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GFP Stable Transfection Facilitated the Characterization of Lung Cancer Stem Cells

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Abstract

Cancer stem cells (CSCs) are a subset of cancer cells that play key roles in metastasis and cancer relapse. The elimination of CSCs is very important during cancer therapy. To develop drugs that target CSCs, the isolation and identification of putative CSCs are required. Some of the characteristics of CSCs are assessed by cell survival assays. In such experiments, the density of the cells seeded on the plates may affect the experimental results, leading to potentially inaccurate conclusions. In this study, a new assay to facilitate the characterization of CSCs has been developed by stable transfection of GFP, using the A549 lung cancer cell line as a model. A putative CSC line, A549 sphere cells, was obtained by culturing A549 cells in ultra-low dishes in serum-free medium. To ensure that the putative CSCs were grown under the same conditions as the A549–GFP cells and were not affected by the number of cells seeded, A549 sphere cells were mixed with GFP stably transfected A549 (A549–GFP) cells. The mixture was subjected to flow cytometry assay and inverted fluorescence microscopy to detect changes in the proportion of GFP-positive cells after treatment. A549 sphere cells had a slower proliferation rate and an improved chemoresistance. They also showed differentiation ability. This work suggests that mixing GFP stably transfected cancer cells with putative CSCs may facilitate the identification of CSCs, making it convenient for studies of targeted CSCs.

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Acknowledgments

We thank the staff at Cell Center of Institute of Biochemistry and Cell Biology for their assistance in the experiments. This work was supported by grants from the National Basic Research Program of China (973 Program) (Nos. 2010CB529901 and 2011CB510104), the Important National Science & Technology Specific Project of Hepatitis and Hepatoma Related Program (2008ZX10002-023), the New Innovation Program (2009-ZX-09102-246), the National Natural Science Foundation of China (81172449), and the Zhejiang Sci-Tech University grant (1016834-Y)

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The authors declare that there are no conflicts of interest.

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Correspondence to Xiliang Zha or Haineng Xu.

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Na Li and Yu Yang contributed equally to this work.

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Li, N., Yang, Y., Ding, M. et al. GFP Stable Transfection Facilitated the Characterization of Lung Cancer Stem Cells. Mol Biotechnol 56, 1079–1088 (2014). https://doi.org/10.1007/s12033-014-9788-3

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