Proteomics And Genomics Analysis Of Brain Tumor Stem Cells Derived From Gliomas

ObjectivesRecently, several groups have described the existence of a cancerstem cell population in human gliomas, which raises fundamental issuesconcerning their actual ability to drive cancer growth and recurrence. TheBTSC hypothesis suggests that not all the cells in gliomas have the sameability to proliferate and maintain the growth of the tumor. Only arelatively small fraction of cells in the tumor possess the ability toproliferate and self-renew extensively. Most of the tumor cells lose theability to proliferate and self-renew and they differentiate into tumor cellsthat become the phenotypic signature of the tumor. The presence ofBTSC will have important implications for investigating the tumorigenicprocess in the central nervous system. However, there are many questionsabout BTSC remains unclear, many definitive answers may have to waitfor. (1) BTSCs are lack of specific surface markers. It is critical to stressthe distinction between NSC and BTSC. Singh et al. show that a subset ofCD133+ brain tumor cells have the exclusive ability to form multipotentneuro-spheres in vitro. If CD133+ cells represent true BTSC, it wouldhave significant implications for the understanding and treatment of braintumors. (2) The pathogenesis of BTSC differentiation is still unknown,which is very important to tumorigenic. In this study we combinemethods of proteomics and genomics, including two-dimensionalpolyacrylamide gel eiectrophoresis (2D-PAGE), mass spectrometry (MS),and microarray technology, to analyze differences between BTSC anddifferentiated glioma cells. There are four sections in this study. (1)Isolation, culture, and identification of BTSC in gliomas. (2) Proteomicsanalysis of BTSC derived from glioma cell line. (3) Genomics analysis of BTSC derived from glioma cell line. (4) Screening target genes andproteins from the two databases by proteomics and genomics analysis.Methods(1) Section one: Nine human gliomas specimens, two glioma celllines (C6 and U87) were involved in this study. The methods of isolationincluded primary culture and CD133+ cells culture after magnetic cellsorting by CD133 cell isolation kit. Cells cultures were performed underthe condition of NSC. The BTSCs were identified byimmunocytochemical staining of neurospheres by detected the types ofmolecular markers of nestin, NSE, GFAP, and also by differentiation tests.(2) Section two: 2D-PAGE was performed to separate the proteins of theCD133+ and CD133- U87 cells, which were isolated by magnetic cellsorting. The differential protein spots were analyzed by software analysis,subject to in-gel digestion, and identified by matrix assistant laserdesorption time-of-flight mass spectrometry (MALDI-TOF-MS) andMALDI-TOF/TOF-MS. (3) Section three: The total RNA of the cells wasextracted from CD133+ and CD133- U87 cells, respectively. Theystudied by means of a DNA microarray (Agilent Human 1A oligo). Thedifferential genes were screened out by software analysis. (4) Section four:We used the differential proteins as clues, and retrieved in SwissProt andPubMed, and found gene names, Gene Symbols, and GenBank ID, andthen searched in differential genes database, respectively.Results(1) Section one: Classical neurospheres formed in vitro by normalNSC were detected in two glioma specimens cultures established frompediatric malignant gliomas. They were medulloblastoma and high-gradeglioma. The neurospheres derived from high-grade glioma could havelong-term cultures, while from medulloblastoma could not. Conversely,clone formation was never observed in cultures from adult gliomas. The neurospheres were also detected in C6 and U87 cell lines either byprimary culture and CD133+ cells culture. The neurospheres couldproliferate and increase in their diameters. For most neurospherescontained many cells expressing the NSC marker nestin and relativelyfew cells expressing the neuronal marker NSE and or the astrocyticmarker GFAP. We then assessed neurosphere cells multipotency bydetermining the ability to generate neurons, astrocytes, andoligodendrocytes. The neurosphere cells underwent terminaldifferentiation upon removal of mitogens to the medium, and all of themdifferentiated into GFAP-positive astrocyte-like cells but also into NSE-positive neuron-like ceils. (2) Section two: There were 73 differentiallyexpressed protein spots observed in comparing between CD133+ andCD133- U87 cells by 2D-PAGE (p＜0.05). Spots with＞1.5-foldexpression changes were 46, and＞2-fold were 27, and＞3-fold were 8.Ten spots were increased, while 63 spots were decreased in CD133+ U87cells. Forty-four high-quality peptide mass fingerprinting and 35 proteinswere obtained by MS. (3) Section three: We identified 840 differentialexpression genes, including 759 overexpressed and 81 underexpressedgenes when comparing CD133+ to CD133- U87 cells by means ofa DNAmicroarray. Among them, the ratio of differential expression genes intranslation-regulator-activity group was 14.9％, and the highest in allgene ontology groups (X~2 test, X~2=88.655, p=0.000). (4) Section four:Four genes and 5 related proteins were obtained by searching indifferential proteins and genes databases. They were TCP1-epsilon andChaperonin, related with CCT6A gene; protein c-abl phosphotyrosineacceptor, related with PTK9L; DnaJ (Hsp40) homolog, subfamily B,member 11 precursor, related with DNAJC10 gene; and coiled-coildomain containing 81, related with CCDC2 gene. Conclusions(1) The results show that gliomas contain neural stem-like cells,which are BTSC. (2) The BTSC can be exclusively isolated with the cellfraction expressing the neural stem cell surface marker CD133. (3)Differentially expressed genes and proteins have been obtained byproteomics and genomics analysis, which are important targets to thestudy of BTSC. (4) Four genes and 5 related proteins, correlated withBTSC have been screened from the two databases by proteomics andgenomics analysis. These genes and proteins are most important targets tothe study of BTSC.