Cancer-Initiating Enriched Cell Lines from Human Glioblastoma: Preparing for Drug Discovery Assays

Cancer-Initiating Enriched Cell Lines from HumanGlioblastoma: Preparing for Drug Discovery Assays

Miriam Romaguera-Ros & María Peris-Celda & Jorge Oliver-De La Cruz &

Josefa Carrión-Navarro & Arantxa Pérez-García & Jose Manuel García-Verdugo &

Angel Ayuso-Sacido

Published online: 30 June 2011# Springer Science+Business Media, LLC 2011

Abstract Glioblastoma multiforme (GBM) is the mostlethal type of brain tumour in the adult humans. Thecancer-Initiatingcell (CIC) hypothesis supports the notionthat failures in current approaches to GBM treatment mightbe attributed to the survival of the CIC subpopulation.Recent evidence shows the idea that using CIC-enrichedcell lines derived from human GBM as new targets for drugdiscovery programs, may improve the chance of success-

fully translating the basic research findingsinto clinicaltrials. Although this approach appears promising, manyimportant biological and technical issues (characterizationof functional CIC markers, inter- and intra-tumoral CICheterogeneity, and isolation and maintenance inconsistency)need to be resolved.

Keywords Tumour stem cells . Glioblastoma . Braintumours . Drug discovery assays

Introduction

Glioblastoma multiforme (GBM), a grade IV astrocytoma(according to the World Health Organization classification),is the most common and devastating brain tumor in theadult humans [1]. The average survival of patients with thiscondition does not exceed 15 months regardless oftreatment choice, which ranges from surgery and chemo-therapy alone to combination with radiotherapy [2]. In mostcases, the GBM relapses within a few millimeters of theoriginal resected tumor [3].In the last decade, the scientistshave dedicated an enormous effort in deciphering theunderlying molecular mechanism of GBM [4, 5]. However,the number of findings and successes in the laboratory hasnot yet been translated into significant improvements inclinical settings [6].

The traditional stochastic model of cancer postulatesthat mutant tumour cells have similar potential forregenerating tumor growth. Unlike this model, the basicidea of the cancer stem cell theory is that there is ahierarchy of cells in a given tumor [7].Cancers appear toharbor small cell populations within the tumor mass thathave the ability to initiate and sustain tumor growth. This

Miriam Romaguera-Ros and María Peris-Celda have equal contribution.

M. Romaguera-Ros : J. Oliver-De La Cruz : J. Carrión-Navarro :A. Pérez-García : J. M. García-Verdugo (*)Department of Cell Morphology, Centro de Investigación PríncipeFelipe and RETICS-CIBERNED,AVDA. Autopista del Saler, 16,46012 Valencia, Spaine-mail: [email protected]

M. Peris-CeldaDepartment of Neurosurgery, Hospital la Fe,Valencia, Spain

J. M. García-VerdugoUniversidad de Valencia,Valencia, Spain

A. Ayuso-SacidoNew York Brain Tumor Research Center, Weill Cornell MedicalCollege of Cornell University,New York, NY, USA

A. Ayuso-Sacido (*)Deparment of Cell Morphology, Centro de Investigación PríncipeFelipe, REIG and CIBERNED,AVDA Autopista del Saler 16,46012 Valencia, Spaine-mail: [email protected]

Stem Cell Rev and Rep (2012) 8:288–298DOI 10.1007/s12015-011-9283-1

so-called subpopulation of tumor stem cells or cancer-initiating cells (CICs), was previously identified inleukemia and later in multiple myelomaand a number ofsolid tumors including breast cancer, medulloblastoma andGBM [8–11]. It is believed that glioma CICs mightdevelop from neural stem cells (NSCs) or progenitor cellsin different stages of commitment that become trans-formed by the acquisition of oncogenic mutations. CICsrecapitulate NSCs characteristics: they express stem cellmarkers, generate spheresthat are morphologically indis-tinguishable from NSC-derived neurospheres, show a self-renewing and proliferating capacity and differentiate intocells that express markers from all3 neuronal lineages [8,12, 13].In the adult brain, neural stem and progenitor cellsremain in 2 separate niches, the subventricular zone of thelateral ventricle and the subgranular zone of the hippo-campus [14–17]. Additionally, glial progenitor cells havebeen isolated from different parts of the brain parenchyma[18]. All these cells are potentially the “cells of origin” ofhuman brain tumors.

The little success seen in the current approaches to treatGBM might be attributed to the biological target of thetherapies. CICs isolated from human GBM samples aremore resistant toradiotherapy, since they preferentiallyactivate the DNA damage checkpoint in response toradiation and repare the radiation-induced DNA damage[19]. In addition, they also display differential levels ofresistance to chemotherapy treatment depending on theirgenetic background [20, 21].Current therapies are focusedon tumour masses, however, and based on these works,useof the CIC subpopulation would be refractory for thetreatment [22]. Additionally, the phenotypic characteristicsand genetic aberrations found in traditionally establishedGBM cell lines, which have been used in drug discoveryassays, often bear little resemblance to those found incorresponding primary human brain tumors [23]. Theevidence supports the notion that GBM models based onCICs would be more biologically relevant for exploringglioma biology and for screening new therapeutic agents[24, 25].

However, despite the enormous potential of CICs fromhuman GBM as a new therapeutic target, we need toincrease our knowledge of the biology of these cells inorder to ensure their successful isolation and identifica-tion. Additionally, a number of technical issues regardingculture media, preservation and expansion are in need ofimmediate attention in order to use CICs as a reliablemodel in cell-based assays as well as to allow forcomparative studies. In this review, we address thesemajor biological and technical questions that remain tobe resolved before bringing our experimental observa-tions on CIC-enriched cell lines to drug discovery assayplatforms.

Biological Issues

Inter- and Intra-Tumor Cancer-Initiating Cell Heterogeneity

Cellular heterogeneity is a hallmark of GBM. Almost 3decades ago, in the early ‘80s, Dr. Shapiro published aseries of cytogenetic works showing the existence ofdifferent cell subpopulations within brain tumors. In lightof the cancer stem cell hypothesis, some of thesesubpopulations could possibly comprise the “stem cell” ofhuman GBM [26–28].

In this seminal work, single-cell suspension and monolayercultures from fresh human GBM samples were established.Later, single cells were harvested from the suspension cultureover the first 24 h and cells from the monolayer culture werecollected over the first 75 h. This approach allowed for thekaryotyping of cells during their first division, which was usedthis as the reference set. The rest of the cells in the suspensionand the monolayer culture were plated into colonies of 6–50cells and karyotyped before 10–15 generations. Finally, thekaryotype of each of these clones was matched with thereference set to distinguish those derived from the originaltumor cells from those emerging as tissue culture artifacts.Using this method, they were able to compare the mitoticsubpopulations within each tumor with those in other tumors.They observed that distinct regions from the same tumorcontained a different distribution of cells [28]. Additionally,cells from adjacent regions shared similarities in terms ofchromosome alterations, while cells in distant regions werequite different. Regarding the chromosome number in theGBM cells, they observed a range extending from near-diploid to hyperdiploid. The hyperdiploid cells were rapidlygrowing and drug-sensitive while the near-diploid cellsappearednormal and were drug-resistant. They finally hy-pothesized that the tumor seemed to develop from almostnormal and near-diploid cells that acted as the GBM tumourstem cells [26].

The presence of a subpopulation of cells within the GBMthat is responsible for tumor initiation and maintenance waslater demonstrated by the group of Dr. Dirkset al. on the basisof the isolation of such cells using the CD133 surface marker[12],which has been used in a plethora of studies. However,most recently,other authors have isolated GBM-CICs on thebasis of the expression of a number of different surfacemarkers like LiCAM, SSEA-1, A2B5 or epidermal growthfactor receptor (EGFR), among others [29–33]. Thus, thenear-diploid population of cells identified by Shapiro’s groupmay correspond either with the subpopulation found withinthe CD133+CICs or with other cell fractionsthat also displayCIC-like features. Therefore, there might be differentpopulations of near-diploid cells in a given tumour. Thiswould also explain why CICs might be identified within theCD133- population [34].

Stem Cell Rev and Rep (2012) 8:288–298 289

In the hierarchical cancel stem sell model, it isconceivable that once the original stem or progenitor cellis transformed, this initial homogeneous population of CICsand/or their niches will change within the tumor, i.e. as thetumor continues to expand within a given timeframe. Aftergeneration of the first homogeneous population of CICs, anew network of abnormal vasculature, one of the keyfeatures of GBM, is starting to form with the probablecontribution of a subpopulation of CICs [35, 36]. Thenormal and new vasculature will be used by some CICs tomigrate out though the brain parenchyma. As the CICniches change, the CICs will also suffer different geneticand epigenetic alterations in different parts of the tumor,while the tumor continues to expand within a giventimeframe.Eventually, within a GBM tumor there is aheterogeneous CIC family whose members arose from theoriginal one [37].Additionally, there might be CIC family-heterogeneity in different GBM tumors [38] (Fig. 1).

On the other hand, despite the number of studiesclaiminga specific CIC marker for human GBM, none of them hasdemonstrated to be specificfor all GBMs. This is probablydue to the inherent heterogeneity of either the cell type oforigin or the diversity of acquired genetic alterations, bothinter- and intra-tumor (Fig. 1). We should stop regardingGBM as a unique disease entity and take a more realisticapproach in discovering the identity of CIC entities bycharacterizing and classifyingboth the GBM tumor massand the CIC-enriched lines. However, to address this issue,an adequate number of samples has to be systematicallyanalyzed and compared. In light of the Cancer GenomeAtlas Research Network, afew hundred of GBM samples havebeen collected and analyzed by different groups of research-ers. Based on massive mRNA expression microarrays thesesamples have been subclassified into 4 groups—proneural,neural, classic and mesenchymal—with deep implications intumor grade, evolution and survival and the presence ofspecific genetic and epigenetic alterations [39–41]. Thesegroups may also differ in the cell type of origin. Accordingly,Günther et al. conducted a gene expression profile of 9different CIC-enriched lines derived from human GBM.Consistent with the previous classification, the 9 CIC-enriched lines were grouped into 2 different clusters

characterized by the differential overexpression of proneural(DLL3) and mesenchymal (CD44) gene markers amongothers. Both clusters showed also biological differences interms of growth pattern, differentiation capacity, and tumor-igenic ability when transplanted into nude mice [42].In line

�Fig. 1 Tentative model for tumour initiation. a. First, a cellulartransformation occurs hypothetically close to a blood vessel. b. ThisCIC has self-renewal capacity that allows the CICs expansion. Asubpopulation within the CICs could participate in the angiogenesisprocess. The vasculature might be used by the CICs to migrate outthrough the parenchymal brain.Due to their genetic instability, thesecells can also generate new CICs clones with new genetic andepigenetic mutations as they are exposed to different niches. c. Thispool of cells is able to expand into a fully heterogenous primarytumour mass,composed of different types of CICs and CICs niches.According to the location of thebiopsy, the results of in vitro assayscan vary dramatically

290 Stem Cell Rev and Rep (2012) 8:288–298

with these observations, Lottaz et al. have recentlypublished a new study including 17 CIC-enriched linesfrom human GBM and compared their gene expressionprofile with those from fetal and adult NSCs. Based onthis comparison, they grouped the CIC-enriched lines into2 clusters. The first clusterdisplayed proneural signaturegenes (overexpression of DLL3 and Olig2 among others)and was comparable to fetal NSCs, while the secondcluster, which was closer to adult NSCs, showed amesenchymal expression pattern (overexpression ofCD44 and vascular endhothelial growth factor, amongothers) [43]. Extending this finding, Mazzoleni et al.further classified GBM samples and CIC-enriched linesaccording to EGFR expression. They showed that EGFR+

sorted cells from either GBM tumour tissue or CICsenriched lines are more tumorigenic than EGFR-cells, anddisplayed an mRNA expression pattern more similar to themost malignant subclasses (proliferative and mesenchy-mal) [32].Altogether it seems like different cells of originare responsible for glioma initiation. These cells maychange along the disease giving rise to CIC families for agiven tumor.

Although this new molecular classification seems to beconsistent for either GBM samples or CIC-enriched linesderived from human GBM, further studies are necessary tobetter subclassify these groups and to identify the real CICentities of families within a specific group. These newfindings will have profound implications in elucidating theappropriate target for new therapies. Rather than using agiven CIC-enriched line to assay new drugs, we might haveto identify CIC family-specific targets and select represen-tative CIC-enriched lines from each to be used in drugdiscovery platforms.

Biological Meaning of Used Markers

In the last few years, the cell surface marker prominin-1/CD133 has been used extensively as a marker for theidentification of CICs from different types of tumors.CD133+ cells isolated from human GBMs display stemcell properties in vitro and are capable of exact recapitula-tion of the original tumor when transplanted into animmunodeficient mouse brain [8, 12, 13]. After thediscovery of this subpopulation of CD133+ cells with CICproperties [9, 13], either in vitro or in vivo assays conductedwith primary cells derived from a variety of human tumorsamples provided evidence for the presence of CD133+

CICs in other types of tumors including prostate, lung,liver, ovarian, pancreatic [44] and breast [45], amongothers. However, compelling studies have reported thatsome CD133- glioma cells share similar properties withCD133+ cells, including self-renewal and tumorigenicity,suggesting that CD133 cannot fully define CICs [33, 46–

49]. Therefore, the discovery of new markers for thesuccessful isolation of CICs from GBM will be one majoraim to be addressed in the future years.

In this regard, a number of surface markers have beensuccessfully used to identify a subpopulation with CIC-likeproperties in other tumours, many of which have also beendetected in GBM cells (Table 1). This suggests thepossibility that some of these markers might also be sharedby the CIC subpopulation from GBM and, therefore, mightbe useful to characterize them. Based on a comparisonbetween the detection of surface markers in CICs fromGBM with other types of tumors, we would highlightCD44, CD24, CD90, CD166, and A2B5 among others, asgoodcandidates to investigate their role in the biology ofCICs from human GBM.

CD44 expression by tumor cells has been shown toincrease migration, matrix adhesion, invasion, and metas-tasis in response to thr cellular microenvironment as well asenhance cellular aggregation and tumor cell growth [50–52]. High CD44 expression levels correlates with the GBMmesenchymal type, and CD44high/Id1high have beenrecently identifed as putative CICs depleted by tumorgrowth factor-β receptor inhibitors in a subtype of humanGBMs [31, 39, 53].Interestingly, hyaluronic acid (the CD44ligand) is especially enriched in white matter tracts [54],some of the most frequent routes of glioma invasion [55].In a rat model of glioma, CD24 overexpression resulted inenhanced cellular invasiveness in vivo [56]. CD24 wasinitially identified as a B-cell specific marker and appears tobe involved in cell adhesion, invasion, and metastasis [57,58]. CD24 is an important candidate molecule that ispotentially involved in IGFBP-2-induced invasion of GBMcells [59]. CD90 is expressed mainly in leukocytes andbone marrow-derived mesenchymal stem cells. It is alsoinvolved in cell-cell and cell-matrix interactions [60–62].Over the past decade, alterations in CD166 expression havebeen reported in several human tumors (melanoma [63],prostate cancer [64], breast cancer [65], colorectal carcinoma[66], bladder cancer, and esophageal squamous cellcarcinoma [67]). A2B5, a cell surface ganglioside glialprogenitor marker, is a CIC marker that is also associatedwith tumor recapitulation in xenografts [33]. Nonetheless,in some cases, such as CD133 or EGFR a conversionbetween positive and negative cells has been observed,and both show CIC features, suggesting an importantinfluence of environmental signals in the expression ofsuch a markers [32].

Further experiments on these and other surface markersare needed to identify andcharacterize the CIC subpopula-tions. In addition, considering the extreme complexity ofGBM tumors, it will be necessary to explore, not onlysurface markers but also cytoplasmic and nuclear markersthat are more related with CIC-associated biological


functions as well as mutations, epigenetic modificationsand chromosomal alterations and use integrativeapproaches to identify and classify these cells.

Technical Issues

CIC-Enriched Cell Line Culture Conditions

Regardless of the target cells, maintainingthe genetic stabilityof the isolated clones in order to establish cell lines that can bepotentially used for drug discovery is a major inconvenient.The genetic aberrations found in traditionally establishedGBM cell lines over passages might be, in part, responsiblefor failures to translate the results into clinical trials. Shapiro etal. found that near-diploid cells showed lower doublingtimeand higher stability than hyperdiploid cells when cultured invitro [26]. Other groups have also reported the stability ofCD133+ CIC-enriched cell lines in serum-free culture [12,68] while others do not recommend the use of these CIClines beyond 20 passages [69]. Future studies focused on thegenetic stability of different CIC-enriched cell lines in earlypassages are necessary to evaluate whether genetic stabilityis a general or cell line-dependent feature. If the latter is true,each CIC family may require different specific growth mediaand culture conditions to ensure their stability.

In this regard, accumulating knowledge on the CICmicroenvironment from human GBM will help in improv-ing the culture conditions. The oxygen tension in regularcell cultures is an important parameter that does not mirrorphysiological conditions [70–72]. Cells are exposed tooxygen tensions of around 20%, whereas the mean tissueoxygen tension is much lower, ranging from 1% to 5% inthe brain [70–73] (Fig. 1). Interestingly, oxygen tensionalters both the CIC marker expression and the propierties,so Platet et al. studied whether oxygen influenced CD133expression in glioma cell cultures and found a higherpercentage of CD133+ cells at <3% O2 compared to 20%O2 [74]. Soeda et al. showed that a low oxygen tension(1% O2) promotes self-renewal and expansion of CD133+glioma CIC enriched for CXCR4, CD44low, A2B5 andCD24 surface markers with reduction in CD44high-expressing cells, and maintenance of undifferentiated CICs[75]. This result highlights the importance of the biologicalmeaning of these markers in the context of specificmicroenvironments. CD133, in addition to some othermarkers, might label different cell typesin different cultureconditions.

Culture Media

Establishing adequate CIC culture conditions will be one ofthe main achievements in their identification and character-T

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ization (Fig. 2). The use of serum-containing medium in cellculture has long been controversial and is considered “non-physiological” since cells are not usually exposed to serum.With this aim, Dr. Fine’s lab tried to test how different invitro conditions affect primary tumor cells. Since similaritiesbetween CICs and NSCs have been previously proposed, theauthors established 2 different culture conditions, one ofwhich was optimal for propagation and non-differentiation ofnormal NSCs (NBE conditions: serum-free neurobasal mediasupplemented with basic FGF2 and EGF), and the otherhaving optimal culture conditions for the growth of gliomaand majority ofother cancer cell lines (Dulbecco modifiedEagle’s minimal essential medium[DMEM] containing 10%fetal bovine serum [FBS]). This way, morphology, prolifer-ation rate, potential for multi-lineage differentiation, clono-genicity, and telomerase activity differences were observedfor the same GBM sample cultured in these 2 differentconditions. Moreover, these intrinsic biological differencesare supported by the distinct expression profiles of both celltypes. The authors demonstrated that NBE-cultured cells, butnot serum-cultured cells, share remarkable similarity tonormal NSCs and harbor the genetic aberrations foundwithin the primary tumor. Considering these results, Lee etal. concluded that tumor cell growth under standard serum-containing cell culture conditions gives rise to CICloss,allowing for the expansion of cells that differ bothgenetically and biologically from the primary tumors fromwhich they were derived. This observation highlighted therelevance of cell culture conditions in studyingthe biology ofhuman cancer [68].

In spite of these observations, recent studies havedemonstrated that addition of EGF and FGF is notnecessary for CIC growth. Weisset al. proposed that CICswould be able to grow without exogenous mitogen

stimulation. Using different culture conditions, the authorsdemonstrated that GBM CICs proliferated in the absence ofEGF and FGF, maintaining the in vitro characteristicspreviously ascribed to CICs. Moreover, they pointed outthat increasing cell proliferation due to EGF and FGFaddition could give rise to amplification of a subgroup ofcells that are responsive to exogenous mitogens but are nottrue CICs. In addition, growth factors increase the rate ofgenomic instability events over the time [76]. These2studies show how the culture conditions may dramaticallyinfluence the experimental results and, therefore, in thecorrect characterization of CIC biology. In this regard, theCIC microenvironment of the glioma may provide strictregulated signals necessary to maintain the undifferentiatedstate of this cell subpopulation, thereby preserving theirproliferative and multipotential capacities [68, 77]. Thesesignals might differ byGBM type, so we might need todesign a variety of media to fit the requirements of distinctCIC subpopulations.

Monolayer Versus Suspension Cultures

To determine the most reliable and accurate model to studyGBM, some recent studieshavecompared CIC-adherent lineswithneurosphere cultures from human GBM. Using a laminisubstrate, Dirkset al., expanded 6GBM cell-adherent lines forat least 1 year with parallel cultures of neurospheres ongelatin or untreated plastic. They observed increasedefficiency of establishing and propagating glioma cells inadherent conditions (5 passages in neurospheres vs >10in adherent) and sustained expression of undifferentiationmarkers. Indeed, the neurospheres showed an increasedlevel of apoptosis as well as greaterexpression ofdifferentiation markers (Glial fibrillary acidic protein

Fig. 2 Schematic representation of primary culture of GBM. In theinitial passage (or passage 0) there are different CICs and normal cellsfrom the parenchymal brain, growing attached to the culture dish orforming spheres. The culture media only allows the expansion ofsome specific CIC clones responsive to the growth factors. This clonalselection is reflected in passage 1. During successive passages, the

cryopreservation and disaggregation methods are two important issuesto take into account. In addition, due to their genetic instability, cellscan suffer and accumulate new mutations through the passages in aCICs enriched lines-dependent manner, obtaining a cell culture thatdiffers dramatically from the original source


[GFAP], O4 and Tuj-1) [69],although quantitative analy-sis of these markers has been criticized by Vescoviet al.[78]. They have also suggested that the addition of lamininalters the precursors growth conditions. Consistent withresults reported with somatic neural stem cells, thedifferences in growth conditions could explain the lackof differentiation marker expression [79].

CIC-adherent lines have demonstrated genomic alter-ations characteristic of GBM and high capacity to formtumors in immunocompromised mice. Injection of just 100cells formed tumors in 2 out of 6 lines, and the efficiency iscomparable to CD133+ cells directly isolated from tumors[13, 69]. The authors also correlated different phenotypes inadherent cell lines before differentiation assays usingthesame markers present in the histology of the original humantumor.

Regarding future drug discovery assays, some disadvan-tages of neurosphere cultures have been identified: inaccu-rate quantification of cell proliferation due to variable celldeath, the difficulty in identifying the precise cellular target(progenitors and differentiated cells in neurospheres),andneurosphere fusion, which could confound the analysis.Many of these problems seem to be overcome through theuse ofadherent GBM cell lines.

In essence, both monolayer and suspension culture haveadvantages and disadvantages, and the use of either onewill depend on the rationale of the research.

Sphere Disaggregation and Cryopreservation

When CIC-enriched cell lines are cultured in eithermonolayer or suspension, caltures,the need for a largernumber of cells is a priority if we want to run multipleassays. However, this is not always possible for differentreasons, 2 of them being the lack of effective methods todisaggregate them and poor preservation both of whichhave astrong influence on survival ratio (Fig. 2).

The procedure to isolate and culture CICs from humanGBM is based on our previous knowledge of NSCmanagement. Indeed, CIC sphere disaggregation protocolsare derived from those used with NSCs, that combineenzy-matic treatment with either trypsin or triplex, among otherenzymes, with mechanical trituration using polished Pasteurpipettes. However, CIC spheremorphology and cell adhe-sion are not homogeneous. Some CIC-enriched lines growdirectly as floating spheres, others behave as adherentspheres, and many others consist of adherent spheres thateventually detach from the surface. This behavior might bedue to the differential composition of the extracellularmatrix secreted by the cancer cells that also surrounds thecells within the spheres. Extracellular matrix differencesmay also explain why CIC-enriched lines respond differ-entially to disaggregation treatment. In general, the survival

ratio after isolation and disaggregation treatments is quitelow and differs between samples. In addition, some clonesmight be missed due to a higher sensitivity to themechanical procedure itself, and the lower number of cellsin early passages makesit difficult to simultaneouslyconduct several experiments. Further experiments areneeded to identify the matrix composition in a number ofCIC-enriched cell lines, that will allow for the developmentof more efficient enzymatic cocktails and, more reproduc-ible and effective disaggregation protocols.

Considering the low number of cells that is usuallyisolated from each sample, increasing the survival ratioafter long-term preservation of CIC-enriched cell linesbecomes a major concern. In order to overcome thisimportant issue,Chong et al. developed a modified methodof vitrification that preserves the biological and geneticproperties of brain tumor neurospheres from adult GBM[80]. The procedure consists of treating the cells with aseries of freezing solutions that containincreasing concen-trations of cryoprotectant (such as dimethyl sulfoxide[DMSO], ethylene glycol and sucrose). Later, they aretransferred into borosilicate glass capillaries, snap-frozenand stored in liquid nitrogen.

Vitrification was compared with standard cryopreserva-tion methods such as slow freezing in a freezing containerusing DMEM/F12 with 10% DMSO or 10% DMSO and90% FBS. After thawing and culturing for1, 5 and 10 daysthey saw that standard freezing with only 10% DMSOresulted ingreater cell death and smaller neurospheres.Standard freezing with 90% FBS produced the bestviability and preservation of the spheres structural mor-phology but also showed signs of differentiation 5 and10 days after thawing. Finally, vitrification with low serumconcentration maintained sphere sizeand their undifferenti-ated state and resulted in decreased cell death.

Vitrified tumor spheres maintained the expression ofstemness marker expression (Nestin, Sox-2, CD133,Mushashi-1, Bmi-1, Nanog and Oct-3/4) and differenti-ation (Tuj1, myelin-associated oligodendrocyte basicprotein andGFAP). Moreover, they were able to demon-strate formation of secondary sphere that kept their self-renewal potential, genotypic profiles, and capacity togenerate tumors in NOD/SCID mice that replicate gliomapathophysiology [80].

In short, this method maintains stem-like properties,multi-potentiality, and genotypic profiles and thus is aneffective solution to the long-term storage problem.

Drug Discovery with CICs from Human GBM

Current therapies for the treatment of GBM include surgery,radiation and concurrent and adjuvant temozolomide [81].


However, the median survival between groups treated with orwithout temozolomide differs by just few months, meaningthat the benefit of the first-linetreatment is still quite modest.Interestingly, in the last decade, a plethora of molecularalterations has been reported in gliomas, which opens the doorto new therapeutic approaches including the discovery of newmolecules targeting specific oncogenic signaling pathways(for an extensive review see Sathornsumette and Rich, 2008)[6]. On the other hand, the CIC paradigm points to thesecells as being the entities responsible for the initiation andmaintenance of tumor masses, as they are highly chemo- andradio-resistant and are likely responsible for malignantglioma recurrence, making the CICs and/or their niches themost promising targets for fighting this tumor type.Therefore, it is reasonable to think that new generation drugsshould target oncogenic molecular alterations described inthe CIC subpopulation or progenitor cells with dedifferenti-ation capacity. In this sense, a number of studies have alreadyprovided either invitro or in vivo evidence of successful CICtargeting in GBM (see Lamszuz and Günther, 2010) [82].However, most published studiesused CD133+ CIC-enrichedlines. Considering that different types of GBM cells of originmay exist, their evolution along the disease course and thetechnical issues related to heterogeneous CIC isolation andculture, the results using CD133 might not be extrapolated toall CICs. Indeed, successful targeting of CICs other thanCD133+ remains to be proven. Although the possibility ofhaving a unique compound or combination of activecompounds against all GBM CICs does exist, an alternativeapproach that is more likely to succeed involves completingCIC classification before identifying CIC-specific targets. Todo so, we first need to resolve a number of technical issuesrelated toCICheterogeneity, isolation, and culture. Addition-ally, following the same approach that was successfully usedfor GBM tumour mass analysis and classification, we haveto carry out comparative studies usinga high number ofsamples

Conclusions

The theory that CICs might be the appropriate target todefinitely control the growth of GBM is very promising andenthusiastic. However, this principle has not yet beenproven. Considering the potential intra- and inter-tumorCIC heterogeneity, the apparent absence of a specificmarker or set of markers to identify CICs in GBM maysuggest that they are not represented by one particularphenotype. Indeed, CICs from human GBM may begrouped into CIC families, coming from different cells oforigin, showing different genetic backgrounds, and differ-ing in their requirements for isolation and in vitromaintenance. Therefore, it will be necessary to explore the

biological functions as well as mutations, epigeneticmodifications, and chromosomal alterations of CICs anduse integrative approaches to identify and classify thesecells. Meanwhile, the use of CIC-enriched cell lines ratherthan traditionally established GBM cell lines in drugdiscovery programs may be an important milestone insuccessfully translating the basic research findings intoclinical trials. Nonetheless, we still need to work out anumber of technical issues and optimize and standardizeprotocols to allow for better classification and comparativestudies of an important number of both intra- and inter-tumor CIC entities and families. Working with such a highnumber of CIC-enriched lines will be essential to eitherrescue some drugs that might work on specific GBMsubtypes or try out new generation drugs that target specificintermediates of the oncogenic pathways.

Acknowledgment We are especially grateful to Dr. Escobedo-Luceafor her technical advice. Miriam Romaguera-Ros is recipient of aPredoctoral Fellowship from The FIS program (FI05/0087), Ministeriode Sanidad, Spain. Jorge Oliver De La Cruz and Arantxa Pérez Garcíaare recipients of a Predoctoral Fellowship from The FPU program(AP2008/02823 and AP2009/1317), Ministerio de Educacion yCiencia, Spain. This work was supported in part by grants fromGeneralitat Valenciana (GV/2009/68) (AAS), The Gent x GentFoundation (AAS) and Fondo de Investigaciones Sanitarias (FIS) delinstituto de Salud Carlos III (PI10/01069)(AAS).

Conflict of interest All authors have read and have approved of themanuscripts in their final form. Al authors declare that there is notconflict of interest with any material included in the manuscript.

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