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从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Published: October 02, 2020
doi:
10.3791/61775
, , , ,
1London Regional Cancer Program, 2Department of Anatomy & Cell Biology,Western University, 3Department of Oncology,Western University, 4Lawson Health Research Institute

Summary

该实验方案描述了从乳腺癌细胞和组织样品中分离BCSC以及可用于评估BCSC表型和功能的 体外 和 体内 测定。

Abstract

乳腺癌干细胞（BCSC）是具有遗传或获得性干细胞样特征的癌细胞。尽管它们的频率很低，但它们是乳腺癌发生、复发、转移和治疗耐药性的主要贡献者。了解乳腺癌干细胞的生物学必须确定治疗乳腺癌的新治疗靶点。乳腺癌干细胞是根据独特的细胞表面标志物（如CD44、CD24）和醛脱氢酶（ALDH）的酶活性）的表达进行分离和表征的。这些 ALDH高CD44+CD24– 细胞构成了 BCSC 群体，可通过荧光激活细胞分选 （FACS） 进行下游功能研究。根据科学问题，可以使用不同的 体外 和 体内 方法来评估BCSC的功能特征。在这里，我们提供了一个详细的实验方案，用于从乳腺癌细胞的异质群体以及从乳腺癌患者获得的原发性肿瘤组织中分离人类BCSC。此外，我们还重点介绍了下游体 外 和 体内 功能测定，包括集落形成测定、乳腺球测定、3D 培养模型和可用于评估 BCSC 功能的肿瘤异种移植测定。

Introduction

了解人类乳腺癌干细胞（BCSC）的细胞和分子机制对于解决乳腺癌治疗中遇到的挑战至关重要。BCSC概念的出现可以追溯到21世纪初，当时发现少量CD44 + CD24-/低乳腺癌细胞能够在小鼠中产生异质肿瘤1，2。随后，观察到具有高酶活性的醛脱氢酶（ALDH高）的人乳腺癌细胞也表现出类似的干细胞样性质3。这些BCSC代表能够自我更新和分化的细胞群，有助于本体肿瘤的异质性1，2，3。越来越多的证据表明，进化保守信号通路的改变推动了BCSC的存活和维持4，5，6，7，8，9，10，11，12，13，14.此外，细胞外在微环境已被证明在决定不同的BCSC功能中起着关键作用15，16，17。这些分子途径和调节BCSC功能的外部因素有助于乳腺癌复发，转移18和对治疗耐药性的发展19，20，21，治疗后BCSC的残留存在对乳腺癌患者的总体生存构成重大挑战22，23.因此，这些因素的临床前评估对于确定BCSC靶向疗法非常重要，这些疗法可能有利于实现更好的治疗结果和提高乳腺癌患者的总生存期。

几种体外人乳腺癌细胞系模型和体内人异种移植模型已被用于表征BCSCs24，25，26，27，28，29。细胞系在每次连续传代后不断重新填充的能力使其成为进行基于组学和药物基因组学研究的理想模型系统。然而，细胞系通常无法概括在患者样本中观察到的异质性。因此，用患者来源的样本补充细胞系数据非常重要。以最纯净的形式分离BCSC对于实现BCSC的详细表征非常重要。 实现这种纯度取决于BCSC特有的表型标记的选择。 目前，ALDH高CD44+CD24– 细胞表型最常用于使用荧光活化细胞分选 （FACS） 从大量乳腺癌细胞群中区分和分离人 BCSC，以获得最大纯度1，3，26.此外，可以使用体外和体内技术评估分离的BCSCs的特性，例如自我更新，增殖和分化。

例如，体外集落形成测定可用于评估单个细胞在不同治疗条件下自我更新以形成50个或更多细胞集落的能力30。乳腺球测定也可用于评估乳腺癌细胞在锚定非依赖条件下的自我更新潜力。该测定法测量单细胞在无血清非贴壁培养条件下每次连续传代时以球体（BCSC 和非 BCSC 的混合物）生成和生长的能力31。此外，三维（3D）培养模型可用于评估BCSC功能，包括细胞-细胞和细胞-基质相互作用，这些相互作用密切概括了体内微环境，并允许研究潜在的BCSC靶向疗法的活性32。尽管体外模型的应用多种多样，但仅使用体外测定很难模拟体内条件的复杂性。这一挑战可以通过使用小鼠异种移植模型来评估体内BCSC行为来克服。特别是，这些模型可作为评估乳腺癌转移33，研究疾病进展期间与微环境的相互作用34，体内成像35以及预测患者特异性毒性和抗肿瘤药物功效的理想系统34。

该协议详细描述了从大量异质乳腺癌细胞群中以最大纯度分离人ALDH高CD44 + CD24-BCSC。我们还详细介绍了三种体外技术（集落形成测定、乳腺球测定和 3D 培养模型）和可用于评估 BCSC 不同功能的体内肿瘤异种移植测定。这些方法适合于有兴趣从人类乳腺癌细胞系或原发性患者来源的乳腺癌细胞和肿瘤组织中分离和表征BCSC的研究人员使用，以了解BCSC生物学和/或研究新的BCSC靶向疗法。

Protocol

直接从同意的乳腺癌患者那里收集患者来源的手术或活检样本是根据机构伦理委员会批准的批准的人类伦理协议进行的。所有用于生成患者来源的异种移植模型的小鼠均被维持并饲养在机构批准的动物设施中。使用小鼠的患者来源的异种移植模型的肿瘤组织是根据机构动物护理委员会批准的批准的伦理方案生成的。 1. 细胞系的制备 在生物安全柜的无菌条件下执行所有…

Representative Results

所描述的方案允许从异质的乳腺癌细胞群中分离人BCSC，无论是来自细胞系还是从解离的肿瘤组织中。对于任何给定的细胞系或组织样品，产生均匀的单细胞悬液以最大纯度分离BCSC至关重要，因为污染的非BCSC群体可能导致不同的细胞反应，特别是如果研究目的是评估靶向BCSC的治疗剂的功效。 应用严格的分选策略将最大限度地减少污染非BCSC的存在，并导致收集乳腺癌细胞比例的能力具有干细胞样?…

Discussion

乳腺癌转移和对治疗的抵抗已成为全世界妇女死亡的主要原因。乳腺癌干细胞（BCSC）亚群的存在有助于增强转移26，43，44，45，46和治疗耐药性21，47，48。因此，未来治疗的重点应旨在根除BCSC以达到更好?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢实验室成员的有益讨论和支持。我们对乳腺癌干细胞和肿瘤微环境的研究由加拿大癌症研究学会研究所和美国陆军国防部乳腺癌计划（赠款#BC160912）资助。V.B.由西部博士后奖学金（西部大学）支持，A.L.A.和V.B.都得到加拿腺癌协会的支持。C.L.得到了加拿大政府颁发的Vanier加拿大研究生奖学金的支持。

Materials

7-Aminoactinomycin D (7AAD) BD 51-68981E suggested: 0.25 µg/1×106 cells
Acetone Fisher A18-1
Aldehyde dehydrogenase (ALDH) substrate Stemcell Technologies 1700 Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer's instructions for ALDH substrate preparation
Basement membrane extract (BME) Corning 354234 Sold under the commercial name Matrigel
Cell culture plates: 6 well Corning 877218
Cell culture plates: 60mm Corning 353002
Cell culture plates: 96-well ultra low attachment Corning 3474
Cell strainer: 40 micron BD 352340
Collagen Stemcell Technologies 7001 Prepare 1:30 dilution of 3 mg/mL collagen in PBS
Collagenase Sigma 11088807001 1x
Conical tubes: 50 mL Fisher scientific 05-539-7
Crystal violet Sigma C6158 Use 0.05% crystal violet solution in water for staining
Dispase Stemcell Technologies 7913 5U/mL
DMEM:F12 Gibco 11330-032 1x, With L-glutamine and 15 mM HEPES
DNAse Sigma D5052 0.1 mg/mL final concentration
FBS Avantor Seradigm Lifescience 97068-085  
Flow tubes: 5ml BD 352063 Polypropylene round-bottom tubes
Methanol Fisher 84124
mouse anti-Human CD24 antibody BD 561646 R-phycoerythrin and Cyanine dye conjugated Clone: ML5
mouse anti-Human CD44 antibody BD 555479 R-phycoerythrin conjugated, Clone: G44-26
N,N-diethylaminobenzaldehyde (DEAB) Stemcell Technologies 1700 Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer's instructions DEAB preparation
PBS Wisent Inc 311-425-CL 1x, Without calcium and magnesium
Trypsin-EDTA Gibco 25200-056
Mammosphere Media Composition
B27 Gibco 17504-44 1x
bFGF Sigma F2006 10 ng/mL
BSA Bioshop ALB003 04%
DMEM:F12 Gibco 11330-032 1x, With L-glutamine and 15 mM HEPES
EGF Sigma E9644 20 ng/mL
Insulin Sigma 16634 5 µg/mL
3D Organoid Media Composition
A8301 Tocris 2939 500 nM
B27 Gibco 17504-44 1x
DMEM:F12 Gibco 11330-032 1x, With L-glutamine and 15 mM HEPES
EGF Sigma E9644 5 ng/mL
FGF10 Peprotech 100-26 20 ng/mL
FGF7 Peprotech 100-19 5 ng/mL
GlutaMax Invitrogen 35050-061 1x
HEPES Gibco 15630-080 10 mM
N-acetylcysteine Sigma A9165 1.25 mM
Neuregulin β1 Peprotech 100-03 5 nM
Nicotinamide Sigma N0636 5 mM
Noggin Peprotech 120-10C 100 ng/mL
R-spondin3 R&D 3500 250 ng/mL
SB202190 Sigma S7067 500 nM
Y-27632 Tocris 1254 5 µM
DOWNLOAD MATERIALS LIST

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Tags

Breast Cancer Stem CellsCell IsolationSingle Cell SuspensionALDH AssayColony Forming AssayFunctional CharacterizationTumorigenicityMetastasis
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Bhat, V., Lefebvre, C., Goodale, D., Rodriguez-Torres, M., Allan, A. L. Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples. J. Vis. Exp. (164), e61775, doi:10.3791/61775 (2020).
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