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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

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Research Article

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

DOI: 10.3791/61775

October 2, 2020

, , , ,

1London Regional Cancer Program, 2Department of Anatomy & Cell Biology,Western University, 3Department of Oncology,Western University, 4Lawson Health Research Institute

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In This Article

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

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

  1. 在生物安全柜的无菌条件下执行所有细胞培养和染色程序。使用无菌细胞培养皿/烧瓶和试剂。
  2. 在补充有胎牛血清（FBS）和每个细胞系特异性的必要生长因子的定义培养基中用5%CO2 将人乳腺癌细胞维持在37°C。





  11. &B

Figure 1

Figure 2






Figure 3





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特征，显示出将它们与大量癌细胞区分开来的细胞表型。表现出增强的 ALDH 酶活性、表达高水平的细胞表面标志物 CD44 和低/阴性表达的 CD24 的人乳腺癌细胞具有 ALDH高CD44+CD24- 表型，可归类为 BCSC。散装群体中BCSC的比例可能因细胞系或患者而异（图2），并且通常取决于疾病阶段，更具侵袭性的乳腺癌通常显示更高比例的BCSCs26，36，37。

分离的BCSC可用于进行不同的 体外 和 体内 测定，其中其行为和功能可以与大量和/或非BCSC群体的行为和功能进行比较。例如，单个乳腺癌细胞自我更新和产生50个细胞集落的能力可以通过集落形成测定来评估（图3A）。BCSC在锚地无关的实验条件下自我更新的能力可以通过乳球测定进行评估，其中可以分析可变球体数量，大小和球起始能力，并与BCSC的存在和功能相关联（图3B）。确定不同乳腺癌细胞系或乳腺肿瘤样本的接种细胞密度以获得最佳结果非常重要。这在进行SLDA时尤其重要，因为较高的细胞密度可能导致细胞聚集，从而导致对细胞活动的误解。

在BME中培养乳腺癌细胞允许BCSC形成3D结构，概括 体内 条件（图3C）。在其他微环境细胞类型（如成纤维细胞、内皮细胞和/或免疫细胞）存在下对乳腺癌细胞进行 3D 培养，具有研究微环境在 BCSC 3D 生长中的作用的额外能力38，39。生成3D类器官所需的特定细胞数量可能因细胞系或患者肿瘤来源而异，因此在任何大规模实验之前优化培养条件和细胞数量非常重要。

最后，体内小鼠异种移植模型可用于了解与非BCSC或大细胞群相比，BCSC在体内的生长（图4）自我更新，分化和/或肿瘤起始能力的差异。通常，在存在外源性因子或治疗剂的情况下观察到的体外细胞反应不代表体内环境，这表明体外观察应尽可能与体内研究相辅相成。使用体内异种移植模型，保留了细胞异质性和肿瘤结构，因此这些模型可以作为密切模拟人类患者微环境的系统。体内可以进行LDA以确定给定混合癌细胞群（BCSC或非BCSC）中肿瘤起始细胞的比例40，41。应优化使用的细胞稀释范围，并将取决于目标细胞群中启动细胞的频率。理想情况下，这些稀释液应包括导致100%肿瘤形成的剂量，低至没有肿瘤形成的细胞剂量，并且在两者之间有一个合理的范围。原发样本中肿瘤起始细胞的频率可能是可变的，并且在乳腺肿瘤具有非常少的数量或肿瘤起始细胞群异质的情况下，进行LDA可能特别具有挑战性42。在这些情况下，注射大量细胞更适合理解乳腺癌生物学。

Figure 4
图 4:用于评估 BCSC 功能的体内异种移植测定。如图1所示，通过FACS分离MDA-MB-231乳腺癌细胞，并按照方案第9.1至9.8节（5 x 105细胞/小鼠;4只小鼠/细胞群）所述注射到雌性NSG小鼠的右胸乳脂垫中。显示原发性乳腺肿瘤生长动力学适用于 ALDHhi CD44+CD24- （■） 人群与 ALDH 低 CD44低/-CD24+ （□） 人群。数据表示为±平均值 S.E.M. * = 肿瘤大小与同一时间点各自的 ALDH 低 CD44低/-亚群显著不同（P < 0.05）。这个数字改编自克罗克等人26。请点击此处查看此图的大图。

Discussion

作者没有什么可透露的。

Disclosures

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。

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

干细胞溶液 1：30 稀释液
7-氨基放线菌素 D （7AAD）BD51-68981E建议：0.25 &微量;g/1x106 细胞
丙酮FisherA18-1
醛脱氢酶 （ALDH） 底物Stemcell Technologies1700作为 ALDEFLOUR 检测试剂盒的一部分进行商业销售;按照制造商的说明进行 ALDH 底物制备
基底膜提取物 （BME） 康宁354234以商业名称 Matrigel 出售
细胞培养板：6 孔康宁877218
细胞培养板：60 mm康宁353002
细胞培养板：96 孔超低吸附康宁3474
细胞过滤器：40 微米BD352340
胶原蛋白技术7001制备 3 mg/mL 胶原蛋白的 PBS
胶原酶Sigma110888070011x
锥形管：50 mLFisher scientific05-539-7
结晶紫SigmaC6158使用 0.05% 结晶紫水溶液进行染色
DispaseStemcell Technologies79135U/mL
DMEM：F12Gibco11330-0321x，含 L-谷氨酰胺和 15 mM HEPES
DNAseSigmaD50520.1 mg/mL 终浓度
FBSAvantor Seradigm Lifescience97068-085 
流管：5mlBD352063聚丙烯圆底管
甲醇Fisher84124
小鼠抗人 CD24 抗体BD561646R-藻红蛋白和花青染料偶联 克隆：ML5
小鼠抗人 CD44 抗体BD555479R-藻红蛋白偶联，克隆：G44-26
N，N-二乙氨基苯甲醛 （DEAB）Stemcell Technologies1700作为 ALDEFLOUR 检测试剂盒的一部分进行商业销售;遵循制造商的说明 DEAB 制备
PBSWisent Inc311-425-CL1x，不含钙和镁
胰蛋白酶-EDTAGibco25200-056
乳腺球 培养基成分
B27Gibco17504-441x
bFGFSigmaF200610 ng/mL
BSABioshop ALB00304%
DMEM：F12Gibco11330-0321x，含 L-谷氨酰胺和 15 mM HEPES
EGFSigmaE964420 ng/mL
胰岛素Sigma166345 & 微型;g/mL
3D 类器官培养基组成
A8301Tocris2939500 nM
B27Gibco17504-441x
DMEM：F12Gibco11330-0321x，含 L-谷氨酰胺和 15 mM HEPES
EGFSigmaE96445 ng/mL
FGF10Peprotech100-2620 ng/mL
FGF7Peprotech100-195 ng/mL
GlutaMaxInvitrogen35050-0611x
HEPESGibco15630-08010 mM
N-乙酰半胱氨酸SigmaA91651.25 mM
神经调节蛋白和 β;1Peprotech100-035 nM
烟酰胺SigmaN06365 mM
NogginPeprotech120-10C100 ng/mL
R-spondin3R&D3500250 ng/mL
SB202190SigmaS7067500 nM
Y-27632Tocris12545 µ米

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