Experimental Study On Synergistic Targeting Restraint Effect Of ATRA/BCNU Against Human Brain Tumor Stem Cells

OBJECTIVE The aims of the first part of the paper are to establish a culture system to isolate and propagate brain tumor stem cells (BTSCs) within human gliomas, to study their self-renew, proliferation and dedifferentiation characteristics, to determine the tumor initiating efficiency of BTSCs in brains of BALB/c-nu nude mice. Through these experiments, we may have a profile of BTSCs within gliomas and make a foundation for the next part of study. One of the two main purposes of the second part of this investigation is to determin whether ATRA/BCNU can restrain BTSCs more than BCNU alone in vitro. Another main aim of this part of study is to determine what molecular mechanisms induce the restraint and whether apoptosis is one of the mechanisms.METHODS In this study, samples from 34 gliomas of different grades were obtained from patients undergoing microsurgical tumor resections. Tumor masses were acutely dissociated and triturated into single cells, which were then seeded into serum-free DMEM/F12 medium containing B27 (1:50), basic fibroblast growth factor (bFGF, 20μtg/L), epidermal growth factor (EGF, 20μg/L). After the primary BTSs or neurospheres generated, they were mechanically dissociated and passaged in the above serum-free medium, then BTSCs was induced in mitogen-free DMEM/F12 medium supplemented with 10% fetal bovine serum. The expressions of stem cell antigens such as CD133 and Nestin, and mature neurocyte antigens such as NeuN, GFAP and CNPase of BTSs before and after differentiation were assayed by immunocytochemistry. Magnetic cell sorting assay of BTSCs based on CD133 expression was performed to isolate CD133+ tumor cells. The CD133 positive and negative cell were transplanted into brains of BALB/c-nu nude mice to determine the tumor initiating efficiency of BTSCs. Immunohistochemistry of the samples of transplanted tumors were also performed for the expression of CD133. Then in the second part, we use monoclone and MTT test to determine whether ATRA/BCNU can restrain BTSCs more than BCNU alone in vitro and use AO/EB staining and flow cytometry to prove apoptosis be a crucial role in the restraint. After that, we use RT-PCR test to investgate the molecular mechanisms of restraint.RESULTS In each specimen in this series, only a minority of glioma cells showed unique capacity of self-renew and proliferation. These BTSCs generated free-floating neurosphere-like BTSs in the serum-free medium used in this study in vitro. BTSs generated from BTSC attached to poly-L-lysine coated coverslips and were induced to differentiate when the serum-supplemented medium was added. The differentiated cells were morphological different and express mature neurocyte antigens such NeuN, GFAP and CNPase. Magnetic cell sorting assay of BTSCs based on CD133 expression was competent for isolate CD133+ BTSCs. After transplanted into brains of BALB/c nude mice, CD133 positive BTSCs established tumors, whereas CD133 negative tumor cells always failed. Then in the second part, the fact that ATRA/BCNU can restrain BTSCs more than BCNU alone in vitro is determined through monoclone and MTT test and apoptosis is proved to be a crucial role in this process by using of AO/EB staining and flow cytometry. After that, the fact that the down regulation of Survivin is one of molecular mechanisms induce the restraint is proved by using of RT-PCR test.CONCLUSIONS Only a small proportion of cells composing glioma masses are stem cells with the ability to self-renew and multilineage differentiation. The serum-free medium containing bFGF and EGF used in this study is competent for isolation and culture of BTSCs. BTSCs propagate in this medium and generate numerous tumor spheres which could be passaged for a prolonged time. BTSCs show extensive proliferation and selfrenew copacity and abnormal multilineage differentiation potential. They also possess tumor initiating ability in vivo in nude mice. CD133 is a specific surface marker of BTSC from tumor entity. ATRA/BCNU can restrain BTSCs more than BCNU alone in vitro and apoptosis is proved to be a crucial role in this process. Finally, the down regulation of Survivin is one of molecular mechanisms induce the restraint. The BTSCs may provide both a platform for basic research and a target for clinical treatment of glioma and medulloblastoma.