一种混合的3D组织工程<em>体外</em> /<em>在硅片</em在具体的突变背景预测药物的有效性>肺肿瘤模型
1Department of Tissue Engineering and Regenerative Medicine (TERM),University Hospital Wuerzburg, 2Department of Cardiothoracic Surgery,University Hospital Wuerzburg, 3Department of Bioinformatics,University Wuerzburg, 4Translational Center Wuerzburg,Fraunhofer Institute Interfacial Engineering and Biotechnology IGB
Summary
We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.
Abstract
在本研究中，我们结合一个体外三维肺肿瘤模型与在硅片模型基于特定突变背景，以优化药物的反应的预测。该模型是上再现有关的细胞外基质的组成和架构，包括基底膜组织特异性特征的猪去细胞支架产生的。我们标准化的协议，允许在14天内，包括药物治疗三天人造肿瘤组织产生。我们的文章提供了三维的几个详细描述读出的筛选技术，例如增殖指数的Ki67染色的，凋亡的M30-ELISA测定从上清液和上皮的评估至间质转变 （EMT），这是有用的工具，用于评估的有效性治疗化合物。我们可以证明相比于2D文化在我们的三维肿瘤模型减少扩散是RELAT编临床情况。尽管该下扩散，模型正确地根据该生物标记状态如由肺癌细胞系HCC827的比较预测EGFR -targeted药物反应（EGFR -mutated，KRAS野生型）和A549（EGFR野生型，KRAS –突变）用酪氨酸激酶抑制剂 （TKI）吉非替尼治疗。调查更先进的肿瘤细胞的药物反应，我们诱导的EMT由长期治疗的TGF-β-1波形蛋白/泛细胞角蛋白免疫荧光染色所评估。甲流生物反应器是用来调整文化，生理条件，提高了组织的产生。此外，我们表现出药物反应在吉非替尼治疗或TGF-β-1刺激的整合-细胞凋亡，增殖指数和EMT -为布尔在硅片模式。此外，我们解释了与特定突变的背景和计数肿瘤细胞如何药物反应抗阻力erstrategies可以预测的。我们有信心， 我们特别是其在硅片扩张体外方法3D提供了更现实的条件临床前药物测试比二维细胞培养的附加 价值。
Introduction
医药行业在癌症治疗领域的临床阶段面临着高达95％的高流失率造成了巨大的成本1-5。对于这个逆差的一个原因是目前潜在的新的化合物的功效在对癌症细胞系的2D细胞培养物或动物模型中的大型放映被评估的事实。动物模型具有较高的复杂性，但也有老鼠和人之间的6,7重要区别。在过去十年中，已经生成使用不同的方法的3D癌症模型弥合的癌细胞系的2D培养和体内肿瘤6,8,9复杂之间的差距。三维环境对细胞分化和也对信号的影响已经在一些研究中已显示年前（ 例如 ，由米娜比斯尔）10,11。今天，许多3D细胞培养模型可用，如球体文化，水凝胶或微流控芯片12-16。虽然THESE型增强复杂相比传统二维培养系统，它们大多缺乏该已知具有肿瘤支撑效应并且还影响药效的组织的微环境。
为了解决这个问题，我们产生基于称为SISmuc（ 小肠-下层+粘膜 ）的生物支架，其由脱细胞猪空肠衍生的三维肿瘤模型。从而，组织架构和诸如不同胶原以及基底膜结构ECM的重要组成部分被保留17。这种独特的特征是肿瘤模型生成从上皮出现并包括实体肿瘤的约80％的癌中是至关重要的。此外，在我们的组织工程化的肿瘤模型中的细胞增殖率相比，在2D培养取得的人为的高率降低。如增殖是在评估药物疗效的重要参数，药物测试是在我们的模型中启用更多类似在体内条件下肿瘤17。
为了评估我们的模型来正确预测生物标记物相关的药物的疗效，我们对在他们的EGFR -biomarker状态不同的两种不同的肺癌细胞系这里本数据的潜力。这种突变状态已经开始在小细胞肺癌患者常规测定。靶向治疗与TKIs的如EGFR -inhibitor抗肿瘤轴承相比，含铂类化疗18-21活化EGFR突变呈现出优异的成果吉非替尼。
我们建立了相关评价化合物功效几个读出的技术。此外，TGF-β-1刺激后，我们能够调查在肿瘤细胞中启动该EMT过程，这被认为是在恶变22,23的一个重要步骤，并连接到药物resistan化合物的动作CE 24。
三维肿瘤模型允许监测细胞特异性反应针对性的治疗，化学疗法，或具有良好反差的药物组合。为了进一步增强和加速药物筛选和遇到阻力，这是通过一个在电脑模拟的补充。基于一些实验，肿瘤反应，可以在硅片方面的成果进行全方位的药物及其组合预测。
Protocol
1.二维（2D）细胞培养商购获得肿瘤细胞系HCC827（DSMZ）。文化RPMI-1640肺腺癌细胞株HCC827（EGFR突变，KRAS野生型），辅以20％FCS。换液，每2 – 3天。分裂细胞每周两次。用于细胞到达通道20到。 市售获得肿瘤细胞系A549（DSMZ）。培养的肺癌细胞株A549（EGFR野生型，KRAS突变的）的RPMI-1640添加有10％FCS中。如上所述执行文化。用于细胞到达通道20到。 （ – 6?…
Representative Results
在SISmuc支架（ 图2A至C）的基础上，我们建立了一个三维肿瘤的测试系统（ 图2D）的产生，刺激和治疗的标准化操作协议。此模型使增殖指数和使用M30-ELISA分别示于图1和图3中 ，细胞凋亡的定量的测定。 图3示出了A549和HCC827模型代表H＆E染色和吉非替尼一个代表性的细胞凋亡测定。在这里提出的模型是用它成功…
Discussion
/在硅片肿瘤测试系统的生物标志物指导的治疗预言我们已经建立了体外组合。 在体外模型评价化合物的动作的不同的重要方面如在一个特定的突变背景也可以在计算机芯片 17模拟肿瘤细胞增殖和凋亡的变更。在这里，我们提出了三维肿瘤模型的生成和包括增殖和凋亡的量化和在硅片模型建立的预测复合测试的标准化协议。三维肿瘤模型是基于肿瘤细胞?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由该中心的维尔茨堡大学医院的多学科临床研究（IZKF，补助BD247）和拜仁飞度程序（授予海克Walles）赞助。
Materials
| Bioreactors | Chair of Tissue Engineering and Regenerative Medicine, Würzburg (GER) | Bioreactor setup | |
| BioVoxxel Toolbox (ImageJ / Fiji) | Jan Brocher, Thorsten Wagner, https://github.com/biovoxxel/BioVoxxel_Toolbox | ||
| Cell crowns | Chair of Tissue Engineering and Regenerative Medicine, Würzburg (GER) | for static 3D culture | |
| CellDesigner | http://www.celldesigner.org/ | – | This software was used for drawing the network. |
| citrate buffer stock solution (10x) | in house production | 42 g/l Citric acid monohydrate, 17.,6 g/l Sodium hydroxide pellets in deionized water, pH 6,.0, stored at RT. | |
| citrate buffer working solution | in house production | 10 % Citrate buffer stock solution in demineralized water, stored at RT. | |
| Citric acid monohydrate | VWR, Darmstadt (GER) | 1002441000 | used for the citrate buffer |
| Cover slips | VWR, Darmstadt (GER) | 631-1339 | |
| DAPI Fluoromount-GTM | SouthernBiotech, Birmingham (USA) | SBA-0100-20 | |
| Databases such as KEGG, HPRD and QIAGEN (Genes & Pathways) | http://www.genome.jp/kegg/pathway.html; http://www.hprd.org/; https://www.qiagen.com/de/geneglobe/ | – | Different known literature databases were used for generating the network topology. |
| Female Luer Lug Style Tee | Mednet, Münster (GER) | FTLT-1 | Bioreactor setup |
| Female Luer Thread Style with 5/16" Hex to 1/4-28 UNF Thread | Mednet, Münster (GER) | SFTLL-J1A | Bioreactor setup |
| Fetal calf serum | Bio&SELL, Feucht (GER) | FCS.ADD.0500 | not heat-inactivated |
| Gefitinib | Absource Diagnostics GmbH, München (GER) | S1025-100 mg | 100 mM stock solution with DMSO |
| Glas flask (Schott, GER) provided with glas hose connection | Weckert, Kitzingen (GER) | custom made | |
| Histofix 4 % (Paraformaldehyd) | Carl Roth, Karlsruhe (GER) | P087.1 | |
| Hose coupling | Mednet, Münster (GER) | CC-9 | Bioreactor setup |
| Incubator for bioreactors | Chair of Tissue Engineering and Regenerative Medicine, Würzburg (GER) | Bioreactor setup | |
| M30 CytoDeathTM ELISA | Peviva, Bromma (SWE) | 10900 | |
| Male Luer Integral Lock Ring | Mednet, Münster (GER) | MTLL230-J1A | Bioreactor setup |
| Moisture chamber | custom made | ||
| Mouse anti Pan-Cytokeratin | Sigma-Aldrich, Munich (GER) | C2562-2ML | Clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2, used 1/100 for immunofluorescence |
| Needlefree Swabable Valve Female Luer | Mednet, Münster (GER) | NVFMLLPC | Bioreactor setup, for sampling, gamma-sterilized |
| O-Ring MVQ 10 red 37*3 mm | Arcus Dichtelemente, Seevetal (GER) | 21444 | O-ring large, Bioreactor setup |
| O-Ring MVQ 70 red 27*2.5 mm | Arcus Dichtelemente, Seevetal (GER) | 19170 | O-ring small, Bioreactor setup |
| PAP pen | Dako, Hamburg (GER) | S002 | |
| Paraffin | Carl Roth, Karlsruhe (GER) | 6642.6 | |
| Peristaltic pump | Ismatec, Wertheim-Mondfeld (GER) | Bioreactor setup | |
| Phosphate Buffered Saline | Sigma-Aldrich, Munich (GER) | D8537-6x500ml | |
| Pump tubing cassette | Ismatec, Wertheim (GER) | IS 3710 | Bioreactor setup |
| Rabbit anti Ki67 | Abcam, Cambridge (UK) | ab16667 | Clone SP6, used for 1/100 for IF |
| Rabbit anti Vimentin | Abcam, Cambridge (UK) | ab92547 | used 1/100 for IF |
| RPMI-1640 medium | Life technologies, Darmstadt (GER) | 61870-044 | warm in 37°C waterbath before use |
| Silicone tube | Carl Roth GmbH, Karlsruhe (GER) | HC66.1 | Bioreactor setup |
| Sodium Hydroxide | Sigma-Aldrich, München (GER) | 30620-1KG-R | used for the citrate buffer |
| SQUAD | http://sbos.eu/docu/docu/SQUAD/doku.php.htm | – | This software was used for performing the semiquantitative simulations. |
| Sterile air filter, pore size 0.2 µm | Sartorius Stedium Biotech, Göttlingen (GER) | 16596-HYK | Bioreactor setup |
| Syringe Luer Lok 5ml | BD Biosciences, Heidelberg (GER) | 309649 | for bioreactor sampling |
| Tissue culture test plates: 6-, 12-, 24-, 96- well | TPP Techno Plastic Products AG, Trasadingen (GER) | 92006, 92012, 92024, 92048 | |
| Transforming growth factor-beta 1 (TGF-β1) with carrier | Cell Signaling, Frankfurt (GER) | 8915LC | stock solution in sterile citrate buffer pH 3.0 |
| Triton X-100 | Sigma-Aldrich, München (GER) | X100-1L | |
| Tween-20 | Sigma-Aldrich, München (GER) | P7949-500ml | for washing buffer of immunofluorescent staining |
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Cite This Article
Göttlich, C., Müller, L. C., Kunz, M., Schmitt, F., Walles, H., Walles, T., Dandekar, T., Dandekar, G., Nietzer, S. L. A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds. J. Vis. Exp. (110), e53885, doi:10.3791/53885 (2016).