Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines
Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines
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DOI：
10.3791/52671
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发表时间：
2015-03-01
影响因子：
1.2
通讯作者：
Castellano, Leandro
中科院分区：
文献类型：
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作者：
Lombardo, Ylenia;de Giorgio, Alexander;Castellano, Leandro
与健康组织类似，许多血液和实体恶性肿瘤现在被认为是分级组织的，具有干细胞样癌细胞的子集，它们可以自我更新，同时产生更多分化的后代。了解和靶向乳腺癌中的这些肿瘤干细胞，与非干细胞肿瘤相比，可能具有更强的化疗和放射抗性，已成为一个重要的研究领域。包括CD44、CD24和ALDH活性在内的标记物可以使用荧光激活细胞分类(FACS)进行评估，以前瞻性地分离当移植到免疫低下的小鼠中时显示出增强的致瘤性的细胞：乳房试验也因其能够回溯鉴定从单个干细胞样克隆发展而来的球状细胞而被广泛使用。在这里，我们概述了从细胞系或原始患者样本中适当培养乳房的方法，它们的传代，以及估计球体形成效率(SFE)的计算。首先，我们讨论在适当计划和解释大气层实验时的主要考虑因素和陷阱。
Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo-and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.