in leukemias , rare tumor cells show extensive proliferative and self - renewal potential and are responsible for maintaining the tumor clone ; similar cells were subsequently described in gliomas [ 24 ] . they were called brain tumor stem cells ( btscs ) , believed to be responsible for tumor growth , recurrence and resistance to therapies and , supposedly , to derive from neural stem cells ( nscs ) transformation and to be related to restricted unipotent or multipotent progenitors [ 57 ] . they share with nscs antigenic properties of stemness , such as nestin , musashi-1 , cd133 , and sox2 expression ; it has been suggested to better identify them on the basis of their functional competence than as cell types . a derivation from dedifferentiated tumor cells that re - acquire stem cell - like properties has also been hypothesized [ 3 , 10 , 11 ] . beside common antigenic properties with nscs , btscs share genetic properties with primary tumor cells . their existence in tumors is indirectly demonstrated by the generation , from tumor samples put in culture , of neurospheres ( ns ) and , by adding 10% of fetal bovine serum ( fbs ) , of adherent cells ( ac ) with different capacities of self - renewal , differentiation , clonogenicity , and tumorigenicity . the demonstration of btscs in vitro may be influenced by the methodological procedures employed and may have different expression modalities [ 12 , 13 ] . for example , using n29 and n32 tumor models , it has been demonstrated that ns formation is not necessary to enrich for tumorigenic cells , due to the fact that ac can display high clonogenicity in vitro , tumorigenicity in vivo , and high expression of cd133 and nestin . recently , two transcription factors have been proposed as putative markers of stemness : sex determining region of y chromosome ( sry)-related high mobility group box2 ( sox2 ) and repressor element-1 silencing transcription / neuron - restrictive silencer factor ( rest / nrsf ) . sox2 belongs to a family of transcription factors expressed in the central nervous system ( cns ) at early stages of development   and it is expressed in malignant gliomas at both mrna and protein level . the second putative marker is rest / nrsf ( rest ) , which represses transcription of several neuronal genes by binding to a dna regulatory motif known as repressor element 1/neuron - restrictive silencer element ( re1/nrse ) . rest is highly expressed in nscs and its transcription is blocked as they exit the cell cycle and differentiate . there is no possibility to demonstrate the existence of btscs in tumors before surgical intervention as to be useful for therapeutic strategies . specific phenotypes have been shown to be related with the derivation of ns from surgical samples [ 19 , 20 ] , but it has not yet been definitively established whether the phenotypical heterogeneity influences   or not   the occurrence of btscs . in a series of gbms we wanted to verify from the genetic and antigenic point of view the relationship between ns with ac and the primary tumor phenotype . the investigation was carried out on 16 partially resected primary hemispheric gbms ( cv1 - 20 ) operated on at the department of neuroscience , neurosurgical unit , university of turin . the mean age of the patients was 62 years ( range : 2376 years ) . the patient stratification was as follows : out of 16  gbm patients , 10 received postoperative standard radiation therapy ( rt ) ( 60  gy total dose in 2730 fractions administered via a linac ) . among the 10 irradiated patients , 8 received standard temozolomide ( tmz ) therapy ( 75  mg / m / daily for 6 weeks ) , followed by adjuvant tmz ( 200  mg / m from day 1 to day 5 every 4 weeks for 612 cycles ) . of the remaining patients , 2 did not receive any treatment because of low karnofsky performance status ( kps ) , and 2 were lost at follow up . all patients are still alive with the exception of 2 patients who died 3 months following surgery . a viable sample from the dissected tumor was divided into three serial slices : one was stored at 80c for molecular genetics and frozen sections , one was fixed in buffered - formalin and embedded in paraffin ( ffpe ) and one was washed , minced , and enzymatically dissociated for expansion in culture . all patients provided informed consent allowing for the use of tumor specimens for molecular and cell culture studies . haematoxylin and eosin stain ( h&e ) was performed on 4  m paraffin sections . ihc was performed on 4  m paraffin sections using a ventana full benchmark automatic immunostainer ( ventana medical systems inc . , heat - induced epitope retrieval ( hier ) was performed in tris - edta , ph 8 ( ventana medical systems ) . double immunohistochemistry was performed using nestin , gfap , and cd34 antibodies with ultraview universal alkaline phosphatase red detection kit as chromogen ( ventana medical systems ) .   if was performed on frozen sections , ns and ac . goat anti - rabbit fitc - conjugated igg and rabbit anti - mouse tritc - conjugated igg antibodies were used . observations were made on a zeiss axioskop fluorescence microscope ( karl zeiss , oberkochen , germany ) equipped with an axiocam5mr5c and coupled to an imaging system ( axiovision release 4.5 , zeiss ) . culture conditions were the following : for ns , dulbecco 's modified eagle 's medium ( dmem)/f-12 with 10  ng / ml bfgf ( basic fibroblast growth factor ) and 20  ng / ml egf ( epidermal growth factor ) ; for ac , dmem with 10% fetal bovine serum ( fbs ) . gbm reference cell lines were u87-mg and 010627 ( galli r , dibit san raffaele , milan ) for both ac and ns .   all cell cultures were periodically checked for mycoplasma contamination ( e - myco mycoplasma pcr detection kit , intron biotechnology , korea ) . for population analyses , cells were plated at 200,000  cells / cm , and the resulting spheres were collected every 46 days . the total number of viable cells was assessed at each passage by trypan blue ( sigma , st . briefly , ns were dissociated into single cells and plated into a 96-well plate for the subsphere - forming assay by limiting dilution . single - cell suspensions were diluted and plated at 1 - 2  cells / well . after plating , cells were monitored , and only wells containing a single cell were considered for the analysis . differentiation assays were performed by mitogen withdrawal and the addition of 3% fbs to the culture . it was tested by transplanting ns and ac from each sample into nod scid mice ( charles river , calco , italy ) . two microliters of a 1  10  cells / ml suspension were stereotactically injected into the right striatum according to the procedure described in galli et al . . mouse brains were treated with ffpe using the same procedures as described for primary tumors . all animals were handled in strict accordance with good animal practice as defined by the relevant international ( directive 86/609/eec and recommendation 2007/526/ec from the european community ) and national ( legislative decree 116/92 and law n. 413/1993 ) directives and according to protocols approved by the animal care and use committee of the h. s. raffaele ( iacuc # 316 ) . genomic dna was extracted from frozen tumor samples according to a standard phenol - chloroform protocol . dna extraction from cell lines was performed with the qiamp dna mini kit ( qiagen gmbh , hamburg , germany ) and from peripheral blood by a salting - out procedure . the genetic analyses described below were carried out on primary tumors , ns and ac . highly polymorphic markers were selected based on their heterozygosity and position on the five most frequently deleted chromosomes in gliomas ( table 2 ) . microsatellites were amplified in multiplex reactions by pcr with fluorescently labeled primers ( applied biosystems ) . following electrophoresis on an abi 3130 genetic analyzer ( applied biosystems ) , data were collected by genemapper v4.0 software ( applied biosystems ) . loh was determined by measuring the peak height ratio between the two alleles ( allelic ratio ) in constitutive ( n1/n2 ) and tumor ( t1/t2 ) dna samples . values less than 0.5 and greater than 1.5 were considered evidence of loh . as in previous studies , egfr amplification status ( genebank sequence nm_005228 ) was assessed by fragment analysis and capillary electrophoresis ( ce ) on an abi 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) . the amplification status of the egfr gene was determined by measuring the egfr / inf- ratio . a ratio greater than 2.09 was considered evidence of more than two copies of the egfr gene . mgmt promoter hypermethylation status ( genebank sequence nm_002412 ) was determined by methylation specific polymerase chain reaction ( ms - pcr ) followed by ce as previously reported . sodium bisulfite modification was performed with the methyleasy dna bisulphite modification kit ( human genetic signatures pty ltd , macquarie park , sydney , australia ) . temecula , ca , usa ) , and normal lymphocyte dna samples were used as methylated and unmethylated controls , respectively . following electrophoresis using an abi 3130 genetic analyzer ( applied biosystems ) , data were collected by the genemapper software v4.0 for fragment analysis ( applied biosystems ) . the peak height ratio between peaks for the methylated and unmethylated allele was determined . values greater than 0.1 were scored as evidence of the methylated status of the mgmt gene . the entire coding region of pten ( exons 19 ) ( genebank sequence nm_000314 ) and tp53 ( exons 111 ) ( genebank sequence nm_000546 ) , the tyrosine kinase domain ( tk ) ( exons 1824 ) of egfr ( genebank sequence nm_005228 ) , and codons r132 of idh1 ( genebank sequence nm_005896 ) and r172 of idh2 ( genebank sequence nm_002168 ) were investigated for variations by direct sequencing . amplicons were analyzed by direct dna sequencing on an abi 3130 genetic analyzer using the bigdye terminator version 1.1 cycle sequencing kit . the reported nucleotide and amino acid numbering is relative to the transcription start site ( + 1 ) corresponding to the a of the atg on the corresponding genebank reference sequences . to establish if each putative mutation was somatic ( i.e. , tumor specific ) or germ - line , the corresponding patient 's constitutional dna was also analyzed when available . 
 in silico predictions of the putative functional effects of missense mutations in genes coding for tp53 , pten , and egfr amplification were determined by pmut ( http://mmb.pcb.ub.es/pmut/ ) , polyphen ( http://genetics.bwh.harvard.edu/pph/ ) , and snap ( http://cubic.bioc.columbia.edu/services/snap/ ) programs . the effect of the intronic variants on splicing was evaluated by spliceview ( http://bioinfo2.itb.cnr.it/sun/webgene/ ) and nnsplice ( http://biologyhelp.awardspace.com/desc.php?id=147&type=biotech ) software programs . frozen tissue and cultured cells ( ns and ac ) were homogenized in a lysis buffer ( 50  mm tris - hcl , ph 7.4 , 150  mm  nacl , 1% v / v igepal , 2% sodium dodecyl sulfate ( sds ) , 0.5% sodium deoxycholate , 10  mm edta ) supplemented with protease inhibitor cocktail ( sigma ) , 2  mm sodium orthovanadate , and 10  mm sodium fluoride . whole protein extracts were quantified by bca protein assay kit ( pierce biotechnology , rockford , il , usa ) and equal amounts were resolved in 10% sds - page gels and transferred onto pvdf membranes ( immobilon - p , millipore , bedford , ma ) . membranes were then incubated with the appropriate hrp - conjugated secondary antibodies and proteins were detected by enhanced chemiluminescence ( millipore ) . band intensity was quantified by densitometry using nih image j software ( rsb , nimh , bethesda , md , usa ) . slices from the 16 tumor samples used for histological analyses were serial to those used for cell culture . the following phenotypic features characterized 4 of the 6 samples from which ns generated and 3 of the 9 samples from which ac generated : high cell density , high number of mitoses , a ki-67/mib-1 labeling index ( li ) between 25% and 30% , circumscribed necroses with pseudo - palisading , high vessel density , and perivascular cuffings of tumor cells ( figure 1 ) . nestin was variably expressed in all gbms , but its distribution did not completely overlap with that of gfap . as a matter of fact , nestin expression was higher than gfap expression , considering both the number of positive cells and the same cells ( figures 2(a ) and 2(b ) ) . sox2 and rest , which are diffusely expressed in all gbms , reached the highest intensity in the areas the phenotype of which has been above described and around vessels ( figures 2(c ) and 2(d ) ) . as determined by analysis of frozen sections , cd133 and musashi-1 were sporadically expressed in areas of variable size or in single cells ( figures 2(e ) and 2(f ) ) . ns and ac were observed in cultures from 6 ( cv1 , cv7 , cv10 , cv13 , cv17 , and cv20 ) and 9 ( cv1 , cv2 , cv3 , cv4 , cv6 , cv8 , cv9 , cv10 , and cv17 ) of the 16 samples , respectively ( 37.5% and 56.3% ) . cells derived from cv10 primarily grew as ac , occasionally forming cell aggregates attached to the culture plate . when cv10 aggregates were transferred to a low - adherent culture plate with dmem / f-12 medium and mitogens , they gave rise to ns . in cells derived from cv1 , in addition to ns alternatively , cultures derived from cv17 developed both ns and ac in their respective media conditions . ns cultures were verified to contain true btscs by assessing their long - term self - renewing ability by population analysis . cells grown in culture with growth factors proliferated as nonadherent , multicellular spheres ( figure 3(a ) ) , whereas cells grown with serum proliferated as an adherent monolayer with an initial heterogeneous morphology which later became homogeneous with resemblance to fibroblasts ( figure 4(a ) ) and showing astrocytic , oligodendrocytic , or neuronal differentiation ( figures 4(b)4(d ) ) . growth rates of ns and ac are illustrated in figures 5(a ) and 5(b ) . figure 5(c ) shows the different growth rates between ns and ac of the same cell line . the subsphere - forming assay performed by limiting dilution demonstrated that the clonogenic efficiency of all ns to form subspheres from individual ns cells was 79% .   by differentiation assays ns demonstrated their multipotency showing after 7 days from the addition of 3% serum an astrocytic , oligodendrocytic and neuronal differentiation ( figures 6(a)6(c ) ) . upon intracranial transplantation , ns developed tumors in mice , whereas ac did not developed tumors or showed a reduced tumorigenicity in two cases ( data not shown ) , demonstrating that ns populations included true btscs . nestin was strongly expressed in ns , along with sox2 and rest , whereas cd133 and musashi-1 were only sporadically expressed ( figures 3(b)3(f ) ) . ac , in contrast , showed decreased nestin expression and increased gfap expression when compared with ns . the expression of antigens indicative of stemness was highest in primary tumors and ns , whereas the expression of antigens indicative of differentiation was highest in ac . the derivation of ns and ac from the respective primary tumors was confirmed by genotyping of the 13 microsatellite markers used for loh analysis . genetic alterations in primary tumors and in the matched ns or ac are summarized in table 4 . no significant differences in the frequency of genetic or epigenetic alterations were observed between primary tumors generating cell lines and those not generating cell lines . loh on chromosomes 1p , 9p , 10q , 17p , and 19q , egfr amplification , mgmt promoter hypermethylation as well as pten and tp53 mutations was identified with similar frequency in primary tumors and ns , but not in ac . in primary tumors , pten mutations were detected in 56.2% of cases , whereas only 31.2% of cases showed tp53 mutations . the tk domain of the gene coding for egfr was found to be mutated in only one primary tumor ( cv6 ) generating ac . this previously undescribed mutation was a somatic synonymous nucleotide substitution ( c.2904c > t , p.f968f ) . no point mutations were identified in codons r132 or r172 of idh1 and idh2 genes , respectively .   with respect to loh analysis , the specific spectrum of loh present in primary tumors was detected only in ns . loh of 10q was present in all primary tumors , followed by loh of 9p in 8 of the 16 cases ( 50% ) . the allelic imbalance was partial in all primary tumors and complete in ns ( figure 7 ) . western blotting analysis confirmed a higher prevalence of antigens indicative of stemness in primary tumors and ns when compared to ac , in which antigens indicative of differentiation were more highly expressed . mgmt expression was consistent with the mgmt promoter hypermethylation status ( figure 8(a ) ) . densitometric analyses of the antigens studied in ns , ac , and the corresponding primary tumors are reported in figures 8(b ) and 8(c ) . basically , it has been demonstrated that different subsets of btscs with different proliferative , clonogenic , and tumorigenic properties exist in gbms . moreover , chemosensitivity of highly infiltrative gbms has been demonstrated to be associated with a stem cell - like phenotype , that is , with the expression of nestin , vimentin , and musashi-1 [ 3 , 29 ] . in a study no difference was found among several samples from the same tumor as for their btsc content , but in another study the existence of at least two types of btscs from different regions of the same tumor has been demonstrated ; they showed different genetic anomalies and tumorigenic potentials , even though the cells were considered to derive from common ancestors . the demonstration of btscs largely depends on the technical procedures , but in most studies only approximately 30% of gbms generate ns . selecting differently glioma initiating cells , for example by autofluorescence , these can be found in all glioma types .   in our gbm series , the development of ns in culture is largely dependent on the occurrence in primary tumors of what is known as the most malignant phenotype of gmb , composed of small hyperchromatic and rapidly dividing cells , with vessel hyperdensity and circumscribed necroses . in the present study , the most important observation is that the frequency of the genetic and epigenetic alterations is very similar between primary tumors and ns , showing a strong genotypic concordance . this is particularly remarkable in the case of loh profiles , which are present with the same spectrum in both primary tumors and ns , but not in ac . the allelic imbalance was partial in primary tumors and complete in ns . in ac , no detectable genetic or epigenetic alteration similar to that of the corresponding primary tumor was observed , with the exception of occasional pten mutations . the mgmt hypermethylation status and the egfr amplification status showed a similar frequency between primary tumors and ns , but not between primary tumors and ac . the same is true for the mutation spectrum of tp53 and pten genes . in 3 of our cases , both ns and ac developed from the same tumor sample indicating that cells with different potential can coexist in the same tumor . interestingly , in 1 of these 3 cases , ac generating ns showed the same molecular profile as ns . the prevalence of nestin upon gfap and the expression of stemness antigens in perivascular cuffings and in areas with vessel hyperdensity of primary tumors is in line with the preference of btscs for perivascular niches [ 3234 ] . the latter recapitulate normal nscs and the vasculature of developing nervous system [ 35 , 36 ] ; on the other hand , btscs in gliomas mediate vascular proliferation via vegf [ 37 , 38 ] . the different behavior of ns and ac may be due to showing the former full stemness properties and appearing the latter as the product of cell differentiation . ns could be transformed nscs from germinal zones , subventricular zone ( svz ) , or subgranular zone ( sgz ) , or from dedifferentiated elements of the astrocytic lineage , whereas ac could represent a differentiating cell type . intermediated behaviors have been observed in our material , as it has been already shown . the generation of ns from a tumor sample mostly when it shows the mentioned phenotype may suggest that btscs arise from or are contained in the novel , proliferating and dedifferentiated clones which develop following mutation accumulation or epigenetic events during malignant transformation and substitute the predecessors via competitive selection . this hypothesis , previously considered [ 3 , 8 , 10 , 11 ] , regards dedifferentiated cells as regressed to an embryonic state reacquiring a stem cell - like status . embryonic regression has been previously invoked , for example , for reactive astrocytes reexpressing nestin [ 40 , 41 ] . our observations concern a very important debated question , that is , the cell composition of a starting tumor tissue . it is not clear whether a major role is played by the occurrence of btscs in the tissue or by culture conditions . on the other hand , a clear distinction between ns and ac is still debated concerning self - renewal , clonogenicity and tumorigenicity , and intermediate stages between them exist ; terms such as semiadherent cells have been coined for them . this is confirmed by the behavior of cv1 , cv10 , and cv17 of our series . stemness , therefore , might not be a property of a specific cell type , but a status which encompasses the early steps of the cytogenesis . it is conceivable that in the process of differentiation during cytogenesis there is a point , represented by progenitors , beyond which stemness ceases . in the same way , considering the process of tumor cell dedifferentiation from more differentiated stages , through the development of new clones there should be a point beyond which stemness reappears . from the different stages of differentiation and dedifferentiation , the high expression of sox2 and rest in primary tumors and ns is a stemness property . in our samples , sox2 not only correlated with the malignancy grade , but it was also overexpressed in the gbm phenotype from which ns could be generated and even amplified as in ns . rest has been shown to display tumor - suppressor activity   and oncogenic functions in medulloblastoma . we did not find in our samples even a slight rest amplification ; however , it is overexpressed in the primary tumor phenotype generating ns .
formation of neurospheres ( ns ) in cultures of glioblastomas ( gbms ) , with self - renewal , clonogenic capacities , and tumorigenicity following transplantation into immunodeficient mice , may denounce the existence of brain tumor stem cells ( btscs ) in vivo . 
 in sixteen cell lines from resected primary glioblastomas , ns showed the same genetic alterations as primary tumors and the expression of stemness antigens . 
 adherent cells ( ac ) , after adding 10% of fetal bovine serum ( fbs ) to the culture , were genetically different from ns and prevailingly expressed differentiation antigens . 
 ns developed from a highly malignant tumor phenotype with proliferation , circumscribed necrosis , and high vessel density . beside originating from transformed neural stem cells ( nscs ) 
 , btscs may be contained within or correspond to dedifferentiated cells after mutation accumulation , which reacquire the expression of stemness antigens .