Cancer stem cells (CSCs) have been implicated in growth, metastasis, recurrence and chemo-/radio-resistance in several cancer types. Despite a plenty of literature about different in vitro techniques to enrich/isolate CSCs, their comparative characterization for stemness is not well established. In the present study, cells obtained following three in vitro assays [clonogenic assay, tumorsphere assay (TSA) and single cell assay (SCA)] were compared for their cancer stem-like cell characteristics in human lung adenocarcinoma (A549) cells. Expression of the pluripotent (OCT4, NANOG) and lung cancer stem cell marker (CD166) genes were studied in these cells. Results showed that in comparison to cells obtained from routine culture (CC), the cells obtained from TSA showed significantly higher expression of OCT-4 and NANOG. These results were further validated with quantification of cell surface cancer stem cell markers i.e. CD44+/CD24- in the cells obtained from different methods, which were higher in TSA and SCA. Additionally, functional characterization of cancer stem-like cells (CSLCs) using ALDH assay showed the highest % of ALDH+ cells in TSA. These results were in agreement with higher resistance of these cells against 5-Fluorouracil suggesting higher fraction of CSLCs in TSA than the other assays. These results showed that TSA provides a better method to enrich CSLCs in A549 lung adenocarcinoma cells.

中文翻译：

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肿瘤球测定法为癌干样细胞富集A549肺腺癌细胞提供了更好的体外方法。

癌症干细胞（CSC）与多种癌症类型的生长，转移，复发和化学/放射抗性有关。尽管有大量文献报道了不同的​​体外技术来富集/分离CSC，但它们对茎干的比较表征尚未得到很好的确立。在本研究中，比较了通过三种体外测定[克隆形成测定，肿瘤球测定（TSA）和单细胞测定（SCA）]获得的细胞在人肺腺癌（A549）细胞中的癌干样细胞特征。在这些细胞中研究了多能性（OCT4，NANOG）和肺癌干细胞标记（CD166）基因的表达。结果表明，与从常规培养（CC）获得的细胞相比，从TSA获得的细胞显示OCT-4和NANOG的表达明显更高。通过定量从不同方法获得的细胞中的细胞表面癌干细胞标记即CD44 + / CD24-进一步验证了这些结果，其中TSA和SCA的表达较高。另外，使用ALDH测定法对癌症干样细胞（CSLC）的功能表征显示，TSA中ALDH +细胞的百分比最高。这些结果与这些细胞对5-氟尿嘧啶的更高抗性一致，表明在TSA中CSLC的比例要高于其他测定。这些结果表明，TSA提供了一种更好的富集A549肺腺癌细胞中CSLC的方法。使用ALDH分析对癌症干样细胞（CSLC）的功能表征显示，TSA中ALDH +细胞的百分比最高。这些结果与这些细胞对5-氟尿嘧啶的更高抗性一致，表明在TSA中CSLC的比例要高于其他测定。这些结果表明，TSA提供了一种更好的富集A549肺腺癌细胞中CSLC的方法。使用ALDH分析对癌症干样细胞（CSLC）的功能表征显示，TSA中ALDH +细胞的百分比最高。这些结果与这些细胞对5-氟尿嘧啶的更高抗性一致，表明在TSA中CSLC的比例要高于其他测定。这些结果表明，TSA为富集A549肺腺癌细胞中的CSLC提供了更好的方法。