Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods

Abstract

The cancer stem cell (CSC) hypothesis has provided insights into the initiation and recurrence of brain tumor. Specific identification and targeted elimination of these CSCs within the tumor mass represents a promising therapeutic strategy for refractory brain tumors. In this study, we attempted to identify CSCs in the rat C6 glioma cell line by three different identification methods. It is interesting to note that single-cell clonal analysis showed most C6 cells are cancer stem-like cells with characteristics of self-renewal, multilineage differentiation potentials in vitro, and tumorigenic capacity in vivo. It is surprising to note that CD133 failed to identify the total cancer stem-like cell population in the C6 line, since both CD133 (+) and CD133 (-) C6 cells have cancer stem-like cell fractions. Moreover, Hoechst 33342 staining, which is used in flow cytometry to isolate the side population (SP), was found to be harmful to C6 cells. Therefore, CD133 (-) and non-SP C6 cells may also harbor cancer stem-like cells. These results imply the limitation of using current identification methods in C6 line and underscore the importance of defining the genetic and molecular basis of CSCs.

Extracellular ATP reduces tumor sphere growth and cancer stem cell population in glioblastoma cells

“…The other purinergic receptors expressed by U87 cells (Supplementary Table 2) were not differentially expressed between spheres and attached cells. reported in glioma tumor spheres is quite broad, with primary cultures of brain tumors or cell lines presenting from 1% to about 50% of CD133 [17,[22][23][24]. Our analysis indicates that, despite variability among experiments, the proportion of cells positive for stemness markers are, with the exception of one experiment, always higher in the NSC medium when compared to the FBS medium.…”

“…The maintenance of a CSC population in spheres, in spite of the presence of 5% serum, may be due to the establishment of a microenvironment that favors the maintenance and growth of CSCs. As already pointed out by Shen et al [23], glioma cell lines grown on serum-containing medium for several years must be able to maintain a CSC population under this condition in order to remain tumorigenic, as is the case of U87. U87 cells grown on serum-containing medium are able to form tumors when injected into the brain of nude mice, indicating that under these conditions, the main feature of CSCs, which is the formation of tumor, is maintained [2,26,27].…”

“…It is also important to observe that for the same number of cells plated, much more spheres where observed in NSC medium when compared to serum-containing medium since the former contains factors that favor CSC proliferation. Additionally, another human glioma cell line, U343 as well as the C6 rat glioma cell line, which is widely used in animal studies and was recently shown to contain CSCs [22,23], presented a drastic reduction in sphere formation when treated with 100 μM ATP in NSC medium (Fig. 3b, c).…”

“…Although the majority of the data with gliomas CSCs were obtained in defined serum-free medium, some glioma cell lines readily form clones in the serumcontaining medium [23]. The maintenance of a CSC population in spheres, in spite of the presence of 5% serum, may be due to the establishment of a microenvironment that favors the maintenance and growth of CSCs.…”

Targeting glioblastoma cancer stem cells: the next great hope?

“…32 Functional assays such as serial implantation in nude mice are considered the gold standard. 22 Other techniques include Hoechst dye exclusion, 38,59,67,122,127 aldehyde dehydrogenase 1 assay, 114 spectroscopy, 76,77,130 and neurosphere cultures. 53,138 Cell sorting is another commonly used potent tool to enrich the population of CSCs (Table 1).…”