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 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.

Keywords: DiI; cancer stem cell; glioma; label-retaining assay; label-retaining cell.

Figure 1.

Populations of DiI-retaining cells and DiI-negative cells with varying proliferative potentials may be differentiated within glioblastoma spheres at 2 weeks subsequent to initial DiI labeling. (A) Diagram illustrating that the change in the cellular fluorescence intensity in initially labeled cells reflects the proliferative state. (B) Representative images showing the dynamic changes in and distribution of DiI fluorescence (lower panel) during cell proliferation and sphere reformation (upper panel) in the NCH421k cell line at the indicated times (Day 1, Day 3, Day 5, Day 8, Day 11 and Day 14). Scale bars: Day 1 and day 3, 10 µm; day 5, 20 µm; day 8, day 11 and day 14, 50 µm. The cells were initially stained with DiI fluorescence through incubation with DiI cell-labeling solution, as indicated in the Materials and methods. (C) Percentage of DiI-retaining cells in the NCH421k, NCH441 and NCH644 cell lines as analyzed by flow cytometry at 2 weeks subsequent to initial DiI labeling. (D) Proliferative potential of DiI-retaining and DiI-negative cells in the NCH421k, NCH441 and NCH644 cell lines as analyzed by the cell counting kit-8 assay. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 compared with the DiI-negative group. OD, optical density.

Figure 2.

DiI-retaining cells possess greater self-renewal capacity in vitro compared with DiI-negative cells. Sphere-forming frequencies of DiI-retaining and DiI-negative cells in the (A) NCH441, (B) NCH644 (C-a) and NCH421k glioblastoma sphere cell line observed in the serial clonogenicity assay. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 compared with DiI-retaining cells at the same passage. **P<0.05 vs. DiI-negative cells at different passages. (C-b) Representative images of the sphere-forming abilities of DiI-retaining and DiI-negative cells in the NCH421k cell line at the indicated passages (secondary, tertiary and quarternary) under phase-contrast microscopy. Scale bar=100 µm.

Figure 3.

DiI-retaining cells possess multipotency in vitro. (A) Expression of GFAP, βIII-tubulin and PDGFRα in DiI-retaining and DiI-negative cells in the NCH421k cell line prior to and following differentiation as assayed by western blot, with β-actin as the internal reference. Quantification of the band densities of (B-a) GFAP, (B-b) βIII-tubulin and (B-c) PDGFRα through normalization to β-actin using ImageJ software. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 compared with expression prior to differentiation in the DiI-retaining group. GFAP, glial fibrillary acidic protein; PDGFRα, platelet-derived growth factor receptor α.

Figure 4.

DiI-retaining cells demonstrate significant resistance to irradiation and temozolomide treatment. Proliferative potentials of the DiI-retaining and DiI-negative cells in the NCH421k, NCH441 and NCH644 glioblastoma sphere lines prior to and following (A) irradiation or (B) temozolomide treatment. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, DiI-retaining cells vs. DiI-negative cells with irradiation/temozolomide in the respective cell lines, or DiI-negative cells prior to irradiation/temozolomide vs. DiI-negative cells following irradiation/temozolomide. OD, optical density.

Figure 5.

DiI-retaining cells demonstrate significantly increased tumorigenicity in NOD/SCID mice compared with DiI-negative cells. Representative images of (A-a) Nestin (left panel); (A-b) GFAP (left panel); (A-c) Ki67 (left panel) and (A-d) cluster of differentiation 31 (left panel) immunohistochemical staining in the xenografts from NOD/SCID mice implanted with DiI-retaining or DiI-negative cells. Scale bar=100 µm. Quantification of the cells positive for (A-a) Nestin (right panel), (A-b) GFAP (right panel) and (A-c) Ki67 (right panel). *P<0.05. (A-d) MVD in the xenografts from NOD/SCID mice implanted with DiI-retaining or DiI-negative cells. *P<0.05. The data are presented as the mean ± standard deviation. (B) Kaplan-Meier survival analysis of the overall survival of NOD/SCID mice harboring tumors from DiI-retaining or DiI-negative cells. NOD/SCID, non-obese diabetic/severe combined immunodeficiency; GFAP, glial fibrillary acidic protein; MVD, microvessel density.