Flow cytometric determination of stem/progenitor content in epithelial tissues: an example from nonsmall lung cancer and normal lung.
Flow cytometric determination of stem/progenitor content in epithelial tissues: an example from nonsmall lung cancer and normal lung.
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上皮组织中的茎/祖细胞含量的流式细胞术测定：非肺癌和正常肺的一个例子。
DOI：
10.1002/cyto.a.22156
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发表时间：
2013-01
期刊：
影响因子：
3.7
通讯作者：
Donnenberg, Albert D.
中科院分区：
文献类型：
--
作者：
Donnenberg, Vera S.;Landreneau, Rodney J.;Pfeifer, Melanie E.;Donnenberg, Albert D.
单细胞分析和细胞分选使得研究“液体”组织及其癌症的发育、生长、分化、修复和维护成为可能。这些方法在实体组织中的应用同样前景广阔，但必须解决一些独特的技术挑战。本报告阐述了多维流式细胞术在非小细胞肺癌和配对正常肺组织中候选干细胞/祖细胞群鉴定中的应用。在手术切除时收集了十七对肿瘤/正常肺样本并立即进行处理。将组织机械和酶解成单细胞悬浮液，并用一组抗体染色，用于负门控（CD45、CD14、CD33、血型糖蛋白 A）、上皮细胞（细胞内细胞角蛋白）的鉴定以及干细胞/祖细胞标记物（CD44、CD90、CD117、CD133）的检测。添加 DAPI 来测量 DNA 含量。福尔马林固定石蜡包埋的组织样本用关键标记物（细胞角蛋白、CD117、DAPI）染色，用于通过流式细胞术检测群体的免疫荧光组织定位。分类的肿瘤和肺标本包含高比例的事件，如果不通过逻辑门控消除这些事件，则会干扰分析。我们展示了双联体、亚二倍体 DNA 事件和细胞角蛋白+事件的包含如何也对造血标记物进行染色，从而降低了定量上皮细胞及其前体细胞的能力。使用肺癌/正常肺数据集，我们提出了一种多维数据分析方法，包括伪影去除、识别要进一步研究的细胞类别（分类器）以及测量这些细胞类别的结果变量。双变量分析的结果显示，肺肿瘤和邻近的无肿瘤肺上干细胞/祖细胞标记物的表达之间存在惊人的相似性。
Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of “liquid” tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung.
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影响因子：
11.2
作者：
Harris MA;Yang H;Low BE;Mukherjee J;Guha A;Bronson RT;Shultz LD;Israel MA;Yun K
通讯作者：
Yun K
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
8.8
作者：
Abeln, E. C. A.;Cover, W. E.;Kuipers-Dijkshoorn, N. J.;Fleuren, G.-J.;Cornelisse, C. J.
通讯作者：
Cornelisse, C. J.
影响因子：
56.9
作者：
FULWYLER, MJ
通讯作者：
FULWYLER, MJ
影响因子：
10.8
作者：
Donnenberg, Vera S.;Landreneau, Rodney J.;Donnenberg, Albert D.
通讯作者：
Donnenberg, Albert D.