从人乳腺癌组织及细胞系微球体形成分析
Summary
Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.
Abstract
类似的健康组织，许多血液和实体恶性肿瘤现在认为是分层组织的，与干细胞样癌细胞自我更新而引起更加分化的后代的一个子集。理解并针对这些癌干细胞在乳腺癌，其可以具有增强化疗和放射抗性相比非干肿瘤散装，已成为一个重要的研究领域。该微球体检测也成为广泛使用的，因为它能够识别追溯sphere-：标志物CD44包括，CD24和乙醛脱氢酶的活性可以用荧光活化细胞分选（FACS）来隔离前瞻性当细胞植入免疫缺陷小鼠显示增强的致瘤性进行评估形成细胞，从单一的干细胞样克隆的发展。在这里，我们列出的微球体从细胞系或小学患者样本，他们的传代，并计算估计范围f显示相应的培养方法orming效率（SFE）。首先我们讨论关键的考虑和陷阱在适当的规划和微球体的实验解释。
Introduction
通过干细胞样肿瘤干细胞为首的肿瘤细胞系的存在，极大地增加我们的肿瘤异质性的理解。而在一些肿瘤的遗传多样性并从基因不同克隆的克隆产物出现，大幅组件出现从后生差异导致：癌细胞可以通过激活特定基因或抑制干，祖和分化状态之间转换（有时可逆）表达程序1 – 3。这可能反映了细胞的内在或外在的因素，这反映当前正从邻近癌，间质或免疫细胞递送调节因子表达与在与旁分泌信号结合其所得自分泌信号传导的细胞中的基因表达程序，microenviromental条件如度缺氧2,4,5。
虽然创新的谱系追踪的方法在前进我们研究推定的癌干细胞在它们的体内利基6能力-如图8所示 ，球形成测定法仍估计乳腺癌细胞的潜力，以行为像干细胞，至少在所用的测定条件一种流行和方便的方法。它经常被用来一起回顾性方法用于净化癌症干细胞，通过对已提出的膜标记物CD44和CD24 9，和酶乙醛脱氢酶（醛脱氢酶）的活性水平10，标志物的表达，以对应于更mesenchymal-和上皮分别为11样癌干细胞。球体的形成方法首先被开发作为神经球测定法，使得能够从单个克隆在非粘附，无血清条件，增加了上皮生长因子（EGF）12推定的干细胞的生长，后来被有效地应用于正常和癌乳腺组织。
<p claSS =“jove_content”>球体形成创始人小区的身份，并且构成该球体的质量混合细胞类型，是相关的，可以从mammo-，或其他球形成测定法进行的推论。长期静态骨真正的干细胞，认为休息G0期，将不会遇到的因素将有利于激活体内的精确组合。所述微球体测定代替使细胞要么蓄势有丝分裂或已经除以13的生长。这些祖细胞，虽然不是一个真正的静态细胞，可能是细胞阶段，与在测定中使用的表皮生长因子和碱性成纤维细胞生长因子（bFGF）促分裂原增殖。然而，它们包含了一系列激活干细胞相关的信令的通路14。另外，它们的形成的速率涉及当通过在有限稀释测定法效力在小鼠异种移植2,15,16测定它们取自组织的致瘤</ SUP>。在这里，我们提供详细的协议，以分离单个细胞，并产生来自人乳腺癌细胞系和乳腺肿瘤的临床样品初级乳腺球。我们还描述了如何执行主微球体的连续传代，以评估自我更新，以及如何计算球体形成效率，允许在不同的接种密度比较（见方案在图1中）。
Protocol
Representative Results
Discussion
中小学微球体的成功评估依赖于细胞被镀在足够低的密度，从单一的克隆微球体的形式，以最小的领域聚集。然而，在密度过低，过少的微球体可能形成区分疗效的统计。播种密度应为使用，因为他们可以有很大的不同在他们的领域形成效率每个细胞系进行优化（那些表达低E-cadherin的可能形成不太稳定，短期微球体培养18）。这种优化阶段应确保从单细胞的克隆生长最初级和次级球形成?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是具有特别提到希拉里工艺和道格拉斯·迈尔斯爵士支持帝国BRC，国家卫生研究院的研究，并采取行动战胜癌症。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| DMEM/F12 | Lonza | CC-3151 | |
| 2mM L-Glutamine | Sigma Aldrich | G8540 | |
| 100U/ml Penicillin & Streptomycin | Sigma Aldrich | P4083 | |
| 20ng/ml recombinant human epidermal growth factor (EGF) | Sigma Aldrich | E9644 | |
| 20ng/ml recombinant human basic fibroblast growth factor (bFGF) | R&D systems | 233-FB-025 | |
| 1x B27 supplement | Invitrogen | 17504-044 | |
| Phosphate buffered saline (PBS); | Thermo Scientific | 12399902 | |
| 0.5% trypsin-0.2%EDTA; | Sigma Aldrich | 59418C | |
| Fetal Calf Serum | First Link UK | 02-00-850 | |
| Trypan Blue | Sigma Aldrich | 93595 | |
| Low attachment 6 well plates | Corning | CLS3814 | |
| Collagenase type 1A | Sigma Aldrich | C9891 | |
| Hyaluronidase | Sigma Aldrich | H3506 | |
| Sterile razor blades | Fisher Scientific | 12443170 | |
| Sterile scalpel | Fisher Scientific | 11758353 | |
| Sterile micro-dissecting scissors | Sigma Aldrich | S3146 |
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