人类乳腺上皮细胞致癌转化的体外评价
1Department of Cell Biology, Physiology and Immunology,Universitat Autònoma de Barcelona, 2Optical Microscopy Core Facilities,IDIBELL, 3Hereditary Cancer Program, Catalan Institute of Oncology,IDIBELL, 4Program in Molecular Mechanisms and Experimental Therapy in Oncology (Oncobell),IDIBELL, 5New Therapeutic Strategies in Cancer Group. Department of Biochemistry and Molecular Biomedicine, School of Biology, Institute of Biomedicine,University of Barcelona (IBUB), 6Department of Biochemistry & Physiology, School of Pharmacy and Food Sciences,University of Barcelona, 7Department of Biosciences, Faculty of Sciences and Technology,University of Vic
Summary
该协议提供实验体外工具，以评估人类乳腺细胞的转化。介绍了后续细胞增殖率、锚地独立生长能力以及3D培养基中细胞系系与地下室膜基质分布的详细步骤。
Abstract
肿瘤生成是一个多步骤的过程，细胞获得能力，允许其生长，生存和传播在敌对条件下。不同的测试试图识别和量化癌细胞的这些特征;然而，他们往往专注于细胞转换的一个方面，事实上，需要多重测试，以适当的表征。这项工作的目的是为研究人员提供一套工具，从广泛的视角评估细胞体外转化，从而有可能得出合理的结论。
持续增殖信号激活是肿瘤组织的主要特征，可以通过计算随着时间的推移实现的种群数量翻倍，在体外条件下可以轻松监测。此外，3D培养物中的细胞生长允许它们与周围细胞的相互作用，类似于体内发生的情况。这能够评估细胞聚集，并结合独特的细胞标记的免疫荧光标记，获得关于肿瘤转化的另一个相关特征的信息：失去适当的组织。转化细胞的另一个显著特征是它们在不附着于其他细胞和细胞外基质的情况下生长的能力，可以通过锚定测定来评估。
提供了详细的实验程序，以评估细胞生长率，在3D培养物中对细胞系系标记进行免疫荧光标记，并测试软糖中与锚系无关的细胞生长。由于乳腺原发性细胞（BPEC）与乳腺癌的相关性，这些方法针对乳原细胞（BPEC）进行了优化;但是，在进行某些调整后，可以应用于其他单元格类型的过程。
Introduction
肿瘤发育需要多个连续事件。2011年，Hanahan和温伯格描述了10种能够转化细胞生长、存活和传播的能力：所谓的”癌症的霍尔克“1。此处描述的方法汇编了三种不同的工具，通过关注肿瘤细胞的一些显著特征来评估体外细胞转化。这些技术评估细胞增殖率、3D培养细胞的行为及其形成具有锚定独立性的菌落的能力。
细胞模型对于体外检验假说至关重要。为研究癌症2、3、4，开发了不同的方法来生成细胞转化的实验模型。由于乳腺癌是全世界女性最常见的癌症，占5岁女性癌症死亡的15%左右，因此，为乳腺上皮细胞提供合适的细胞模型对于进一步研究至关重要。在这篇文章中，我们说明了三种技术的潜力，评估细胞转化使用实验模型的乳房原发性上皮细胞（BPECs）转化最初描述由因斯和他的同事在2007年6日，后来在我们的实验室7实施。该实验模型基于三个靶向基因（SV40大T和小T抗原（此处称为Ttag、hTERT和HRAS）对非转化BPE的基因组的连续变化。 此外，用于BPECs衍生的方法有利于维持乳腺上皮细胞与发光或骨髓标记，导致异质细胞培养，保留一些乳腺生理特征。
在乳腺中，负责牛奶生产的发光乳腺上皮细胞位于流明附近，而骨髓细胞位于发光细胞周围，并照顾导致乳汁到奶嘴的收缩运动。这些细胞系间失去适当的组织是肿瘤转化8的一个特征，可以在免疫 荧光检测3D细胞培养物中独特的血统标记后进行体外评估。肿瘤细胞的另一个主要特征是它们在不附着其他细胞和细胞外基质1的情况下生长的能力。当健康细胞被迫在悬浮过程中生长时， 阿诺基斯 \u2012等机制因与细胞外基质分离而诱发的细胞死亡类型被激活9。细胞死亡的逃避是癌症的显著特征之一，因此，转化的细胞能够灭活阿 诺基 ，以独立于锚的方式生存。这种能力可以在体外评估与锚地无关的测定使用软阿加。此外，肿瘤组织的一个固有特征是其持续的增殖信号能力，这种能力可以通过测量细胞数随时间的增加，不仅在悬浮测定中，而且通过监测单层粘附培养物的生长速度，在体外条件下易于监测。
尽管测试肿瘤原能的最佳模型是接种穆林模型中的肿瘤细胞和原位肿瘤发育评估，但尽可能减少实验程序中使用的动物数量非常重要。因此，进行适当的测试以评估体外转化是重中之重。在这里，我们提供一套工具来评估部分和完全转化的乳房上皮细胞的肿瘤原位，这些细胞在大多数使用细胞转化模型的实验室中都可以轻松实施。
Protocol
在以下实验中使用的人类样本是从在标准程序同意下在克拉尼卡皮拉尔圣 若尔迪 （巴塞罗那）进行的减少乳腺手术中获得的。除非另有说明，所有程序在 II 类生物安全柜中执行。 1. 人类乳腺上皮细胞的体外培养和生长曲线图的积累 乳房原发性上皮细胞（BPECs）的体外培养：细胞传通注：对于 BPEC 衍生和细胞培养，请遵循 Ince 等人描述的说明，…
Representative Results
选择在BPECs中引入三种遗传元素的细胞转化实验模型，产生具有代表性的致生转化结果6、7（图3）。非转化的BPEC （N） 来自无病乳房组织，如因斯及其同事6所述，按照此处所示的协议进行培养。在克服STAGSIS（压力或异常信号诱导-衰老，一种通常在体外乳腺上皮细胞中观察到的现象，在细胞培…
Discussion
本文描述的实验方案为评估体外培养细胞的致癌转化提供了有用的工具。每种技术都评估转换过程的具体方面，因此，在从单个分析中得出结论时，必须特别注意。生长曲线积累是一种在培养细胞用于其他目的时需要已有信息的方法。与其他细胞增殖测定相比，这使得这项技术更便宜、更容易应用。然而，为了获得有效的结果，每次对细胞进行亚栽培时，必须特别注意其计数和播种。增长率的提?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
AG实验室由西班牙核安全委员会资助。T.A. 和 A.G. 是加泰罗尼亚总分类（2017-SGR-503） 认可的研究小组的成员。MT拥有一份由西班牙科学基金会资助的合同[AECC-INVES19022TERR]。G.F. 合同由 Cellex 基金会的赠款资助。
Materials
| 1 ml Serological Pipettes | Labclinics | PLC91001 | |
| 1.5 ml Eppendorfs | Thermo Fisher Scientific | 3451 | Dark eppendorfs are preferred for MTT long-term storage |
| 10 μl Pipette tips w/o filter | Biologix | 20-0010 | |
| 100 ml glass bottle | With cap, autoclavable | ||
| 1000 μl Pipette tips w/ filter | Labclinics | LAB1000ULFNL | |
| 1000 μl Pipette tips w/o filter | Biologix | 20-1000 | |
| 15 ml Conical tubes | VWR | 525-0400 | |
| 2 ml Serological Pipettes | Labclinics | PLC91002 | |
| 200 μl Pipette tips w/ filter | Labclinics | FTR200-96 | |
| 5 ml Serological Pipettes | Labclinics | PLC91005 | |
| 50 ml Conical Tubes | VWR | 525-0304 | |
| Acetone | PanReac AppliChem | 211007 | Used for 3D structure fixation prior to immunofluorescent labelling |
| Agar | Sigma-Aldrich | A1296 | Used for anchorage assay |
| Anti-Claudin 4 antibody | Abcam | 15104, RRID:AB_301650 | Working dilution 1:100, host: rabbit |
| Anti-Cytokeratin 14 [RCK107] antibody | Abcam | 9220, RRID:AB_307087 | Working dilution 1:100, host: mouse |
| Anti-mouse Cyanine Cy3 antibody | Jackson ImmunoResearch Inc. | 115-165-146, RRID:AB_2338690 | Working dilution 1:500, host: goat |
| Anti-rabbit Alexa Fluor 488 antibody | Thermo Fisher Scientific | A-11034, RRID:AB_2576217 | Working dilution 1:500, host: goat |
| Autoclave | |||
| BioVoxxel Toolbox | RRID:SCR_015825 | ||
| Cell culture 24-well Plate | Labclinics | PLC30024 | Used for 3D cultures in Matrigel. Flat Bottom |
| Cell culture 6-well Plate | Labclinics | PLC30006 | Used for anchorage assay |
| Cell incubator (37 ºC and 5 % CO2) | |||
| Cell Strainers | Fisherbrand | 11587522 | Mesh size: 40 μm |
| CellSense software | Olympus | Used to image acquisition | |
| Centrifuge | |||
| Cholera Toxin from Vibrio cholerae | Sigma-Aldrich | C8052 | Used to supplement cell culture medium |
| Class II Biological Safety Cabinet | Herasafe | HAEREUS HS12 | |
| Confocal inverted Microscope | Leica | TCS SP5 | |
| Cover glasses | Witeg Labortechnik GmbH | 4600122 | 22 X 22 mm, thickness 0.13 – 0.17 mm |
| DAPI | 2-(4-amidinophenyl)-1H -indole-6-carboxamidine | ||
| Fetal Bovine Serum | Biowest | S1810 | Used to inactivate trypsine action |
| Fiji software (ImageJ) | National Institutes of Health | RRID:SCR_002285 | Free download, no license needed |
| Glass Pasteur Pipettes | |||
| Glass slides | Fisherbrand | 11844782 | |
| Goat Serum | Biowest | S2000 | Used for immunofluorescence of 3D structures |
| Heat-Resistant Gloves | Used for agar manipulation after autoclave | ||
| Heater bath (37 ºC) | Used to temper solutions prior to cell subculture | ||
| Heater bath (42 ºC) | Used to keep agar warm | ||
| Heating plate | Used for Matrigel dehydration | ||
| Humid chamber | Used for the incubation of antibodies during immunofluorescence | ||
| Ice | Used during Matrigel manipulation | ||
| Ice-box | |||
| Inverted Optic Microscope | Olympus | IX71 | |
| Matrigel Matrix | Becton Dickinson | 354234 | Store at -20 ºC and keep cold when in use. Referred to as basement membrane matrix |
| Methanol | PanReac AppliChem | 131091 | Used for 3D structure fixation prior to immunofluorescent labelling |
| Micropipette | p1000, p200 and p10 | ||
| Microsoft Office Excel | Microsoft | RRID:SCR_016137 | Used to calculate population doubling and to obtain growth rate equation |
| MilliQ water | Referred to as ultrapure water | ||
| Nail Polish | Used to seal samples after mounting | ||
| Parafilm M | Bemis | PM-999 | Used to cover antibody solution during incubation |
| PBS pH 7.4 (w/o calcium & magnesium) | Gibco | 10010-056 | Sterile. Used for cell subculture |
| PBS tablets | Sigma-Aldrich | P4417 | Dilute in milliQ water. No sterility required. Used for immunofluorescence |
| Pipette Aid | |||
| Primaria T25 flasks | Corning | 353808 | Used for BPEC culture |
| Scepter Automated Cell Counter | Millipore | PHCC20060 | Alternatively, use an haemocytometer |
| Scissors | Used to cut pipette tips and parafilm | ||
| Sterile filters 0.22 μm | Millipore | SLGP033RS | Used to filter MTT solution |
| Thiazolyl Blue Tetrazolium Bromide (MTT) | Sigma-Aldrich | M2128 | Store at -20 ºC |
| Triton X-100 | Sigma-Aldrich | T8787 | Used for immunofluorescence of 3D structures |
| Trypsin-EDTA 10X | Biowest | X0930 | Dilute in PBS to obtain 3X solution |
| Vectashield Antifade Mounting Medium | Vector Laboratories | H-1000 | |
| WIT-P-NC Culture Medium | Stemgent | 00-0051 | Used for primary BPEC culture |
| WIT-T Culture Medium | Stemgent | 00-0047 | Used for transformed BPEC culture |
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Cite This Article
Repullés, J., Terradas, M., Fuster, G., Genescà, A., Anglada, T. In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells. J. Vis. Exp. (163), e61716, doi:10.3791/61716 (2020).