Identification Of Cancer Stem-like Cells In Rat C6 Glioma Cell Line And Insights Into Their Biological Characteristics

Background: Gliomas, especially high grade glioblastoma, are highly malignant central nervous system tumors with early relapse after surgery and resistances to radiotherapy and chemotherapy. Despite advances in surgical management and relevant adjuvant treatments, the 5-year survival rate is still just 20% and it remains one of the greatest challenges for neurosurgeons. The cancer stem cell hypothesis implies that only a relatively small fraction of cells, termed cancer stem cell (CSC), possess the ability to proliferate and self-renew extensively, and give rise to progenies of different linkages. This rare fraction of cells is regarded as the true source for tumor initiation, progression, and metastasis as well as relapse after routine treatments. As strong support for this concept, several authors have already identified CSC from human brain tumors of different types. Therefore the CSC hypothesis has provided insights into the initiation and recurrence of brain gliomas and targeted elimination of these CSC represents a promising therapeutic strategy for refractory brain tumors.Objective: 1. To identify cancer stem-like cells in rat C6 glioma cell line and explore their biological characteristics. 2. To compare the effects of current popular CSC identification methods in identifying C6 cancer stem-like cells. 3. To ascertain whether C6 line is relevant in modeling human brain CSC biology.Methods: 1. Serum-free culture medium (DMEM/F-12 supplemented with bFGF, EGF and B27) was used as the culture system to isolate the tumor-sphere forming cells in C6 line; The primary tumor spheres were dissociated into single cells by limiting dilution method, and assessed for subsphere formation to determine their self-renewal capacity; Subspheres from one mother cell were cultured in serum-containing medium (DMEM+10%FBS) to induce differentiation. Immunofluorescence and immunohistochemistry assay were used to determine the expression of differentiation makers; After proliferation in serum-containing medium, C6 cells were injected subcutaneously into the backs of BALB/c-nude mice to determine their in vivo tumorigenecity. 2. Floating primary tumor spheres in serum-free medium were collected and cultured in alternate serum-containing/free medium to determine whether the percentage of tumor-sphere forming cells was stable in C6 line; Flow cytometry and immunohistochemistry assay were used to determine the expression of stem cell marker CD133 and differentiation makers in different culture conditions. 3. Magnetic cell separation was used to isolate CD133 positive cell fractions from the CD133 negative ones; The purity of the separated cells were assessed by Western Blot and immunofluorescence assay; BrdUrd staining was used to determine their proliferative state; Serum-free culture medium, serum-containing culture medium, and nude mice transplantation were used to determine their self-renewal capacity, multi-linkage differentiation capacity, and in vivo tumorigenic capacity respectively. 4. Hoechst 33342, which is used in Flow cytometry to isolate the side population (SP), was used to incubate the C6 cells for different durations. Its toxicity to C6 cells and the expression level of ABCG2 were examined. After incubation, C6 cells were assessed for their self-renewal capacity and indefinite proliferation capacity.Results: 1. >60% C6 cells were capable of forming tumor spheres in serum-free medium, and >80% of the tumor-sphere cells were capable of self-renewal, producing neuronal and glial progenies in serum-containing medium, and being tumorigenic in vivo. Adherent C6 cells co-expressed CD133 and NSE/GFAP in serum-containing medium, which is rarely seen in serum-free medium. 2. Floating primary tumor spheres cells adhered again in serum-containing, and the percentage of tumor-sphere forming cells in C6 line was relatively stable despite alternation in culture medium; Flow cytometry and immunohistochemistry assay showed a 30% CD133 positive rate in serum-containing medium cultured adherent C6 cells and it was >85% in the serum-free medium cultured tumor spheres. 3. Both CD133 positive and negative cell fractions contained cancer stem-like cells, although with different proliferation and tumorigenicity capacity, as well as different tumor-sphere growth pattern. 4. Hoechst 33342 staining was toxic to most C6 cells, depriving them of their CSC properties; C6 cells also contained 1% Hoechst 33342 negative (SP), while ABCG2 positive cells, which preserved CSC properties after staining.Conclusions: 1. Rat C6 glioma cell line contained cancer stem-like cells, which shared phenotypic similarity to human primary brain CSC. 2. Identifying C6 cancer stem-like cells by tumor-sphere culture system showed a much higher CSC percentage than by CD133 marker (>60% vs. 30%), The toxicity of Hoechst 33342 to C6 cells would deprive those non-SP C6 cells of their CSC properties. 3. Although future efforts should be aimed at finding a more comprehensive and reliable CSC identification method, however, in view of the CSC properties they preserved, the C6 cancer stem-like cells is still an convenient in vitro brain tumor model to study the human brain CSC biology.