title: Label-retaining assay enriches tumor-initiating cells in glioblastoma spheres cultivated in serum-free medium.
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 ofin vitroproliferation, clonogenicity and multipotency and forin vivotumorigenicity, 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 cellsin vitrocompared 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.
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Lingcheng
authors.initials: L
authors.lastname: Zeng
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Taohui
authors.initials: T
authors.lastname: Ouyang
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Zhao
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Suojun
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Jiasheng
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
authors.forename: Ting
authors.initials: T
authors.lastname: Lei
keywords: DiI; cancer stem cell; glioma; label-retaining assay; label-retaining cell