (LUSC)6。经手术切除的人原发性肺癌的基因组分析显示, 某一 histotype 内的肿瘤也能表达多种分子扰动, 进一步促进其临床进展和混淆患者预后。肺癌的显著异质性对合理的设计、临床前试验和有效的治疗策略的实施都是一个重大挑战。因此, 有必要扩大可听话的实验性肺癌模型的汇辑, 以研究这种疾病的不同细胞来源、分子亚型和分期。

使用动物模型的各种方法在体内研究肺癌, 每个都有各自的优缺点, 这取决于所关心的生物学问题。基因工程小鼠模型 (GEMMs) 可以针对特定祖细胞类型的特异基因改变, 导致肿瘤在免疫宿主7中取得进展。虽然非常强大和临床相关的, 潜伏期, 变异性和/或肺部肿瘤发病率与 GEMMs 可能会禁止某些定量测量和检测晚期转移在遥远的器官8。一个补充的方法是使用同种异体模型, 藉以肺癌细胞从老鼠肿瘤直接获得或首先被获取作为建立的细胞线在文化中被重新介绍入自体寄主。类似, 肺癌移植是由人体细胞系或病人衍生的肿瘤样本建立的。人细胞系移植或患者衍生的移植 (PDXs) 一般维持在免疫缺陷小鼠, 因此排除完全免疫监测9。尽管有这个缺点, 它们提供了一个途径来传播限制数量的人类 biospecimens 和研究人类癌细胞的基本的体内性质, 这编码为更复杂的基因组畸变比 GEMM 肿瘤。

同种异体移植和异种移植的一个有用的特性是, 他们可以使用传统的限制性细胞稀释方法, 用于量化10恶性细胞中肿瘤起始细胞 (抽搐) 的频率。在这些实验中, 在动物的侧面注入了一定数量的细胞, 并根据肿瘤的发生率来估计抽搐频率。然而皮下肿瘤可以是更低氧11并且可能不模型主要生理制约肺肿瘤微环境。气管内向小鼠肺内输送上皮干细胞或祖细胞是研究肺再生和气道干细胞生物学12的方法。然而, 这种技术的植入率可能相对较低, 除非肺部首先受到生理形式的伤害, 如病毒感染13,14。炎症基质细胞的支持和 (或) 肺基底膜的破坏, 可改善移植细胞在远端气道15的相关干细胞龛位的保留。纤维化诱导剂也可以预条件肺, 以增强植入诱导多能细胞16和间充质干细胞17。类似的气道损伤形式是否会影响植入率, 肿瘤的启动能力和肺癌细胞的产生还有待系统地评估。

本文通过对损伤小鼠肺的预处理, 描述了提高原位肺癌细胞植入效率的方法。LUAD 出现在远端呼吸道与这些癌症的一个重要的子集发展纤维间质基质18 , 往往与不良预后19。博莱霉素是一种天然非核糖体杂交肽-聚酮体, 已广泛用于诱导小鼠肺纤维化20。博莱霉素的气道灌注首先促进肺泡上皮的磨损和炎症细胞的招募, 包括巨噬细胞、中性粒细胞和单核细胞21。其次是在远端呼吸道组织重塑, 基底膜重组22,23和细胞外基质 (ECM) 沉积24。单个博莱霉素注射液的影响是瞬变的, 在大多数研究中30天后纤维化解决25。利用同种异体移植和异种移植模型, 我们对博莱霉素小鼠呼吸道进行预适应试验, 可以显著提高肺 LUAD 细胞的服用率。

Protocol

所有实验都是按照耶鲁大学机构动物护理和使用委员会 (IACUC) 批准的协议进行的。 1. 试剂的设置/准备。 博莱 霉 素注意: 根据全球统一的化学品分类和标签制度, 博莱霉素被归类为 GHS08 的健康危害。 用化学罩准备博莱霉素。并用重悬 15 U 入5毫升无菌磷酸盐缓冲盐水 (PBS)。 整除100-200 µL 的溶液成玻璃瓶, 并冻结在-20 摄氏度, 以供将来使用…

Representative Results

为了提高 LUAD 癌细胞植入到小鼠肺中的效率, 我们制定了一项协议, 首先使用博莱霉素的呼吸道, 其次是原位肿瘤细胞注射 (图 1)。我们证实, 即使在免疫裸小鼠, 博莱霉素诱导瞬变纤维化14天, 证明了气道结构的损失和增加胶原沉积 (图 2)。这些小鼠的毛纤维化在博莱霉素注射液后50天解决 (图 2; 右面板) 与以…

Discussion

在肺癌和其他慢性疾病的肺36之间有明显的临床相似之处。尤其是特发性肺纤维化 (IPF) 患者对发展肺癌的偏爱增加, 而这个协会独立于吸烟史37,38。森林小组的特点是逐步破坏肺结构和受损的呼吸功能, 通过沉积 ECM39。同时, 在手术切除后, 早期的无细胞肺癌患者并发森林小组的结局较差40。多数 NSCLC ?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项研究的经费来自国家癌症研究所 (R01CA166376 和 R01CA191489 D.X.) 和国防部 (W81XWH-16-1-0227 D.X. 阮) 的赠款。

Materials

Bleomycin Sigma B5507-15UN CAUTION Health hazard GHS08
Exel Catheter 24G Fisher 1484121 Remove needle. For intratracheal injection
Ketamine (Ketaset inl 100 mg/mL C3N 10 mL) Butler Schein 56344 To anesthetize mice
Xylazine Butler Schein 33198 To anesthetize mice
Ketoprofen, 5,000 mg Cayman Chemical 10006661 Analgesic
Puralube Veterinary Ophthalmic Ointment BUTLER ANIMAL HEALTH COMPANY LLC 8897 To prevent eye dryness while under anesthesia
D-Luciferin powder Perkin Elmer Health Sciences Inc 122799 For luminescent imaging. Reconstitute powder with PBS for a working concentration of 15mg/mL. Protect from Light
Rodent Intubation stand Braintree Scientific RIS-100 Recommended stand for intratracheal injection
MI-150 ILLUMINATOR 150W MI-150 DOLAN-JENNER INDUSTRIES MI-150 / EEG2823M To illuminate and visualize trachea
Graefe Forceps, 2.75 (7 cm) long serrat Roboz RS-5111 For intratracheal injection
Syringe Luer-Lok Sterile 5ml BD / Fisher 309646
Satiny Smooth by Conair Dual Foil Wet/Dry Rechargeable Shaver Conair – To shave mice
Bonn Scissors, 3.5" straight 15 mm sharp/sharp sure cut blades Roboz RS-5840SC
15 mL conical tube BD / Fisher 352097
1.5 mL centrifuge tubes USA SCIENTIFIC INC 1615-5500
Vial Scintillation 7 mL Borosilicate Glass GPI Fisher 701350
Filter pipette tips (200 μL) USA SCIENTIFIC INC 1120-8710
Phosphate Buffered Saline Life Technologies 14190-144
0.25% Trypsin-EDTA Life Technologies 25200-056
DMEM high glucose Life Technologies 11965-092
RPMI Medium 1640 Life Technologies 11875-093
Fetal bovine serum USDA Life Technologies 10437-028
Penicillin-Streptomycin Life Technologies 15140-122
Amphotericin B Sigma A2942-20ML
Trypan Blue Stain 0.4% Life Technologies 15250-061
Countess Automated Cell Counter Life Technologies AMQAX1000
Flask T/C 75cm sq canted neck, blue cap Fisher / Corning 353135
IVIS Spectrum Xenogen Bioluminiscence Perkin Elmer Health Sciences Inc 124262 For in vivo bioluminescence imaging
Living image software Perkin Elmer Health Sciences Inc 128113 For in vivo bioluminescence analysis
XGI-8 Gas Anesthesia System Perkin Elmer Health Sciences Inc 118918 For Isoflurane anesthesia
BD Ultra-Fine II Short Needle Insulin Syringe 1 cc. 31 G x 8 mm (5/16 in) BD / Fisher BD328418 For retro-orbital luciferin injection
Syringe 1ml BD / Fisher 14-823-434 For intraperitoneal injections
26 G x 1/2 in. needle BD / Fisher 305111 For intraperitoneal injections
4% Paraformaldehyde VWR 43368-9M CAUTION Health hazard GHS07, GHS08. For fixing tissue
Pipet-Lite Pipette, Unv. SL-200XLS+ METTLER-TOLEDO INTERNATIONAL 17014411
Mayer's Hematoxylin ELECTRON MICROSCOPY SCIENCES 517-28-2
Eosin Y stain 0.25% (w/v) in 57% Fisher 67-63-0
Masson Trichrome Stain Kit IMEB Inc K7228 For masson trichrome stain to visualize collagen
Superfrost plus glass slides Fisher 1255015
6 well plate Corning C3516
Universal Mycoplasma Detection Kit ATCC 30-1012K
OCT Embedding compound ELECTRON MICROSCOPY SCIENCES 62550-12 For embedding tissue for frozen sections
Leica CM3050 S Research Cryostat Leica CM3050 S To section tissue for staining analysis
Keyence All-in One Fluorescence Microscope Keyence BZ-X700
ImageJ US National Institutes of Health IJ1.46 http://rsbweb.nih.gov/ij/ download.html
Prism 7.0 for Mac OS X GraphPad Software, Inc. –
Athymic (Crl:NU(NCr)-Foxn1nu) mice Charles River NIH-553
NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice Jackson Laboratories 5557
B6129SF1/J mice Jackson Laboratories 101043
NIH-H2030 cells ATCC CRL-5914
368T1 generously provided by Monte Winslow (Standford University) –
PC9 cells Nguyen DX et al. Cell. 2009;138:51–62 –
H2030 BrM3 cells Nguyen DX et al. Cell. 2009;138:51–62 –
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Tags

Lung CancerTumor-initiating CellsBleomycinOrthotopic EngraftmentPreclinical ModelFibrosisAirway Pre-conditioningCell TransplantationTracheal InjectionMouse Model

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Stevens, L. E., Arnal-Estapé, A., Nguyen, D. X. Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment. J. Vis. Exp. (136), e56650, doi:10.3791/56650 (2018).
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