利用微阵列研究微环境对癌症细胞表型的影响
Summary
这里介绍的方法的目的是展示如何制造微环境微阵列 (MEMA) 并用于询问数千种简单组合微环境对培养细胞表型的影响。
Abstract
由于可溶性生长因子和基质相关蛋白在体内微环境中的混合混合,理解微环境对细胞表型的影响是一个难题。此外,用于体外微环境建模的现成试剂通常利用未完全定义的蛋白质的复杂混合物,并遭受批次到批次的变异。微环境微阵列 (MEMA) 平台允许评估数千种微环境蛋白的简单组合,以在单个测定中评估其对细胞表型的影响。MEMEA在井板中制备,允许添加单独的配体来分离含有阵列细胞外基质(ECM)蛋白质的孔。可溶性配体与每个印刷的 ECM 的组合形成了独特的组合。典型的 MEMA 测定包含超过 2,500 个独特的组合微环境,细胞在单个检测中暴露。作为测试案例,乳腺癌细胞系MCF7被镀在MEMA平台上。分析这种测定确定了增强和抑制这些细胞的生长和增殖的因素。MEMA 平台非常灵活,可扩展用于癌症研究以外的其他生物学问题。
Introduction
在二维(2D)单层塑料上培养癌细胞系仍然是癌症研究人员的主要工作之一。然而,微环境因其影响细胞表型的能力而日益得到认可。在癌症中,肿瘤微环境已知影响多种细胞行为,包括生长、存活、入侵和对治疗1、2的反应。传统的单层细胞培养通常缺乏微环境影响,这导致开发更复杂的三维(3D)检测来生长细胞,包括市售的纯化基底膜提取物。然而,这些纯化的基质通常使用复杂,并遭受技术问题,如批次变异性3和复杂组合物3。因此,很难将功能分配给可能影响细胞表型3的特定蛋白质。
为了弥补这些限制,我们开发了微环境微阵列 (MEMA) 技术,该技术将微环境简化为细胞外基质 (ECM) 和可溶性生长因子蛋白4、5 的简单组合.MEMA 平台能够识别影响细胞行为的显性微环境因素。通过使用数组格式,可以在单个实验中测定数千个微环境因子的组合。此处描述的 MEMA 询问了 2,500 种不同的独特微环境条件。印在井板的ECM蛋白形成生长垫,细胞可以赖以培养。可溶性配体被添加到单个孔中,在细胞暴露的每个不同点上创建独特的组合微环境(ECM + 配体)。细胞被培养数天,然后固定,染色和图像,以评估细胞表型作为暴露于这些特定的微环境组合的结果。由于微环境是简单的组合,因此很容易识别驱动细胞中主要型型变化的蛋白质。MEMEA已经成功地用于识别影响多细胞表型的因素,包括那些驱动细胞命运决定和对治疗4、5、6、7的反应的因素。这些反应可以在简单的2D实验中验证,然后可以在更全面地概括肿瘤微环境复杂性的条件下进行评估。MEMA 平台高度适应各种细胞类型和端点,前提是提供良好的型型生物标志物。
Protocol
注:图 1所示的流程图概述了整个 MEMA 流程(包括估计时间) 。该协议详细介绍了 8 孔板中 MEMEA 的制造。该协议可适用于其他板或幻灯片。 1. 蛋白质、稀释剂和染色缓冲液的制备 将 EMS、配体和细胞因子小瓶与室温 (RT) 和短暂离心器进行平衡。如产品数据表所示,添加适当的 RT 缓冲区的适当体积。按照制造商关于库存集中的建议进行操作…
Representative Results
为了简化微环境对细胞生长和增殖的影响,并确定促进或抑制细胞生长和增殖的条件,乳腺癌细胞系MCF7被播种在协议中描述的一组8 8孔MEMA上。这种测定使细胞接触48种不同的EMS和57种不同的配体,共2736个组合微环境条件。在培养71小时后,用EdU脉冲细胞,固定,渗透,并沾染DAPI,EdU检测反应,抗纤维蛋白抗体,抗H3K9me3抗体。细胞在高含量显微镜上成像。图像被上传到一个Omero服务器10,分段使?…
Discussion
“维数”和语境的重要性一直是体外培养系统发展的动力,通过它们与微环境的相互作用,以及体外细胞的能力,作为癌细胞表征的工具。模仿体内环境的文化系统是寻求改善这些文化系统的动力。然而,体外系统仍然是癌症研究的重要工具,正是因为它们能够将复杂的体内情况提炼为简化的12型。
虽然 2D 系统可以包括 EVM 和配体,但它们传统上缺乏?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH网络蜂窝签名共同基金图书馆(LINCS)赠款HG008100(J.W.G.、L.M.H.和J.E.K)的支持。
Materials
| Aushon 2470 | Aushon BioSystems | Arrayer robot system used in the protocol | |
| Nikon HCA | Nikon | High Content Imaging system designed around Nikon Eclipse Ti Inverted Microscope | |
| BioTek Precision XS liquid Handler | BioTek | liquid handling robot used in the protocol | |
| Trizma hydrochloride buffer solution | Sigma | T2069 | |
| EDTA | Invitrogen | 15575-038 | |
| Glycerol | Sigma | G5516 | |
| Triton X100 | Sigma | T9284 | |
| Tween 20 | Sigma | P7949 | |
| Kolliphor P338 | BASF | 50424591 | |
| 384-well microarray plate, cylindrical well | Thermo Fisher | ab1055 | |
| Nunc 8 well dish | Thermo Fisher | 267062 | |
| Paraformaldehyde 16% solution | Electron Microscopy Science | 15710 | |
| BSA | Fisher | BP-1600 | |
| Sodium Azide | Sigma | S2002 | |
| Cell Mask | Molecular Probes | H32713 | |
| Click-iTEdU Alexa Fluor | Molecular Probes | C10357 | |
| DAPI | Promo Kine | PK-CA70740043 | |
| ALCAM | R & D Systems | 656-AL | ECM |
| Cadherin-20 (CDH20) | R & D Systems | 5604-CA | ECM |
| Cadherin-6 (CDH6) | R & D Systems | 2715-CA | ECM |
| Cadherin-8 (CDH8) | R & D Systems | 188-C8 | ECM |
| CD44 | R & D Systems | 3660-CD | ECM |
| CEACAM6 | R & D Systems | 3934-CM | ECM |
| Collagen I | Cultrex | 3442-050-01 | ECM |
| Collagen Type II | Millipore | CC052 | ECM |
| Collagen Type III | Millipore | CC054 | ECM |
| Collagen Type IV | Sigma | C5533 | ECM |
| Collagen Type V | Millipore | CC077 | ECM |
| COL23A1 | R & D Systems | 4165-CL | ECM |
| Desmoglein 2 | R & D Systems | 947-DM | ECM |
| E-cadherin (CDH1) | R & D Systems | 648-EC | ECM |
| ECM1 | R & D Systems | 3937-EC | ECM |
| Fibronectin | R & D Systems | 1918-FN | ECM |
| GAP43 | Abcam | ab114188 | ECM |
| HyA-500K | R & D Systems | GLR002 | ECM |
| HyA-50K | R & D Systems | GLR001 | ECM |
| ICAM-1 | R & D Systems | 720-IC | ECM |
| Laminin | Sigma | L6274 | ECM |
| Laminin-5 | Abcam | ab42326 | ECM |
| Lumican | R & D Systems | 2846-LU | ECM |
| M-Cad (CDH15) | R & D Systems | 4096-MC | ECM |
| Nidogen-1 | R & D Systems | 2570-ND | ECM |
| Osteoadherin/OSAD | R & D Systems | 2884-AD | ECM |
| Osteopontin (SPP) | R & D Systems | 1433-OP | ECM |
| P-Cadherin (CDH3) | R & D Systems | 861-PC | ECM |
| PECAM1 | R & D Systems | ADP6 | ECM |
| Tenascin C | R & D Systems | 3358-TC | ECM |
| VCAM1 | R & D Systems | ADP5 | ECM |
| vitronectin | R & D Systems | 2308-VN | ECM |
| Biglycan | R & D Systems | 2667-CM | ECM |
| Decorin | R & D Systems | 143-DE | ECM |
| Periostin | R & D Systems | 3548-F2 | ECM |
| SPARC/osteonectin | R & D Systems | 941-SP | ECM |
| Thrombospondin-1/2 | R & D Systems | 3074-TH | ECM |
| Brevican | R & D Systems | 4009-BC | ECM |
| Elastin | BioMatrix | 5052 | ECM |
| Fibrillin | Lynn Sakai Lab OHSU | N/A | ECM |
| ANGPT2 | RnD_Systems_Own | 623-AN-025 | Ligand |
| IL1B | RnD_Systems_Own | 201-LB-005 | Ligand |
| CXCL8 | RnD_Systems_Own | 208-IL-010 | Ligand |
| IGF1 | RnD_Systems_Own | 291-G1-200 | Ligand |
| TNFRSF11B | RnD_Systems_Own | 185-OS | Ligand |
| BMP6 | RnD_Systems_Own | 507-BP-020 | Ligand |
| FLT3LG | RnD_Systems_Own | 308-FK-005 | Ligand |
| CXCL1 | RnD_Systems_Own | 275-GR-010 | Ligand |
| DLL4 | RnD_Systems_Own | 1506-D4-050 | Ligand |
| HGF | RnD_Systems_Own | 294-HGN-005 | Ligand |
| Wnt5a | RnD_Systems_Own | 645-WN-010 | Ligand |
| CTGF | Life_Technologies_Own | PHG0286 | Ligand |
| LEP | RnD_Systems_Own | 398-LP-01M | Ligand |
| FGF2 | Sigma_Aldrich_Own | SRP4037-50UG | Ligand |
| FGF6 | RnD_Systems_Own | 238-F6 | Ligand |
| IL7 | RnD_Systems_Own | 207-IL-005 | Ligand |
| TGFB1 | RnD_Systems_Own | 246-LP-025 | Ligand |
| PDGFB | RnD_Systems_Own | 220-BB-010 | Ligand |
| WNT10A | Genemed_Own | 90009 | Ligand |
| PTN | RnD_Systems_Own | 252-PL-050 | Ligand |
| BMP3 | RnD_Systems_Own | 113-BP-100 | Ligand |
| BMP4 | RnD_Systems_Own | 314-BP-010 | Ligand |
| TNFSF11 | RnD_Systems_Own | 390-TN-010 | Ligand |
| CSF2 | RnD_Systems_Own | 215-GM-010 | Ligand |
| BMP5 | RnD_Systems_Own | 615-BMC-020 | Ligand |
| DLL1 | RnD_Systems_Own | 1818-DL-050 | Ligand |
| NRG1 | RnD_Systems_Own | 296-HR-050 | Ligand |
| KNG1 | RnD_Systems_Own | 1569-PI-010 | Ligand |
| GPNMB | RnD_Systems_Own | 2550-AC-050 | Ligand |
| CXCL12 | RnD_Systems_Own | 350-NS-010 | Ligand |
| IL15 | RnD_Systems_Own | 247-ILB-005 | Ligand |
| TNF | RnD_Systems_Own | 210-TA-020 | Ligand |
| IGFBP3 | RnD_Systems_Own | 675-B3-025 | Ligand |
| WNT3A | RnD_Systems_Own | 5036-WNP-010 | Ligand |
| PDGFAB | RnD_Systems_Own | 222-AB | Ligand |
| AREG | RnD_Systems_Own | 262-AR-100 | Ligand |
| JAG1 | RnD_Systems_Own | 1277-JG-050 | Ligand |
| BMP7 | RnD_Systems_Own | 354-BP-010 | Ligand |
| TGFB2 | RnD_Systems_Own | 302-B2-010 | Ligand |
| VEGFA | RnD_Systems_Own | 293-VE-010 | Ligand |
| IL6 | RnD_Systems_Own | 206-IL-010 | Ligand |
| CXCL12 | RnD_Systems_Own | 351-FS-010 | Ligand |
| NRG1 | RnD_Systems_Own | 378-SM | Ligand |
| IGFBP2 | RnD_Systems_Own | 674-B2-025 | Ligand |
| SHH | RnD_Systems_Own | 1314-SH-025 | Ligand |
| FASLG | RnD_Systems_Own | 126-FL-010 | Ligand |
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Cite This Article
Smith, R., Devlin, K., Kilburn, D., Gross, S., Sudar, D., Bucher, E., Nederlof, M., Dane, M., Gray, J. W., Heiser, L., Korkola, J. E. Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer. J. Vis. Exp. (147), e58957, doi:10.3791/58957 (2019).