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Cancer Research

从妇科和乳腺癌肿瘤获得癌症干细胞球

Published: March 01, 2020
doi:
10.3791/60022
, , , , , , ,
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

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。 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 在65°C下在绝对乙醇中搅拌聚-HEMA，制?…

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）。 <p cla…

Discussion

该协议详细说明了从癌细胞系和原发性人体样本中获取肿瘤球的方法。肿瘤球在亚群中富集，具有干细胞样特性36。CSC中的这种富集取决于无锚定环境中的生存能力，而分化的细胞则依赖于附着在基质37上。由于肿瘤细胞在低粘附环境中的初级电镀，在施加悬浮液本身并不确保浓缩，我们提供策略来评估自我更新（球形形成能力和自我更新），分化能力（衍生粘…

Disclosures

The authors have nothing to disclose.

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

Absolute ethanol Merck Millipore 100983
Accutase Gibco A1110501 StemPro Accutas Cell Dissociation Reagent
ALDH antibody Santa Cruz Biotechnology SC166362
Annexin V FITC BD Biosciences 556547
Antibiotic antimycotic solution Sigma A5955
BCA assay Thermo Scientific 23225 Pierce BCA Protein Assay Kit
Bovine serum albumin Sigma A9418
CD133 antibody Miteny Biotec 293C3-APC Allophycocyanin (APC)
CD24 antibody BD Biosciences 658331 Allophycocyanin-H7 (APC-H7)
CD44 antibody Biolegend 103020 Pacific Blue (PB)
Cell strainer BD Falcon 352340 40 µM
Collagenase, type IV Gibco 17104-019
cOmplete Mini Roche 118 361 700 0
Dithiothreitol Sigma 43815
DMEM-F12 Sigma D8900
DNAse I Roche 11284932001
ECC-1 ATCC CRL-2923 Human endometrium adenocarcinoma cell line
Epidermal growth factor Sigma E9644
Fibroblast growth factor basic Sigma F0291
Haemocytometer VWR HERE1080339
HCC1806 ATCC CRL-2335 Human mammary squamous cell carcinoma cell line
Insulin, transferrin, selenium Solution Gibco 41400045
MCF7 ATCC HTB-22 Human mammary adenocarcinoma cell line
Methylcellulose AlfaAesar 45490
NaCl JMGS 37040005002212
Poly(2-hydroxyethyl-methacrylate Sigma P3932
Putrescine Sigma P7505
RL95-2 ATCC CRL-1671 Human endometrium carcinoma cell line
Sodium deoxycholic acid JMS EINECS 206-132-7
Sodium dodecyl sulfate Sigma 436143
Tris JMGS 20360000BP152112
Triton-X 100 Merck 108603
Trypan blue Sigma T8154
Trypsin-EDTA Sigma T4049
��-actin antibody Sigma A5316
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Cancer Stem CellsCancer Stem Cell SpheresSphere-forming AssayPolyHEMANon-adherent Suspension CultureEpidermal Growth FactorBasic Fibroblast Growth FactorSphere-forming CapacityAdherent SpheresHemocytometerGynecological CancerBreast Cancer

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Laranjo, M., Carvalho, M. J., Serambeque, B., Alves, A., Marto, C. M., Silva, I., Paiva, A., Botelho, M. F. Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors. J. Vis. Exp. (157), e60022, doi:10.3791/60022 (2020).
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