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从妇科和乳腺癌肿瘤获得癌症干细胞球

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Research Article

从妇科和乳腺癌肿瘤获得癌症干细胞球

DOI: 10.3791/60022

March 1, 2020

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1Institute of Biophysics, Faculty of Medicine,University of Coimbra, 2Coimbra Institute for Clinical and Biomedical Research (iCBR), area of Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine,University of Coimbra, 3CNC.IBILI/Center for Innovative Biomedicine and Biotechnology (CIBB),University of Coimbra, 4Clinical Academic Center of Coimbra (CACC), 5Universitary Clinic of Gynecology, Faculty of Medicine,University of Coimbra, 6Gynecology A Service,Coimbra Hospital and Universitary Center, 7Institute of Pharmacology & Experimental Therapeutics, Faculty of Medicine,University of Coimbra, 8Institute of Experimental Pathology, Faculty of Medicine,University of Coimbra, 9Cytometry Operational Management Unit, Clinical Pathology Service,Coimbra Hospital and Universitary Center, 10Polytechnic Institute of Coimbra, ESTESC-Coimbra Health School,Laboratory Biomedical Sciences

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In This Article

Summary Abstract Introduction Protocol Representative Results Discussion Disclosures Acknowledgements Materials References Reprints and Permissions

Summary

该方法的目的是使用功能测定和表型表征特征，通过流式细胞学和西式细胞测定法，以强有力的方式，用球形形成方案识别癌细胞系和原发性人类肿瘤样本中的癌症干细胞（CSC）。杂交。

Abstract

癌症干细胞（CSC）是一个小群，具有自我更新和可塑性，负责肿瘤发生，抗治疗和复发性疾病。此总体可以通过表面标记、酶活动和功能配置文件进行识别。由于型型异质性和CSC可塑性，这些方法本身是有限的。在这里，我们更新球形形成协议，从乳腺癌和妇科癌症中获得CSC球体，评估功能特性、CSC标记和蛋白质表达。球体在悬浮培养中低密度下进行单细胞播种，使用半固体甲基纤维素培养基避免迁移和聚集。这种有利可图的方案可用于癌细胞系，但也可用于原发性肿瘤。三维非粘附悬浮培养，被认为模仿肿瘤微环境，特别是CSC-niche，辅以表皮生长因子和基本成纤维细胞生长因子，以确保CSC信号。为了对CSC进行强有力的识别，我们提出了一种互补的方法，将功能和体位评价结合起来。球形形成能力、自更新和球体投影区域建立 CSC 功能属性。此外，表征包括流动细胞学评估标记，代表CD44+/CD24-和CD133，和西斑点，考虑ALDH。提出的方案还针对原发性肿瘤样本进行了优化，遵循样本消化程序，可用于转化研究。

Introduction

癌症群体是异质的，由于基因表达的差异，细胞呈现不同的形态、增殖和入侵能力。在这些细胞中，少数群体存在名为癌症干细胞（CSC）1，具有自我更新的能力，重述原发性肿瘤利基的异质性，并产生异常不同的祖细胞，对静息控制2没有充分反应。CSC属性可以直接在临床实践中翻译，因为与事件有关，如肿瘤原性或对化疗的抗药性3。CSC的识别可以导致靶向疗法的发展，可能包括表面标记的堵塞，促进CSC分化，阻塞CSC信号通路成分，利基破坏和表观遗传机制4。

CSC的分离已在细胞系和原发性肿瘤5、6、7、8的样本中进行。CSC描述的功能剖面包括克隆能力、侧群和肿瘤层形成9。CD44高/CD24低表型一直与乳腺CSC相关，乳腺CSC已被证明是体内肿瘤，并且已经与上皮到中位体过渡5，10相关联。高ALDH活性也与茎和上皮到中位性过渡（EMT）在几种类型的实体肿瘤11。ALDH表达与抗药性化疗和CSC表型体外12，13，14，15，16。其他几种标记物已链接到不同类型肿瘤的CSC特性，如CD133、CD49f、ITGA6、CD163、4等，如表1所述。

肿瘤球由三维模型组成，用于CSC的研究和扩展。在此模型中，细胞系和血液或肿瘤样本中的细胞悬浮液在培养中，辅以生长因子，即表皮生长因子（EGF）和基本成纤维细胞生长因子（bFGF），无胎儿牛血清，在非粘附条件17。抑制细胞粘附导致分化细胞的阿诺基斯死亡18。球体来自分离细胞的克隆生长。为此，细胞以低密度分布，以避免细胞融合和聚集19。另一个策略包括使用半固体甲基纤维素20。

球形协议在CSC隔离和扩展中越来越受欢迎，由于时间、成本和技术、盈利和可重复的原因21，22。尽管一些关于球体形成反映CSC的保留，但干细胞有在非粘附条件下生长的倾向，其特征表型类似于原生微环境21。从实体肿瘤中分离CSC的方法都没有完全的效率，这突出表明开发更具体的标记物或方法和标记的组合的重要性。

在本协议中，我们详细介绍了CSC与球形协议的隔离，具有非粘附条件下单细胞生长原理以及产生分化表型的能力。此过程的原理图表示在图 1中。我们还描述了CSC的表面标记和ALDH表达的表征，包括乳腺和妇科肿瘤细胞系和原发性肿瘤样本。

Protocol

该协议符合科英布拉医院和大学中心（CHUC）肿瘤库的道德准则，并经CHUC卫生伦理委员会和葡萄牙国家数据保护委员会批准。

1. 连续细胞培养的球形形成协议和派生附着种群

注:在严格的无菌条件下执行所有程序。

  1. 通过在生长表面涂上聚聚物（2-羟基乙酰-甲胺酮（聚-HEMA）来制备非粘附悬浮培养瓶或板
    1. 在65°C下在绝对乙醇中搅拌聚-HEMA，制备15mg/mL溶液。涂层细胞培养瓶或板 50 μL/cm2.
    2. 在干燥炉中37°C干燥。如有必要，将板包裹并在室温下存放。
  2. 球体培养介质 （SCM） 的准备
    1. 在超纯水中制备2%的甲基纤维素溶液，并在高压灭菌器中消毒。甲基纤维素往往更容易通过冷却而溶解;因此，将粉末分散在水中80°C，搅拌至冷却23。
    2. 准备两次集中的 SCM（库存解决方案）解决方案。SCM工作溶液含有DMEM-F12，辅以100 mM putresin、1%胰岛素、转移素、硒和1%抗生素抗霉菌溶液（10，000 U/mL青霉素、10毫克/mL链霉素和25微克/mL氨基林B）。
    3. 要制备SCM，将SCM库存溶液的等量与甲基纤维素的2%溶液混合。
    4. 在使用前通过添加10纳克/mL表皮生长因子（EFG）和10纳克/mL基本成纤维细胞生长因子（bFGF）完成该介质。
    5. 如果使用更挑剔的细胞系，用0.4%牛血清白蛋白补充培养基，这可能是一个优势。
  3. 从 MCF7 或 HCC1806 乳腺癌或 ECC-1 或 RL95-2 子宫内膜癌细胞（或其他选择的癌细胞系）的烧瓶开始，80% 到 90% 汇合。
  4. 丢弃细胞培养基，用磷酸盐缓冲盐水溶液（PBS）清洗，用胰蛋白酶-EDTA分离细胞（75 cm2细胞培养瓶为1至2 mL）。
  5. 加入细胞培养基（75厘米2细胞培养瓶为2至4 mL），在200 x g下离心5分钟，以丢弃酶。
  6. 将颗粒悬浮在已知体积的细胞培养培养和移液器上和下，以确保单个细胞悬浮。为此，可以使用40μm细胞滤网。
  7. 计算血细胞计中的细胞，并计算细胞悬浮液的细胞浓度。利用此步骤，确保观察单个细胞悬浮液。仔细的细胞计数对于准确量化治疗的效果至关重要。
  8. 暂停SCM完整介质中确定数量的细胞悬浮液，并转移到多HEMA涂层培养皿中。作为播种密度的参考值，请考虑 500 到 2000 个细胞/cm2。
    注:强烈建议优化每个细胞系的种子密度和培养时间24。
  9. 在37°C和5%CO2下孵育2天，不干扰板材。
  10. 通过在细胞培养基中加入10纳克/mL EFG和10纳克/mL bFGF，重新确定生长因子的浓度。每两天重复此步骤。
  11. 在37°C和5%CO2孵育，直到电镀后5天（根据细胞系，这可以从3天到12天不等），以获得球体，这表明悬浮球状细胞菌落的形态。
  12. 使用或收集球体，通过移液，用于实验。
  13. 要获得派生的附着体，将球体置于标准培养条件中，分别用于所使用的细胞系。1至2天后，可以观察到粘附球周围生长的细胞的单层，其形态类似于细胞起源系。

2. 人类肿瘤样本的球形形成协议

注:将人体样本用于研究目的必须符合每个国家的立法，并经相关机构道德委员会批准。

  1. 准备含有DMEM/F12的运输介质，辅以10%胎儿牛血清（FBS）和2%抗生素抗霉菌溶液（10，000 U/mL青霉素，10毫克/mL链霉素和25微克/mL五聚他素B）。

Figure 1

Representative Results

球形形成协议允许从多个子宫内膜和乳腺癌细胞系（图2A）或从人类肿瘤样本中温和酶消化组织后，在悬浮液中获得球菌落（图2E）。在这两种情况下，在电镀几天后，在悬浮液中获得单克隆球菌落。子宫内膜和乳腺癌球体在电镀后1至2天产生与细胞原系形态相似的细胞单层（图2A）。

独特的血统和组织起源可以通过球形形成能力、自我更新和投影区域进行比较。在图2B-D的图表中可以观察到乳腺癌细胞系的代表性结果。激素受体阳性乳腺癌MCF7细胞表现出更高的球形形成能力，自我更新和投影面积比三阴性乳腺癌细胞HCC180614。对于两个细胞系，一小部分细胞的镀层（小于3%）能够产生强调癌症干细胞作为肿瘤细胞异质性内的少数人群的球体。癌症干细胞自我更新的专利是显着细胞系的球体自我更新的显著不同价值。球体投影区域作为球体尺寸的粗略测量，与线粒周期数相关，并显示两个谱系的不同时间间隔。

虽然只有一小部分细胞能够在体外形成肿瘤球，并保留携带干细胞特性的自更新能力，但若干标记物与这种表型有关。

提出的流式细胞测定方案允许采用多功能的实验方法，考虑表面抗原（见表1）。如图3A-B所示的代表性结果涉及CD44/CD24和CD133膜标记物，这些标记被建议与一种更像癌症的干细胞样表型相对应。对从子宫内膜RL95-2和ECC-1细胞系获得的球体进行分析，可以识别四个群体（图3A）。从子宫内膜RL95-2获得的球体包括CD44高/CD24-比亲细胞系35大三倍。在ECC-1球体的情况下，CD44高/CD24-对应于主要人群，这也是CD133正，而CD44低/CD24-CD44低/CD24+和CD44-/CD24=具有负或低CD133表达。

在温和的球体采集和精心制备蛋白质样品后，也可以通过西方标记来评估表面和细胞内标记物。图3C显示了ALDH变化的典型结果，ALDH变化是一种标记物，其活性增加或增强蛋白表达与癌症干细胞表型13、14球体和与子宫内膜ECC1细胞系相关的衍生粘附细胞有关。

Figure 2
图2:子宫内膜和乳腺癌细胞、球体和衍生粘附体。（A）.子宫内膜（RL95-2和ECC-1）和乳房（MCF7和HCC1806）癌细胞系、各自的子宫内膜（ES1）和乳房（MS1）球体和衍生粘附体（G1）的代表性图像。RL95-2、ECC-1、MCF7和HCC1806癌细胞系的代表性图像在200倍（刻度柱 = 50 μm）下得到。ES1 RL95-2 和 ES1 ECC-1 的代表性图像以 200 倍的放大倍数（刻度柱 = 50 μm）获得。MS1 MCF7 和 MS1 HCC1806 的代表性图像以 200 倍（刻度条 = 100 μm）的放大倍数获得。G1 RL95-2 和 G1 ECC-1 的代表性图像以 200 倍的放大倍数（刻度柱 = 50 μm）获得。G1 MCF7 和 G1 HCC1806 的代表性图像以 200 倍（刻度柱 = 100 μm）的放大倍数获得。（B-D）.乳腺癌球体的球形形成能力、自我更新和球体投影面积MCF7和HCC1806。（E）.从人类子宫内膜肿瘤样本中获得的球体的代表性图像。这些图像的放大倍数为 200 倍（比例尺 = 50 μm）。此图的一部分已由以前的出版物修改，并经发布者14许可。请点击此处查看此图的较大版本。

Figure 3
图3:子宫内膜癌细胞中癌症干细胞标记物的综合评价。（A）.RL95-2细胞系和RL95-2球体细胞的CD44/CD24标签的代表性图。（B）.从RL95-2和ECC-1细胞系获得的球细胞（ES1）的CD133标签代表性直方图。密度表示细胞计数的度量值。CD44+/CD24-， CD44低/CD24-，CD44低 /CD24+和 CD44-/CD24=人群分别被涂成绿色、 粉红色、 蓝色和黄色。C. ECC-1细胞系、球体（ES1）和衍生附着体（G1）中的ALDH表达。免疫蛋白表示相关实验条件的ALDH和行为蛋白表达。ALDH表达与抗体ALDH1/2一起评估，该抗体检测小鼠、大鼠和人类来源的等体ALDH1A1、ALDH1A2、ALDH1A3和ALDH2。这个数字的一部分已经修改了以前的出版物，并经出版商13的许可。请点击此处查看此图的较大版本。

表1:妇科和乳腺癌干细胞标记物列表。

标记 干细胞来源 引用
CD24 卵巢癌 60
CD29 乳腺癌 61
CD44 卵巢癌 62，63
CD44/CD24-/低 乳腺癌 13，5，3，64
CD44=/CD24-/低/ESA 乳腺癌 65
CD44/ALDH1+/hi 乳腺癌 66
CD44/CD24-/低/ABCG2 乳腺癌 67
CD44/CD24-/低/ALDH1 乳腺癌 43，68，69
CD44/CD24-/低/EPCAM 乳腺癌 5
CD44/CD24-/低/SSEA-3 乳腺癌 70
CD44/CD49f/CD133/2 乳腺癌 71
CD44/CD133/ALDH1+/hi 乳腺癌 69
CD44/CD117 卵巢癌 7
CD44/MyD88 卵巢癌 72，73
CD44/E-卡塞林-/CD34- 卵巢癌 74，75
CD44/CD24/Epcam 卵巢癌 76，77
CD44/CD24- 卵巢癌 78，79
CD44/CD166 卵巢癌 80
CD44/CD24 子宫颈癌 81
CD49f 乳腺癌 4
子宫颈癌 82
CD117 或 c-Kit 子宫内膜癌 83
卵巢癌 62，84
CD133 乳腺癌 4，85
卵巢癌 62，86
子宫内膜癌 13，87，88，89
子宫颈癌 82
CD133高/CXCR4高/ALDH1Hi 乳腺癌 90
CD133/ALDH1 乳腺癌 91
卵巢癌 60，92
CD133/CXCR4 子宫内膜癌 93
ABCG2 乳腺癌 65
子宫颈癌 82，81
ALDH-1 乳腺癌 4
子宫内膜癌 94，95
子宫颈癌 82
CXCR4 或 CD184 乳腺癌 96
EpCAM/CD49f 乳腺癌 97
EpCAMhi/PROCRhi/SSEA-3 乳腺癌 70
GD2/GD3/GD3S喜 乳腺癌 98
ITGA6 乳腺癌 4
PROCR 乳腺癌 43

注:本表提供了由各种妇科和乳腺癌干细胞表达的表面表;大多数这些标记也由一系列其他组织表达。此列表的目的不是包括报告的所有标记。

表2:在典型的流式细胞测量实验中包括的管列表，以评估CD24/CD44表型。该表显示了共同染色实验所需的最小样本管集，包括必要的控制。

管 条件 抗原-荧光酸
1 未染色的单元格 没有
2 单色CD44 CD44-PE
3 单色 CD24 CD24-APC
4 双染色 CD44/CD24 CD44-PE 和 CD24-APC

注:本实验可将附件V-FICT添加到管4中，并加入相应的控制管，以封闭附件五负细胞，并排除凋亡中的最终细胞。

Discussion

作者没有什么可透露的。

Disclosures

该方法的目的是使用功能测定和表型表征特征，通过流式细胞学和西式细胞测定法，以强有力的方式，用球形形成方案识别癌细胞系和原发性人类肿瘤样本中的癌症干细胞（CSC）。杂交。

Acknowledgements

这项研究由葡萄牙妇科学会通过2016年研究奖和CIMAGO资助。数控。IBILI由葡萄牙科学和技术基金会（UID/NEU/04539/2013）提供支持，并由FEDER-竞争（POCI-01-0145-FEDER-007440）共同资助。科英布拉医院和大学肿瘤中心（CHUC）肿瘤库，经CHUC卫生伦理委员会和葡萄牙国家数据保护委员会批准，是该院妇科服务所跟踪的患者子宫内膜样本的来源。图1是使用Servier医学艺术制作的，可从www.servier.com。

Materials

牛甲基）
无水乙醇Merck Millipore100983
AccutaseGibcoA1110501StemPro Accutas 细胞解离试剂
ALDH 抗体Santa Cruz BiotechnologySC166362
Annexin V FITCBD Biosciences556547
抗生素抗真菌溶液SigmaA5955
BCA 检测Thermo Scientific23225Pierce BCA 蛋白检测试剂盒
血清白蛋白SigmaA9418
CD133 抗体Miteny Biotec293C3-APC别藻蓝蛋白 （APC）
CD24 抗体BD Biosciences658331别藻蓝蛋白-H7 （APC-H7）
CD44 抗体Biolegend103020太平洋蓝 （PB）
细胞过滤器BD Falcon35234040 µ
M 胶原酶，IV 型Gibco17104-019
cOmplete MiniRoche118 361 700 0
二硫苏糖醇Sigma43815
DMEM-F12SigmaD8900
DNAse I罗氏11284932001
ECC-1ATCCCRL-2923人子宫内膜腺癌细胞系
表皮生长因子SigmaE9644
成纤维细胞生长因子碱性SigmaF0291
血细胞计数器VWRHERE1080339
HCC1806ATCCCRL-2335人乳腺鳞状细胞癌细胞系
胰岛素、转铁蛋白、硒溶液Gibco41400045
MCF7ATCCHTB-22人乳腺癌细胞系
纤维素AlfaAesar45490
NaClJMGS37040005002212
聚（2-羟乙基甲基丙烯酸酯SigmaP3932
腐胺SigmaP7505
RL95-2ATCCCRL-1671人子宫内膜癌细胞系
脱氧胆酸钠JMSEINECS 206-132-7
十二烷基硫酸钠Sigma436143
Tris JMGS20360000BP152112
Triton-X 100默克108603
台盼蓝SigmaT8154
胰蛋白酶-EDTASigmaT4049
ß-肌动蛋白抗体SigmaA5316

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