Label-retaining assay enriches tumor-initiating cells in glioblastoma spheres cultivated in serum-free medium

Zeng,Lingcheng; Zhao,Yiqing; Ouyang,Taohui; Zhao,Tianyuan; Zhang,Suojun; Chen,Jian; Yu,Jiasheng; Lei,Ting

doi:10.3892/ol.2016.4690

Label-retaining assay enriches tumor-initiating cells in glioblastoma spheres cultivated in serum-free medium

Authors:
- Lingcheng Zeng
- Yiqing Zhao
- Taohui Ouyang
- Tianyuan Zhao
- Suojun Zhang
- Jian Chen
- Jiasheng Yu
- Ting Lei
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Affiliations: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
Published online on: June 8, 2016 https://doi.org/10.3892/ol.2016.4690
Pages: 815-824
Copyright: © Zeng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Label-retaining cells, which are characterized by dormancy or slow cycling, may be identified in a number of human normal and cancer tissues, and these cells demonstrate stem cell potential. In glioblastoma, label-retaining assays to enrich glioma stem cells remain to be fully investigated. In the present study, glioblastoma sphere cells cultured in serum‑free medium were initially stained with the cell membrane fluorescent marker DiI. The fluorescence intensity during cell proliferation and sphere reformation was observed. At 2 weeks, the DiI‑retaining cells were screened by fluorescence‑activated cell sorting and compared phenotypically with the DiI‑negative cells in terms of in vitro proliferation, clonogenicity and multipotency and for in vivo tumorigenicity, as well as sensitivity to irradiation and temozolomide treatment. It was observed that DiI‑retaining cells accounted for a small proportion, <10%, within the glioblastoma spheres and that DiI-retaining cells proliferated significantly more slowly compared with DiI‑negative cells (P=0.011, P=0.035 and P=0.023 in the of NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines). Significantly increased clonogenicity (P=0.002, P=0.034 and P=0.016 in the NCH441, NCH644 and NCH421k glioblastoma sphere cell lines) and three-lineage multipotency were observed in DiI‑retaining cells in vitro compared with DiI‑negative cells. As few as 100 DiI-retaining cells were able to effectively generate tumors in the immunocompromised mouse brain, whereas the same number of DiI‑negative cells possessed no such ability, indicating the increased tumorigenicity of DiI‑retaining cells compared with DiI‑negative cells. Furthermore, DiI‑retaining cells demonstrated significant resistance following irradiation (P=0.012, P=0.024 and P=0.036) and temozolomide (P=0.003, P=0.005 and P=0.029) compared with DiI‑negative cells in the NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines, respectively. It was concluded that label-retaining cells in glioblastoma spheres manifest clear stem cell features and that the label‑retaining assay may be utilized to further enrich glioma stem cells cultured under serum‑free conditions for additional study.

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