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Isolation of colorectal cancer stem-like cells

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Abstract

This study is aimed at isolating colorectal cancer stem-like cells in vitro using a neurosphere assay method employed in isolating gliobastoma multiforme tumor cells. This was followed with confirmation of the isolated cells by flow cytometry, pluripotent genes expression and in vivo tumorigenicity assay. Using this culture assay, stem-like and non-stem-like CRC cells were isolated and expanded in vitro from purchased Balb/c mice induced with CT26 colorectal cancer (CRC) cell line. The procedure includes an initial mechanical dissociation and chemical digestion of tumor tissue and subsequently plating the resulting single cell suspension in serum-free medium (SFM) or serum-containing medium (SCM). This selectively permits growth of cancer stem-like cells in SFM and eliminates non-stem-like cancer cells through the process of anoikis or apoptosis. CRC stem cells derived cultures proliferated as non-adherent spheres in vitro in different shapes and sizes. These cells expressed cell surface markers previously reported for tumor stem cells, including CD44, CD133, CD166 and CD26 and formed tumors when implanted in severe combined immunodeficient mice in a concentration dependent manner. Importantly, the stem-like cells had self-renewal properties with significantly higher expression of the pluripotent stem cell genes NANOG, OCT4, and SOX2 compared to the adherent non-stem cells. Collectively, the results of this study indicate that SFM is a defined culture medium that enriches for CRC stem-like cells and represents a suitable in vitro model for the study of CRC stem-like cells. This finding may be useful in developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation within colorectal cancer.

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Acknowledgments

This study is funded by Tianjin science and technology commission of science and technology projects. Project No: 12ZCDZSY16800. The authors wish to extend profound gratitude to Li Wen and Li Shan Shan for their tremendous support.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Tianjin University of Traditional Chinese Medicine, 312 Anshan West Road, Nankai District, Tianjin, 300193, People’s Republic of China

    Eunice Dotse & Yuhong Bian

  2. Noguchi Memorial Institute for Medical Research, University of Ghana, LG 581, Legon, Accra, Ghana

    Eunice Dotse

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  1. Eunice Dotse
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  2. Yuhong Bian
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Correspondence to Yuhong Bian.

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Dotse, E., Bian, Y. Isolation of colorectal cancer stem-like cells. Cytotechnology 68, 609–619 (2016). https://doi.org/10.1007/s10616-014-9806-0

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  • DOI: https://doi.org/10.1007/s10616-014-9806-0

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