Impact of cell dissociation on identification of breast cancer stem cells
Impact of cell dissociation on identification of breast cancer stem cells
复制标题
细胞解离对乳腺癌干细胞鉴定的影响
DOI：
10.3233/cbm-130300
复制
发表时间：
2012-01-01
影响因子：
3.1
通讯作者：
Wang, Yugang
中科院分区：
文献类型：
--
作者：
Quan, Yi;Yan, Ying;Wang, Yugang
背景技术背景：肿瘤干细胞（cancer stem cells，CSC）的鉴定主要依赖于细胞表面的特异性标记。这种方法为我们在体外研究CSCs的特性提供了可能。方法：采用3种常用的消化液和2种不同的消化温度对MCF-7细胞进行消化，检测CD 44（+）CD 24（-）CSCs亚群的表达。结果：胰蛋白酶是研究肿瘤干细胞的常用试剂，胰蛋白酶能显著降低肿瘤干细胞表面标志物的抗原性，使部分CD 44（+）CD 24（-）肿瘤干细胞亚群裂解为CD 44（-）CD 24（-）非干细胞癌细胞（NSCCs）。同时也增加了CD 44（-）CD 24（-）亚群的乳腺球形成效率。当细胞在37摄氏度消化时，这种切割效应尤其严重。而Accutase，一种纯化的胶原酶/中性蛋白酶混合物，提供了解离效率和抗原retention.CONCLUSION的最佳平衡：两者合计，这些结果表明，酶消化过程中起着重要作用，通过流式细胞仪鉴定的CSC与表面标记，这表明研究人员需要重新考虑这个过程认真。
BACKGROUND: Fluorescence-activated cell sorting was commonly used for identification of cancer stem cells (CSCs), which relied on specific cell surface markers. And this approach makes it possible for us to study characteristics of CSCs in vitro. However, the pattern of membrane protein including surface makers might be vitally influenced during the dissociation of the adherent cells, thus it might heavily impact the quantity and quality of CSCs identified by flow cytometry.METHODS: To address this question, in present study, three commonly used digestive reagents and two different temperatures were performed in MCF-7 cells to assay CD44(+)CD24(-) CSCs subpopulation. The potential of sorted CD44(+)CD24(-) cells from different digestion to form mammosphere in culture was also compared.RESULTS: The results showed that trypsin, a commonly used reagent in CSCs studies, most aggressively reduced antigenicity for surface markers and make part of CD44(+)CD24(-) CSCs subpopulation cleaved into CD44(-)CD24(-) non-stem cancer cells (NSCCs). And it also increased the mammosphere formation efficiency of CD44(-)CD24(-) subpopulation. This cleavage effect is especially serious when cells are digested at 37 degrees C. While accutase, a purified collagenase/neutral protease cocktail, provides the best balance of dissociation efficiency and antigen retention.CONCLUSION: Taken together; these results indicate that enzymatic digestion process plays an important role in identification of CSCs with surface marker via flow cytometer, suggesting that researchers need to reconsider this process seriously.