Phenotypic analysis of common cancer stem cell markers and cell sorting in lung cancer cell line A549

doi:10.3969/j.issn.2095-4344.2017.25.009

Abstract

Abstract:

BACKGROUND: It has been widely reported that the most types of cancer are probably originated from cancer stem cells (CSCs) which are subpopulations of tumor cells. Recent studies also suggested that lung cancer could arise from CSCs. However, the phenotypic characteristics of CSCs in lung cancer have not been precisely described.
OBJECTIVE: To systematically analyze the expression of the most common CSC markers in cell line A549.
METHODS: A549 cells were cultured for 1 week under the condition of conventional high glucose. After that, flow cytometry was used to assess the expression of putative stem cell markers, including CD133, CD24, CD44 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) and characterized using their sphere-forming capacity in serum free medium supplemented with several growth factors.
RESULTS AND CONCLUSION: A549 cells expressed the CSC markers CD44 and CD24 at 64.23% and 58.62%, whereas the expression of both ABCG2 and CD133 was around 0.9%. Double-positive CD44/133 populations were rare. CD44⁺/CD24⁺ and CD44⁺/CD24^- subpopulations respectively exhibited 54.64% and 23.38% expression. CD44⁺/CD24⁺ and CD44⁺/CD24^- subsets were sorted by FACS. Both isolated subpopulations formed spheres in the serum free medium supplemented with basic fibroblast growth factor and epidermal growth factor. However, there was no significant difference in the sphere formation efficiency among CD44⁺/CD24⁺ and CD44⁺/CD24^- subsets as well as A549 cells. Our findings suggest that CD44 and CD24 cannot be considered as potential markers for isolating lung CSCs in cell line A549, and further investigation using in vivo assays is required.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Lung Neoplasms

CLC Number:

R394.2

Peng Zhuo, Dang Xiao-yan, Zhang Qiu-hong, Zhang Rui, Yang Shuan-ying. Phenotypic analysis of common cancer stem cell markers and cell sorting in lung cancer cell line A549[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3989-3994.

Figures/Tables 1

2.1 A549细胞的常见肿瘤干细胞抗原分析为了评估A549细胞内的肿瘤干细胞存在情况，实验使用流式细胞技术分析了最常见的肿瘤干细胞的表面标志包括CD133，CD44，CD24和ABCG2的表达情况。如表1所示，实验发现A549细胞中度表达CD44和CD24，而CD133和ABCG2的表达水平极低，均在1%以下。鉴于CD44为很多类型的肿瘤包括肺癌肿瘤干细胞的表型之一，其与CD24联合使用进行肿瘤干细胞的纯化[16-17，25]，如CD44+/CD24+为结肠癌的肿瘤干细胞[19]，而CD44+/ CD24-细胞群为乳腺癌和前列腺癌的肿瘤干细胞[16，18]，因此对于A549细胞，实验同时使用荧光双抗体标记技术考察了A549细胞中CD44+ CD24+和CD44+CD24-两群细胞的比例，结果发现CD44+ CD24+双阳性细胞的比例达到(54.64± 3.30)%，CD44+ CD24-的细胞比例为(23.38±2.70)%。表1列出了A549细胞的常见肿瘤干细胞的表达平均水平以及CD44+CD24+和CD44+CD24-等各群细胞的比例情况，其中因A549细胞中CD133的表达比例很低，所以CD133+CD44+双阳性的比例很少。从流式细胞图中可以直接看出CD133，CD44，CD24和ABCG2的表达水平，同时也能直接获得CD44+CD24+和CD44+ CD24-等各群细胞的比例情况(图1)。 2.2 CD44+/CD24+及CD44+/CD24-细胞的分选和鉴定结果因CD44+/CD24+细胞和CD44+/CD24-细胞为目前报道较多的常见肿瘤肿瘤干细胞亚群[16，18-19]，而A549细胞中这两类细胞群均占有一定程度的比例，因此实验分选出CD44+/CD24+细胞和CD44+/CD24-两个亚群细胞进行分析。通过荧光抗体双标A549细胞后，细胞上流式细胞仪进行分选。分选出的细胞继续培养1周，两群细胞的增殖情况均良好，细胞在形态上和A549细胞群并无显著性差异。培养1周后，为了鉴别其表型的稳定性，实验再次对这两类细胞的CD44抗原和CD24抗原进行了荧光双标后上流式细胞仪确认，结果表明，分选出的CD44+/CD24+细胞群，培养1周后，其CD44和CD24双阳性的细胞比例维持在高水平98.72%(图2)；而CD44+/CD24-细胞群在培养1周后，其CD44+/CD24-的比例仍然维持在98.16%的高水平。实验结果表明，分选出的CD44+CD24+双阳性细胞和CD44+CD24-的细胞群表型稳定，可以进行下一步的肿瘤干细胞特性比较分析。 2.3 CD44+/CD24+，CD44+/CD24-和A549细胞的肿瘤成球能力比较肿瘤干细胞悬浮成球实验是目前比较理想的鉴定肿瘤干细胞的方法，尽管有一些报道表明，对于肿瘤干细胞要形成悬浮肿瘤球，培养环境中不一定需要特定的生长因子[26-28]。但是实验仍然采用了经典的添加碱性成纤维细胞生长因子和表皮生长因子的方法对于CD44+/CD24+及CD44+/CD24-两类细胞亚群的成球能力进行了考察。结果显示，CD44+/CD24+及CD44+/CD24-以及A549细胞群中的少部分细胞能够形成肿瘤球，球的大小相似，体积无显著性差异(图3)。进一步考察统计它们形成肿瘤球的效率，结果显示，各细胞群的成球效率均在5%以下，CD44+/ CD24+及CD44+/CD24-细胞亚群及A549细胞肿瘤球形成效率之间差异无显著性意义(图4)，提示分选出的CD44+/ CD24+及CD44+/CD24-两类细胞群相对于A549细胞并没有包含数量更多的肿瘤干细胞。"

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