| Partâ… . Cellular Biological Behaviors of Brain Tumor Stem Cells: A Comparative Study with Neural Stem CellsBackground and Objective Abnormal differentiation is central to the formation and progression of gliomas, the most common neoplasm of central nervous system in human. Understanding of the differentiation profile of brain tumor stem cells (BTSCs), the key ones among tumor cell population, through comparison with neural stem cells (NSCs) would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Singh et al. observed that, under differentiation condition, all tumor spheres grew attached as monolayer, lacking expression of undifferentiated cell marker CD133 and nestin. However, the fact that only a small proportion of cells within tumor spheres are true stem cells makes it necessary to isolate and focus on tumor stem cells if the exact biological behaviors of this special cell fraction are to be fully understood. So, in this study, we try to culture and purify BTSCs from newly surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property.Methods Brain tumor spheres were cultured, with the technique used in neural stem cell culturing, from both primary and recurrent mixed gliomas harbored in the same patient. Brain tissue from a 1-month-old embryo was subjected to the same procedure as that mentioned above to get neurospheres. Both BTSCs and NSCs were purified with magnetic cell sorting system and evaluated with flow cytometry. Induction of differentiation was carried with culture medium added with 10% fetal bovine serum. The morphological changes taking place in both BTSCs and NSCs were observed under phase-contrast microscopy, and the expressing tendency of differentiation-related surface markers, like CD133, nestin, GFAP,β-TubulinIII, were detected with immunofluorescent staining and flow cytometry.Results 1)Tumor spheres were produced from both primary and recurrent glioma samples. 2)BTSCs (CD133+glioma cell), as well as NSCs, were purified effectively with magnetic cell sorting system. 3)In differentiation assay, NSCs changed morphologically along it's routine way, whereas, BTSCs underwent rather complicated course: changing from round at the beginning to short fusiform, polygon and long fusiform by day 7, after then, cells gradually pulled back their process, huddling together and reforming suspending tumor sphere 21 days after subject to differentiation condition. 4) The differentiation assay with immunoflurescent staining and flow cell cytometry highly coincided with the morphological changes mentioned above. In BTSCs, the expressing rate of CD133 dropped sharply from 80.76% at the beginning to 16.33% on day 3 and to the lowest point of 3.65% by day 7, but bounded up from day 10. During the same period, the expression of nestin was also examined, with the similar results. Inversely, the expressing rates of GFAP andβ-tubulinâ…¢increased gradually till day 7, but decreased after then. Same experiments were also performed on NSCs, however, the expressing rates of CD133 and nestin kept decreasing, till to undetectable level on day 10; at the same time the expression of GFAP andβ-tubulinâ…¢were remarkably increased without turn point. 6) Detection of cell cycling with flow cytometry showed that all BTSCs from both primary and recurrent glioma were hypodiploid, while NSCs were all normal diploid. After differentiation, NSCs were still normal diploid, however, the population of BTSCs were more complicated: in BTSCs from primary tumor mass, cells were mainly hypodiploid with minority diploid; in BTSCs from recurrent lesion, cell were comprised of the following three types: hypodiploid(54.81%), hyperploid (44.2%) and diploid. It was also revealed that cells of S and/or G2-M phase in BTSCs were much richer than those in NSCs regardless of differentiation status. When BTSCs from primary and recurrent lesions were compared, more cells of S and/or G2-M phase were detected in recurrent BTSCs.Conclusions BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma. Brain tumor stem cell is a functional concept, the malignant progression of BTSCs actually represent the evolving of gliomasPartâ…¡The Molecular Cytogenetic Profile of Brain Tumor Stem Cells: A Comparative Study with Neural Stem CellsBackground and Objective This difference in differentiation exhibited by BTSCs and NSCs indicates internal signaling pathway regulating differentiation has been severely shifted by accumulated gene mutation(s) in BTSCs; in other words, genes giving rise to differentiation have been suppressed by genes keeping stem cell undifferentiated. To determine the genes responsible for the dedifferentiation through cytogenetic methods and other methods is of great significance in probing the molecular mechanism of brain tumor initiation. The recently developed array based comparative genomic hybridization (array CGH) has resulted in a significant increase of sensitivity of detecting DNA copy number gains and losses from a resolution of 10-20 Mb, which is routinely achieved with traditional CGH, to an average of 1 Mb. The additional advantage of array-based CGH is that the mapping information of BAC clones printed on the arrays are already known, therefore a direct link between the affected regions and candidate genes can be quickly established. In this part, array-based CGH technique was employed to the end of searching for genes that are candidate partners in the malignant transformation of neural stem cells or precursors.Methods DNA was extracted from brain tumor stem cells purified with magnetic cell sorting system with DNA isolation kit (Qiagen, Valencia, CA), DNA from neural stem cells was also collected as control. The array used in this study consists of 2632 human BACs (Spectral Genomics, Houston, TX). The experiments were performed according to the manufacturer's protocols. Data analysis was carried out using the SpectralWare 2.2.23 (Spectral Genomics, Houston, TX). The very interesting gene discovered by array-CGH was further examined with tissue microarray technique to determine it's protein expression in 68 glioma specimens of different malignant grades.Results 1)Among the 2621 clones spotted on the array, 2457 were mapped to non-sex chromosomes. No DNA copy loss or gain was detected in neural stem cells, while large amount of BAC clones were amplified or deleted in both primary and recurrent brain tumor stem cells, of which, 160 clones were found in primary BTSCs, and 182 clones in recurrent BTSCs. Moreover, most of the altered clones were detected simultaneously in both primary and recurrent BTSCs, from which, dozens of candidate genes associated with tumorigenesis were mapped, including CDK7, p18INK, Rb, 4ING3, ING5, CHES1, MYCL1, ERBB4, RAB40C, and so on. INK4-cyclin D/CDK4/6-Rb-E2F is one of the most essential signal pathways regulating cell progression. In this study, p18INK4, a core member of the upper negative regulator INK4, and the famous tumor suppressor Rb was found to be synchronously lost in both primary and recurrent BTSCs. Besides the common changes detected by array CGH, some candidate genes related to tumor formation or malignant progression were found to be exclusively amplified or lost in BTSC-1 and BTSC-2 respectively. Intriguingly, in recurrent BTSC, genes activating oncogene RAS were found to gain DNA copy, while genes inhibiting RAS were lost. 2) We also examined the expression of p18INK in BTSCs, normal brain tissue, and glioma specimens of various grades with tissue microarray. As a result, no p18INK staining was revealed in BTSCs, but p18 nuclear immunoexpression was found in 46(67.6%) of the glioma tissues we studied. p18 immunoreactivity also exhibited a clear tendency to elevate with increasing tumor grade.Conclusions Dozens of candidate genes related to malignant transformation and progression of glioma were selected out through array-based CGH technique. Disruption of INK4-cyclin D/CDK4/6-Rb-E2F signal pathway resulting from DNA copy loss of both p18INK and Rb may account for the abnormal differentiation and malignant transformation of neural stem cells or precursors. Up-regulation of oncogene RAS maybe associated with the malignant progression of brain tumor stem cells we studied. The ambivalent result that p18, as a tumor suppressor, was positively immunostained in most of the glioma specimens does not deny the tumor suppressing role of p18, on the contrary, the up-regulation of p18 expression is possibly due to a negative feedback mechanism. Like other tumor suppressor, the loss of p18 function is responsible for the development of some but not all glioma cases.Partâ…¢Cell-Cell Fusion and the Malignant Progression of Glioma: A Preliminary StudyBackground and Objective Cell-cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. More and more evidences indicate cell fusion may also play a critical role in the development and malignant progression of cancer. Not long ago, GBM, a glioma cell line unable to produce tumor mass when transplanted, reduced dramatically in vitro culture. To save this glioma cell line, we co-cultured it with feeding cells harvested from Balb/c mice. As a result, cells were successfully expanded and saved. Unexpectedly, cells undergoing co-culturing with feeding cells formed tumor mass when injected into immunodeficient mice. At that time, we didn't know why. Here, we conducted a preliminary study to investigate whether cell-cell fusion take place in co-culture of glioma cells and feeding cells consisting mainly of macrophages and lymphocytes.Methods T98G, a human glioma cell line with very poor potential to produce tumor mass in immunodeficient mice, were raised with feeding cells harvested from GFP(green fluorescent protein) transgenic mouse. During the course of co-culturing, mixed culture was checked under phase-contrast microscope and fluorescent microscope. Laser scanning confocal microscope was employed to search for cells that co-express GFP( green) from feeding cells and nestin (red) from T98G glioma cells. The ploidy and the proportional change of cells giving off green fluorescence were analyzed with flow cytometry. 5×106Glioma cells before and after co-culture were injected into immunodeficient mice to compare their tumorigenesis.Results 1)During the co-culturing of feeding cells and glioma cells, round giant cells emerged, and some of the giant round cells present green under fluorescent microscope. 2)After plated to glass coverslips and immunofluorescently stained, cells presented green and red simultaneously were discovered among the mixed culture, moreover, binucleate was observed in some double-color-stained cells. 3)The percentage of"green"cells among co-culture decreased continuously from the beginning of co-culturing, but kept at a very low but relatively stable level of 0.6% 10 days later. 4)Compared with feeding cells, the"green"cells purified from mixed culture were found to contain cells of aneuploidy which accounting for about 8% of total. 5)The incidence of tumor mass formation in immunodeficient nude mice was dramatically improved from 25% before co-culture to 100% after co-culture.Conclusions The phenomenon of hybridization between glioma cells and feeding cells (macrophage or lymphocytes) do exist, the chance of fusion is very small, but the significance is great. Due to fusion, the features possessed by macrophage, like motility, plasticity, and adaptability were added to glioma cells, which resulted in the progression of glioma cells. |
