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Review Article Open Access

Glioblastoma stem-like cells: approaches for isolation and characterization

Erika S. Molina1, Micheli M. Pillat1, Vivaldo Moura-Neto2, Tamara T. Lah3, and Henning Ulrich1,

1Departmento de Bioqumica, Instituto de Qumica, Universidade de S~ao Paulo, S~ao Paulo, Brazil, 2InstitutoEstadual do Cerebro Paulo Niemeyer, Rio de Janeiro, Brazi, 3Department of Genetic Toxicology and CancerBiology, National Institute of Biology, Ljubljana, Slovenia and Faculty of Chemistry and Chemical

Engineering, University of Ljubljana, Slovenia

CONTENTS1. Introduction 1

1.1. Brain tumor glioblastoma multiforme (GBM) 1

1.2. Cancer stem cell (CSC) paradigm 2

1.3. GBM stem-like cells (GSCs) 4

2. Isolation methodologies of GSCs 4

2.1. SP and ABC transporters 5

2.2. Isolation based on biomarkers 5

2.2.1. CD133 5

2.2.2. ALDH 5

2.2.3. Aptamers 5

3. Enrichment of GSC cultures 6

3.1. Effects of medium and substrates 6

3.2. Effects of hypoxia 7

3.3. Effects of endothelial cells 8

3.4. Effects of pericyte cells 9

3.5. Effects of immunosuppressed animals 9

4. Validation of the GSC phenotype 10

4.1. Sphere-formation assay 10

4.2. Tumor formation in vivo 10

4.3. Expression of biomarkers 11

4.4. Chemoresistance assay 11

4.5. Differentiation assay 12

5. Conclusions 12

Acknowledgments 13

References 13

Abstract: Glioblastoma (GBM) stem-like cells (GSCs) represent the most undifferentiated state of malignant cells with distinctbiological characteristics. The fraction of these cells within a glial brain tumor, ranging from 230%, is correlating with theincreasingWHOstage andpoor prognosis of patients' survival. GSCs represent the least vulnerable, thusmost preferential targetcell population tobeexposed to various therapeuticmodalities, although the underlyingmechanismsof this resistance are not yetfully understood. For the development of GSC-targeting therapies, further in depth studies are needed using enriched and stableGSCcell populations. Here, wediscuss the current approaches ofGSC isolation and validation basedon expression of stemnessand oncogenic markers as well as on functional assays. The enrichment of GSC phenotypes in established cell lines and/orprimary tumor cultures, achieved by different strategies, is reviewed, providing a comprehensive comparison of selected studiesand contemplating the characterization of the plethora of variants of reported GBM population exhibiting the GSC phenotype.

Keywords:Brain tumors,Glioblastomamultiforme,Glioblastomastem-like cells, Cancer biomarkers, Tumormicroenvironment.

ABBREVIATIONS:Aldehyde dehydrogenase (ALDH)

Brain tumor initiating cells (BTIC)

Cancer stem cell (CSC)

Epidermal growth factor (EGF)

Epithelial to mesenchymal transition (EMT)

Fibroblast growth factor (FGF)

Glioblastoma multiforme (GBM)

Glioblastoma stem-like cell (GSC)

Iso-dehydrogenase 1 (IDH1)

Neural progenitor cell (NPC)

Neural stem cell (NSC)

O-6-methylguanine-DNA-methyltransferase (MGMT)

Temozolomide (TMZ)

1. INTRODUCTION1.1 Brain tumor glioblastoma multiforme (GBM)

The most common primary brain tumors are derived from

genetic anomalies in glial development in the brain, com-

prising of astrocytoma and oligodendroglioma [1, 2].

Corresponding author: Tel.: C55 11 30918512 E-mail address:henning@iq.usp.br Departamento de Bioqumica, Instituto de

Qumica, Universidade de S~ao Paulo Av. Prof. Lineu Prestes 748,S~ao Paulo, 05508-000 SP, Brazil Tel.: C55 11 30919181Received: September 8, 2014; Revised: October 21, 2014;

Accepted: October 22, 2014

Journal of Cancer Stem Cell Research (2014), 2:e10072014 Creative Commons. All rights reserved ISSN 2329-5872DOI: 10.14343/JCSCR.2014.2e1007http://cancerstemcellsresearch.com

1007; 8/12/2014; 15:21:23

These are graded on a scale with increasing malignancy as

lower grade astrocytoma (WHO I and II), anaplastic astro-

cytoma (WHO III) and glioblastoma multiforme (GBM;

WHO IV), also termed glioblastoma (GBM). GBM is the

most aggressive of these tumors and ranks among the most

desperate of all human cancers. Survival of GBM patients,

although individually variable, has improved from an

average of 10 to only 14 months after diagnosis in the last

5 years, in spite of significant improvements in the standard

care with more targeted therapy (reviewed in [35]). In

addition to several clinical and histopathological para-

meters, aswell as specific therapeutic approaches, patients

responses to therapy are heterogeneous. This is related to

insufficient data for prognostic and survival stratification.

More information about molecular fingerprints of tumors

as well as to better understanding of cellular origin of the

tumor-initiating cells will help to develop individualized

therapies for patients.

The classification of malignant gliomas is changing

rapidly, and novel schemes are being developed based on

their genetic landscapes [6] from next generation sequenc-

ing [7, 8], followed by epigenetics, proteomics and onco-

genic miRNA data [9]. These are superimposed on the

histological origin of brain tumor-initiating cells (BTIC)

and their precursors evolving into glioma stem-like cells,

also termed glioma stem cells (GSC). Thus, in about 90%of cases, GBMs arise de novo (primary GBM)without any

evidence of less malignant precursor lesions. Secondary

GBMs progress from low-grade diffuse astrocytoma or

anaplastic astrocytoma [2], occurring in younger patients

and having a lesser degree of necrosis and significantly

better prognosis. Although primary and secondary GBMs

are nearly indistinguishable in their histology, they differ

in their genetic and epigenetic profiles providing the state-

of-the-art GMB classification. Decisive genetic markers

for secondary GBMs are iso-dehydrogenase 1 (IDH1)

mutations, which are absent in primary GBMs and asso-

ciated with a hypermethylation (particularly of histones)

phenotype. IDH1 mutations are the earliest detectable

genetic alterations in precursors of low-grade diffuse

astrocytomas and in oligodendrogliomas. The former

acquire further TP53 and ATRX mutations, whereas the

latter lose the 1p/19q arm and are mutated in CIC and

FUBP1 genes. In contrast, primary GBM glial progenitor

cells overexpress epidermal growth factor (EGF) recep-

tors and exhibit TP53 and PTEN mutations as wells as

LOH 10p and LOH 10q deviations. Therefore, recent

classification of glioma is based on the tumor metabolome

and epigenetic changes. As an example,methylation of the

O-6-methylguanine-DNA-methyltransferase (MGMT)

promoter silences expression of this gene by 40% in theprimary tumor, andmore than 70% in the secondary GBM[7]. As this enzyme acts by de-methylating alkyl groups,

epigenetic silencing of its expression increases GBM

sensitivity to treatment by DNA-alkylating agents, such

as commonly used temozolomide (TMZ) [10].

GBMs are histologically similar and featured by atyp-

ical mitotic nuclei often associated with necrosis and/or

angiogenesis. The standard treatment against GBM

includes surgery, radiotherapy and, in the last 5 years,

chemotherapy with TMZ [11]. However, since a popula-

tion of the invasive cells is dispersed through surrounding

normal tissue, and GBMs reveal resistance against radio-

and chemotherapy, the effectiveness of the multimodal

treatment is limited, resulting in GBM reoccurrence.

1.2 Cancer stem cell (CSC) paradigm

The observed diversity of molecular fingerprinting might

originate from the genetically different glioma initiating

cells [3] also called cancer stem-like cells, here termed

glioblastoma stem cells (GSCs). Similar cells have been

found in many, although not in all human tumors. These

"roots of cancer" operate in a hierarchical fashion such as

conceptualized for normal stem cells [12]. The authors of

this work reviewed the evolution of the cancer stem cell

(CSC) model, which is going back to the 19th century to

Virchows embryonic-rest hypothesis, where cancer is a

consequence of the activation of dormant stem-like cells

in adult tissues. The basic understanding of the current

cancer stem cell theory is that heterogeneity within the

tumors is not amere consequence of randommutations and

selection, leading to well recognized clonal evolution of

cancer [13], but results from an intrinsic hierarchy of tumor

cells [1416]. CSCs have unlimited capability to divide

symmetrically and asymmetrically. Asymmetric cell divi-

sion occurs for originating progenitor cells together with a

reduction in the differentiation potential. The theory of a

hierarchical model of tumorigenesis has been widely

accepted, thus stating that only a small fraction of tumor

cells - the CSCs, are capable of initiating tumor growth or

renewing the tumor in the same or other organ after

incomplete surgical removal [17, 18]. When injected

orthotopically, CSCs are capable of forming tumors in

animal. Likewise, CSCs can directly or indirectly contrib-

ute to generationofmetastasis [19].However, the existence

and identification of these cells remains an open question,

as all the existing in vitro and in vivo using animal models

in fact represent artificial environments. Consequently it

cannot be ruled that the so namedCSCs are just cells which

have adapted to altered growth conditions.

The above-described CSC model has a "static" hierar-

chical structure, largely based on the notion that normal

stemcells undergo oncogenic transformation.However, in

the past few years a number of publications have chal-

lenged this concept by demonstrating that in the tumor

environment progenitors, differentiated cells and even

transformed tumor cells may acquire the ability of self-

renewal through their de-differentiation, e.g. a reversal of

differentiation [20, 21], reviewed in [22], or by other

mechanisms, discussed in the following [23]. Moreover,

the finding that epithelial to mesenchymal transition

(EMT), which is a reversible process and can transiently

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induce stem-like cells phenotypes, was crucial to expand

static the CSC model to the fluid CSC model [12], also

termed the CSC plasticity model. This model proposes

CSC plasticity, meaning that by dedifferentiation of a

variety of cell types within growing tumor mass even

various populations of CSCs may evolve by acquiring

additional mutations or epigenetic modifications. Several

authors propose that various CSC subclones can exist as

the result of evolutionary pressure of therapies, giving rise

to more aggressive secondary CSCs [24]. Taken together,

the clonal evolution and CSC hierarchical theory merge

into a so called fluid plasticity or convergence CSCmodel.

This results in several subclones of CSC within the same

tumors, depending on epigenetic and mutational events in

specific cells with self-renewal capabilities. These sec-

ondary aggressive CSCs may become dominant and drive

tumor formation or simply co-existswith other phenotypes

of CSCs. Such concepts represent challenges in order to

develop novel therapeutic approaches that would more

efficiently target the cancerous clonal population with

stem cell-like characteristics.

1.3 GBM stem-like cells (GSCs)

Recent research has revealed that GBM stem-like cells

play important roles in GBM pathogenesis. The first

evidence that GBM display a cellular hierarchy with

self-renewing and tumor-initiating cell types located at

the apexwas provided by Singh et al [25, 26] andGalli et al

[27] followed by further relevant studies on this topic

(reviewed in [2830]. The origin of GBM stem cell has

been widely discussed and is now based on the fact that

genome analyses revealed the transcriptional fingerprints

of neural stem cells (NSC), their progenitors, as well as of

mesenchymal cells [31, 32]. Tumor initiating cell based

classification comprises four subtypes, such as classical,

mesenchymal, neural and proneural transcription profiles,

as summarized by Van et al. [3]. These GBM stem-like

cells (GSCs) self-renew, their hijacking molecular

mechanisms and expressing markers of NSCs [7, 33].

Over the past decade, a wide range of studies have shown

that several signaling proteins involved in neural devel-

opment also play important roles in GBMpathogenesis, as

highlighted and discussed in the context of developing

treatments for GBM [34]. GSCs express the intermediate

filament protein nestin, the transcription factors Sox2 and

Oct 3/4, the RNA-binding protein musahi, transcriptional

repressorBmi1of the polycombof proteins, proteins of the

Notch signaling pathway receptor, aldehyde dehydroge-

nase 1 (ALDH1) and among a few others also the exten-

sively studied transmembrane protein, prominin-1

(CD133) (reviewed in [35, 36]). It has been suggested

that the resistance of GBMs towards radiation and che-

motherapy is attributed to the GSC-like population

[28, 37, 38]. Hence, GSCs are proposed to persist as a

distinct population involved in tumor relapse and propa-

gation and significantly impact the poor prognostics of the

GBMpatients. Accordingly, GSCs have been pursued as a

promising target for the development of novel specific

diagnosis/prognosis therapies against GBM [39].

Although GSCs are increasingly studied for molecular

mechanisms, immunophenotyping or targeting purposes,

the GSC markers, functional assays and the culture con-

ditions remain controversial challenging further investiga-

tions [40]. Different methods of GSC enrichment have

been proposed, either from GBM cell lines and/or primary

cultures [41], resulting in GSCs with rather different

phenotypes. Thus, the enrichment of GSCs by itself may

represent an artifact and contribute to misidentification of

these cells and to the discrepancies in their identification

and characterization.

2. ISOLATION METHODOLOGIES OF GSCsFlow cytometry and fluorescence-activated cell sorting

(FACS) provide the opportunity to analyze various para-

meters in a single live cell as well as separating hetero-

geneous cell populations based on these characteristics.

Besides differential epitope expression, morphological

properties, such as cell size and granularity measured as

forward and side scatter parameters are assessed. Similar

to the identification of embryonic and adult stem cells,

measurements of specific clusters of cell surface markers

and enzymes responsible for metabolic activities are the

methods of choice in flow cytometric identification of

tumor stem cells, as illustrated in Figure 1 and detailed

below. However, the reliability of these tumor cell mar-

kers remains questionable. For instance, tumor cells in

general are known for genetic instability, and consequent-

ly proteome profiles ofGSCbiomarkersmay be difficult to

analyze. Furthermore, epigenetic changes, induced by

tumor microenvironment, in particular when created in

the so called stem cell niches, induce expression of multi-

potency genes in GSCs [20, 23, 42]. It is worthwhile

mentioning that phenotypes may change under in vitro

culture conditions (reviewed in [43]).

As discussed above, GSCs are highly plastic. Conse-

quently similar clones with different biomarker finger-

prints without similar characteristics may exist even in the

same tumor tissue. Nevertheless, the strategies for CSC

identification, isolation and targeting discussed below,

have led to our current knowledge and novel strategies

of glioblastoma. To date, multi-parameter flow cytometry

has been developed for detection of a combination of

characteristics, which define a rare population, such as

cancer stem cells or circulating tumor cells in the blood

[44, 45]. Such rare populations can be identified in a single

experiment, following staining for side populations

(Hoechst dye exclusion by ABC transporters), ALDH1

in the presence of a substrate, which becomes fluorescent

following enzymatic cleavage, and CD markers, i.e.

CD133, and exclusion of dead cells (i.e. stained by 7-

aminoactinomycin-D, 7-AAD), such as proposed by

Greve and co-workers [43]. Optimization of fluorescence

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probes as well as more knowledge on selection of molec-

ular targets by screening for expression profiles will

improve the identification and separation of live cells.

Some of the pluripotency-coding genes, supposedly also

expressed by tumor stem cells, are intracellular antigens,

which cannot be targeted in live cells by flow cytometry.

Therefore, molecular beacons were developed for flow

cytometry detection of gene expression of these genes on

the mRNA level [46]. Applications of in vivo flow cyto-

metry and advanced imaging together with an automated

method for identifying and tracking rare cell events are

available for detection of circulating tumor cells in the

blood [47]. Galanzha and Zharov developed a highly sen-

sitive method, by which the entire blood volume (5 L) of a

patient can be analyzed in vivo by photoacoustic flow

cytometry [48]. Further perspectives here are switching

from the identification of entire cells to the detection of

circulating cancer stem cells and associated microvesicles.

Figure 1. GSC represent a promising target for treating GBM. Classical GSC enrichment consists of serial transplantation in vivo and/or

culture with definedmedium supplemented with EGF and FGF-2, whichmight be improved bymimicking themicro-environment niche of GSC

through hypoxia and/or co-cultivation with endothelial cells. GSC isolation by FACS has been based on differential expression of CD133

followed by ALDH (Aldefluor) as more contemplating functional assessment of GSC phenotype. Additionally, aptamers have turned into

promising tools for targeting biomarkers allowing identification of specific binders against GSC populations.

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2.1 SP and ABC transporters

GSCs can be also identified within side population (SP)

assays using flow cytometry. This SP cell population is

obtained by the efflux of Hoechst fluorescent dyes reveal-

ing stem cell-like characteristics. This dye-efflux, presum-

ably due to the exclusion of the dye triggered by an ATP

binding cassette (ABC) transporter, was also identified in

cells from several tumors exhibiting stem-like genes,

tumorigenicity and multi-drug resistance [49, 50]. In the

C6 glioma cell line, SP cells are described as enriched in

stem-like cells [51]. To date, the SP is employed as gating

criteria for GSC enrichment by flow cytometry with

following demonstration of self-renewal, multi-lineage

differentiation and tumorigenicity [52] or decreased

migration [53]. However, as a drawback to GSC enrich-

ment and purification based on Hoechst exclusion, recent

evidences indicate that, in fact, the GBM SP cells do not

contribute to self-renewal or tumor initiation and thus is

not enriched in GSC and not tumorigenic [54] but consists

of brain endothelial cells [55]. In fact, recent evidence

shows that the three present ABC transporters ABCA1,

MRP4 and MRP5 are differentiation instead of stemness

markers [56]. Taken together, SP flow cytometry is not

recommended as GSC enrichment methodology.

2.2 Isolation based on biomarkers

2.2.1 CD133

The surface marker prominin-1 CD133 is widely used as

phenotypic markers for enrichment of CSCs, including

GSCs, although it is also expressed by hematopoietic,

endothelial and neural progenitors cells [5758] showing

that in the GBM SC population, membrane bouding

expression of CD133was restricted to tumor cells bearing

the extracellular CD133 epitope AC133 [59]. Further-

more, CD133 may also be expressed by differentiated

tumor cells with changed conformation as result of dif-

ferential glycosylation, thus masking the epitope recog-

nized by the anti-AC1333 antibody [59, 60]. CD133

expression is discussed controversially, regarding its use

as criterion for GSC enrichment, since GBM tumorigen-

esis is not only driven by CD133-positive cells. Indeed,

some CD133-negative cells comprise the most undiffer-

entiated and aggressive cells placed in the apex of the

hierarchy [61]. In view of that, the polycomb protein

EZH2 has been indicated to be a more specific marker

for CSCs than CD133 [62]. The enrichment of CSCs or

GSCs based on EZH2 expression remains to be proven.

2.2.2 ALDH

Aldehyde dehydrogenases (ALDH) are a family of cyto-

solic NAD(P)C-dependent enzymes that catalyze the oxi-dation of a wide spectrum of endogenous and exogenous

aliphatic and aromatic aldehyde substrates to their corre-

sponding carboxylic acids [63]. High expression of isoen-

zyme ALDH-1 was for long correlated with resistance to

highly reactive alkylating agent based therapies, such as

derivatives of cyclophosphamide. The enzyme detoxifies

alkylating agents by converting them into less reactive

molecules and favoring survival of leukemia tumor [64]

and GBM cell populations [65, 66]. ALDH-1 has been

proposed to alsomediate the resistance of GBM against the

current standard chemotherapy compound TMZ [67]. Con-

sequently, ALDH inhibition enhances cytotoxicity of alky-

lating agents forGSCs [68]. ALDH-2 andALDH-3 prevent

cytotoxicity such as ALDH-1 [65]. However, only ALDH-

1A1 is involved in the metabolic oxidation of retinal, also

knownas retinaldehyde or vitaminAaldehyde, to the active

metabolite retinoic acid (RA) [69]. Retinal promotes dif-

ferentiation of GSCs, although it is not sufficient to antag-

onize differentiation inhibition by growth factors such as

EGF and fibroblast growth factor (FGF)-2 [70]. ALDH-1

has thereby been proposed as a CSC and GSCmarker [71

73]. Interesting, hypoxia induces or up regulates ALDH-1

expression in established and primary GBM cell lines [74].

Controversially, ALDH-1A1expression was also consid-

ered a marker of astrocytic differentiation in brain tumors

correlating with longer survival of GBM patients [75].

Membrane-permeable fluorescent substrates for ALDH,

such as i.e. dansyl aminoacetaldehyde (DAAA) allow

identification and isolation of ALDH positive cells by flow

cytometry [76]. However, the DAAA-based method does

not only require further fractionation steps to yield highly

purified stem cells and dye excitation with UV light could

bemutagenic to the purified cells. A better alternative is the

stable fluorochrome Bopidy aminoacetaldehyde diethyla-

cetal (BAAA-DA), which when converted into the fluo-

rescent ALDH substrate (BAAA) is excited by visible

light, yielding brighter emission than other fluorochromes,

which could be detected using green fluorescence (FL-1)

channel of a standard flow cytometer [77]. Hence, Alde-

fluor assays can be used in combination with other fluo-

rescent labels such as R-phycoerythrin (PE), PE-tandem

conjugates, PerCP, Cyanine-5 (Cy-5) and allophycocyanin

(APC), which are detected in the FL-2, FL-3 or FL-4

channels. Aldefluor assays employ the ALDH inhibitor

diethylamino-benzaldehyde (DEAB) to set up the fluores-

cent background of negative control populations [78]. In

addition, due to the highly expression of the multiple drug

resistance (MDR) pump by stem-like cells [79]. Aldefluor

assays also contain a MDR inhibitor to avoid the efflux of

the fluorescent product better resolving ALDH positive

from negative discrete cell populations [78, 80]. Remark-

ably, all refinement provided by Aldefluor assays are

improving ALDH-activity studies by fluorescence-activat-

ed cell sorting (FACS), supporting them as a tool marker

for isolation of NSCs [81] and GSCs [82, 83] as well as for

other CSCs [84, 85]. ALDH-1A2 and ALDH-2 activities

are also measured by the Aldefluor assay [86].

2.2.3 Aptamers

Despite the ongoing controversial discussion on the diag-

nostic value of tumor stem cell markers and whether stem

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cells are a stable component of the tumor cell population

and notmerely a transient phenotypewithin a certain tissue

niche, attempts have been made to diagnostically and

therapeutically target the above-discussed marker epitopes

by high-affinity and specificity ligands. In general, cell

surface and transmembrane proteins expressed by various

types of tumor stem cells, including CD44, CD47, CD123,

EpCAM (CD326), CD133 and IGF receptor I, as well as

antibodies interfering with notch-receptor signaling have

been used for identification of monoclonal antibodies of

possible therapeutic value (reviewed by Naujokat, 2014).

Aptamers, evolved from combinatorial DNA or RNA

oligonucleotide libraries by an in vitro selection process

called SELEX, systematic evolution of ligands by expo-

nential enrichment) against a target epitope compete with

antibodies for many applications. Their synthetic nature,

not involving animals in any process, as well as the

easiness of chemical modifications for increasing stability

in biological solutions including plasma have turned them

into promising agents for diagnosis and therapy [88,

89, 90]. There are two alternatives in using aptamers for

identification and isolation of GSCs: (1) Selection of

aptamers against their putative biomarkers using purified

or recombinant expressed proteins. (2) Developing apta-

mers against GSC-like cells by Cell SELEX. This recently

developed technique uses intact live cells expressing the

target epitope(s) and exposes them to the combinatorial

library for aptamer selection (reviewed in [91]). The

differences between the epitopes expression on the cell

surface of a tumor-stem like cells, vs. an untransformed or

non-stem cell of the same histologic origin can be

exploited for obtaining tumor stem cell-specific aptamers.

Aptamers were created with success as specific binders

of GBM populations. First, aptamers following reiterative

cycles of Cell-SELEX were identified binding to human

GBM cell lines. Kang and co-workers [92] first used the

procedure to isolate GBM cells, obtaining a set of apta-

mers not interacting with non-neoplastic astrocytes and

other tumor types. Another approach to identify GBM

cells was to raise aptamers against the EGF variant III

(EGFRvIII), one of the most common mutants in GBM

[93]. These aptamers were used as diagnostic tools, along

with highly specific radionuclide molecular imaging of

GBM in vivo [94]. However, they were developed for

targeting a heterogeneous tumor population and not stem

cells within this cell mixture.

The development of nuclease-resistant 20-fluoropyrimi-dine-modifiedRNAaptamers, which recognizeCD133 and

AC133 epitopes with high specificity [95] is a significant

progress towards targeting CSCs. DY647-fluorescence

labelled anti-CD133 aptamers were verified regarding their

diagnostic potential in imaging and flow cytometry. Fur-

thermore, these aptamers were effective in penetrating

tumorspheres and internalized by tumor cells, opening a

possibility for loading of the aptamer with a cytotoxic

compound or si-RNA for down-regulation of oncogenes

relevant for cell survival. Along the same line, Kim et al.

[96] enriched CD133-positive cells by immunobead sep-

aration from mice GSC xenografts, and validated these for

their tumor-initiating capacity in another animal model.

Then, the authors used CD133-negative cells as well as

non-neoplastic neural progenitor cells as negative control

for a subtraction process for removal of all common

aptamer binders to both cell types. As already shown for

the aptamers selected by Shigdar et al. [95], the aptamers

bound to proliferating and tumor-initiating cells with dis-

sociation constants in the subnanomolar range. The authors

concluded that these aptamersmay gain therapeutic impor-

tance, if conjugated to tacytotoxin. Indeed, paclitaxel-

loaded nanoparticles, reducing GBM proliferation [97],

have been shown to cross the blood brain barrier [98]. In

addition, aptamer penetration of brain tumors can be

facilitated by liposomes, such as those used as vehicles

for doxorubicin-loaded aptamers into brain tumors in

animal models [99, 100].

3. ENRICHMENT OF GSC CULTURES3.1 Effects of medium and substrates

Since thefirst experimental studies supporting in vitro self-

renewal andcapacityofGSCs todifferentiate intoneuronal

and glial cells, GSC enrichment has been essentially

performed in vitro using defined medium. Originally,

GSCs were identified by their capacity to form three

dimension cellular clusters, termed tumorspheres, similar

to neurosphere cultures of NSCs and NPCs [101, 102].

Spheres culture is considered to keep the undifferentiated

state of the cells by minimizing stimulation from the

environment, such as adhesion to substrates, but also

avoiding the access of the cells in the core of the sphere

to differentiation factors [103]. Classically, GSCs arive in

adherent cultures with defined medium following 20 to 40

days after plating [25, 27, 104, 105]. Based on this, tumor-

spheres formationhasbeenobtained faster throughanchor-

age-independent culture, such as in dishes previously

treated with poly-Hema (poly2-hydroxyethyl methacry-

late) [105, 106], gelatin [83] and agar [107]. In fact,

tumorigenicity correlates with suspension growth [108].

In addition, three-dimensional culturing models are also

available, including gelatin foam cultures, for the study of

GSCs [109].However, special care shouldbe taken regard-

ing suspension cultures [110], since poly-Hema derived

spheresmight not be truly three dimensional clonederived,

but rather reflect cell aggregates. In fact, the formation of

spheres as predicative property of CSC enrichment has

been questioned, as no correlation was found with the

expression of GSC markers [111]. Indeed, the technique

of tumorsphere cultivation presents some limitations, such

as differentiation and cell death, occurring in the sphere

environment [112]. Alternatively, GSC enrichment has

also been performed using adhesive cultures, whichwould

provide uniform access to growth factors, thus avoiding

differentiation [113, 114]. Phenotypic differences are

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observed within adhesive surfaces [115], such as in dishes

previously treated with matrigel [116], laminin [113], col-

lagen [117, 118] or extracellular matrix [82]. On the other

hand, organotypic cultures in the presence of ECM and

vascular elements are also available, especially for migra-

tory assays, as this model represents a more accurate brain

matrix micro-environment, in which cells migrate [119].

GSC enrichment performed in adhesive as well as non-

adhesive cultures supports a major role for medium com-

position in this process [113, 118]. GSC culture medium,

similar to that for NSCs, is supplemented with 20 ng/ml of

EGF, 20 ng/ml of FGF-2 and sometimes with the 5 ng/ml

of leukemia inhibitory factor (LIF). The mitogenes EGF

and FGF are for long known for inducing renewal and

expansion and notably increasing the frequency of spheres

formed by undifferentiated cells [120, 121]. On the other

hand, LIF has also been shown to enrichGSCs based on the

transforming growth factor-beta (TGF-b)-mediated pro-motion of symmetric expansions inducing self-renewal

and preventing differentiation [122], also increasing the

expression of core stemness transcription factors and thus

the number of NPCs [123]. Furthermore, medium supple-

mented with B27 and/or N2 enhances proliferation of

multi-potent NPCs [124].

Interestingly, the basis behind the use of the defined

medium is that self-renewal and multipotency of stem-like

cells would be induced and sustained with a supplement

medium switch from serum to EGF and FGF [125, 126].

EGF induces proliferation of adult mouse brain NSCs,

generating spheres responsive to FGF-2. Then, upon stim-

ulation by FGF-2, undifferentiated cells proliferate under-

going symmetric or asymmetric divisions and give rise to

either unipotent (neuronal) or bipotent (neuronal/astroglial)

progenitors [127]. The EGF-responsive mammalian

embryonic neural precursor is a stem cell [121]. Later,

other evidences supported that FGF-2 responsiveness was

sufficient to isolate progenitors found in the adult mamma-

lian spinal cord [128] and that NSCs responsive to FGF-2

arise earliest in embryonic development [129]. GSCmarker

expression was increased upon cultivation stimulation with

FGF-2 compared to EGF [130]. In fact, blockade of EGF

receptors reduced sphere growth, even in the absence of

EGF in the culture medium. Both EGF and FGF enhanced

GSCproliferation and sphere size. However, the absence of

bothmitogens inGBMculturewas already demonstrated to

originate multipotent spheres that were capable of self-

renewal and also forming highly invasive tumors [131].

3.2 Effects of hypoxia

Lowering oxygen concentrations promotes GSC enrich-

ment. Although the standard normoxic condition uses 20%of oxygen, this oxygen level is considered as hyperoxic

compared to physiological brain oxygenation. In fact,

hypoxia is the physiological environment of adult brain

tissue, ranging from 1% to 5% of oxygen [132, 133]. Solidtumors including GBM are less oxygenated, ranging from

physiological levels to below 0.1% in necrotic areas [134,135]. Hypoxia regulates gene expression of hypoxia-

inducible factor 1 (HIF-1), a transcription factor composed

by an oxygen-regulated HIF-1a and a constitutive HIF-1bsubunit. HIF-1a has a half-life of few minutes and desta-bilizes in increasing oxygen levels by the action of proline

hydroxylase which utilize O2 in the range of 0.1 to 21% asa cofactor [136]. HIF post-translational modified within

several domains interacts with the von Hippel-Lindau

(pVHL) followed cytoplasmic translocation and ubiqui-

nin-mediated for proteasome degradation [137, 138]. On

the other hand, HIF-1a stabilized in hypoxic conditionstranslocates to the nucleus interacting with co-activators,

such as cAMP, and binding to the hypoxia response

element located (HRE) in the promoter or the enhancer

regions of target genes [139]. To date, three HIF isoforms

have been described, including HIF-2a and HIF-3a.HIF-1a and HIF-2a expression was increased in GSCs

exposed to hypoxic conditions togetherwith other stemcell

markers such as CD133, Bmi-1 and nestin [140]. Whereas

HIF-1a is ubiquitously expressed, including by all hypoxictumor cells, HIF-2a participates in cell reprogrammingtowards a cancer stemcell phenotypemaintaining theGSC.

HIF-1a target genes are regulated in a tissue-specificfashion. More than 100 downstream genes were identified

as regulators ofmultiple physiological responses to oxygen

deprivation, in general shifting the metabolic balance from

oxidative phosphorylation toward glycolysis. HIF-1a isrequired for the proliferation, survival and angiogenesis

[126]. Overstabilization of HIF-1a supports GBM resis-tance to TMZ [141]. Moreover, HIF-1a interaction withthe activated Notch intracellular domain attenuates differ-

entiation-promoting effects of bone morphogenetic pro-

teins (BMPs), in particular of BMP-2. In NSCs and NPCs,

increasing O2 levels degrade HIF-1a, thus promotingdifferentiation or apoptosis [142]. In GBM, knock down

of HIF-1a at the RNA expression level reduced migration/invasion phenotypes beyond the ability to form tumor-

spheres [143]. A proposed mechanism is that HIF-1adrives the initial response to hypoxia whereas HIF-2 adrives the chronic response. The switch from HIF-1a- toHIF-2a-dependent signaling pathways plays divergent,however complementary roles during the hypoxic response

reprograming GBM cells towards stemness, increasing

aggressive tumor growth and invasiveness [144, 145].

However, HIF-2a stability is unaffected in physiologicaloxygen levels.Moreover, evidences support that HIF-2a isessential only by GSCs expressed and not by NPCs [146].

HIF-2a is overexpressed by GSCs acting as a mediator oftumor plasticity and tumorigenesis [126, 146]. A underly-

ing mechanism could be that HIF-2a upregulates expres-sion of stem-related genes such as Oct4 [147].

Remarkably, hypoxic microenvironment contributes to

the GSC phenotype and increases the number of stem-like

cells in tumor populations. Low 1% oxygen level enrichesthe percentage of CD133-expressing cells [148] without

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affectingN-glycosylation of this epitope [149].A culture in

hypoxia for 48 hours resulted in a four-times increase of the

number of CD133C cells [150]. Hypoxia-mediatedHIF-1aactivation results in a time-dependent increase in CD133

mRNA and protein levels with the peak of protein expres-

sion after 48 hours [149, 150]. The enrichment of GSCs

fromGBMprimary cultures or the cell line U-87 under 1%oxygen condition results in tumorspheres smaller than the

ones obtained in conditions of 20% oxygen. In agreement,expression ofKi-67, amarker of proliferation, is reduced in

conditions of 1% oxygen, whereas expression of stemnessmarkers, such as CD133, podoplanin, Bmi-1 and nestin, is

increased. Sox-2 expression, in particular, is increased only

in tumorspheres from primary cultures [140]. In terms of

enrichment, 1% of oxygen increases the stem-like cellsover fivefold with the percentage of CD133-positive cells

being threefold or more enriched. In the same line, oxygen

levels reduced to 7% enhance the stem-like phenotype ofCD133-positive cells [151]. In addition to promoting

proliferation, hypoxic conditions induced angiogenesis

known to be a prerequisite of tumor expansion. Angiogen-

esis is favored by the association of GSCs and endothelial

cells in perivascular niches [152]. Regulatory functions of

such niche on GSCs is proposed in analogy to the ones in

the control of NSC proliferation [153].

The effects of hypoxia on in vitro cell culture have

gained scientific interest. Precise control of oxygen levels

is essential for accurately interpreting obtained results. To

date, hypoxic cell culture might be performed by different

strategies. The most widely used incubator is based on a

chamber flushed with 1% O2/5% CO2/94% N2 [154].However, this chamber suffers frequently from the leakage

of hypoxic conditions. As a main drawback of this

approach, every time the incubator door is opened, a certain

amount of oxygenneeds to be consumed for re-establishing

initial conditions. Alternatively, a hypoxic environment

might be created by putting the cell dishes inside a sub-

chamber with oxygen controller, which allows studies

exploring multiple oxygen levels for each chamber to be

placed inside a regular cell culture incubator.However, this

alternative also implies in discontinuous oxygen levels and

difficulties of reproducing exact hypoxic conditions.

For improving experimental conditions, hypoxic glove

boxes emerged allowing cell manipulation without

affecting oxygen levels. However, as the incubation and

manipulation cells in the same humid station implies

risk of contamination of cultures, the glove box station

allowing hypoxic manipulation might be enclosed in a

hypoxic incubator. Since exposure to room air also

occurs, disrupting hypoxic condition during regular

microscopy, a hypoxia microscope chamber should be

added the workstation. In this sense, appropriate mod-

ular interconnected closed-hoods accommodating

microscopes, centrifuges, cell sorters, bioreactors are

receiving more attention as these ensure unprecedented

stable and continuous hypoxia, completely isolated from

the outside environment. In parallel, easy to use and

low-cost alternatives have been developed, as, for

instance, inflatable chambers made of transparent plastic

materials allowing real-time studies of hypoxia under

microscopy [155]. Finally, as the oxygen level is typ-

ically controlled in the gas phase differs from that

supplied to the cells submerged in the growth medium;

pericellular oxygen level monitoring might be used for

better accuracy. This can be achieved with sensor chips

embedded in a conventional tissue culture flask [156].

3.3 Effects of endothelial cells

Vascularization is a diagnostic hallmark of GBM. At least

five interlinked pathways by which GBM achieves neo-

vascularization, have been described. A step of vascular

co-option between brain and tumor vasculature, is fol-

lowed by angiogenesis as second step. Vasculogenesis is

the third mechanism sustained by the differentiation of

circulating bone marrow-derived cells (BMDCs), known

as endothelial progenitor cells (EPCs). These cells are

suggested to promote a supportive role and might be

incorporated into tumor vasculature. The fourth mecha-

nism of neovascularization, termed vascular mimicry, is

defined as the ability of tumor cells to form functional

vessel-like networks. The fifth mechanism is based on the

trans differentiation of GBM cells into an endothelial

phenotype [157]. However, tumor cells involved in vas-

cular mimicry are endowed with stemness plasticity,

providing a source for transdifferentiation of GSCs

towards the endothelial phenotype. The presence of

GBM-derived vasculatures highlights the plasticity of

GSCs, although the underlying mechanisms remain to be

elucidated [158, 159].

Hypoxia is involved in all steps of the neovascularization

process. HIF activation has been described to up-regulate

VEGF expression and thus to promote angiogenesis [160],

but also to increase levels of SDF-1, which in turn promote

vasculogenesis recruiting CXCR4 positive BMDCs to the

tumor site. However, unlike in normal vascularization, the

emerging vasculature is often abnormal. In these patholog-

ical angiogenesis, the excess of vascular proliferation

together with the lack of structure-giving pericytes con-

tributes to the formation of tortuous and dilated blood

vessels that are poorly organized and hyperpermeable

(reviewed in [161]). Hence, vaso-occlusive and plasma

coagulation impairs the blood supply perpetuating the

hypoxic microenvironment, which in turn regulates HIF

expression and activity thus leading to a vicious circle of

induction of abnormal vasculature growth in GBM [162].

At the same time, hypoxic exosomes secreted byGBMcells

induce endothelial cells to liberate growth factors and

cytokines accelerating tumor growth [163].

In this context, GSCs are maintained within the vas-

cular niche, in which the endothelial cells play an impor-

tant role to sustain the GSC-like phenotype and GBM

propagation. The endothelial secreted factors, which

8 E. Molina et al.

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activate self-renewal pathways, including Hedgehog

[164] and the Notch pathway [165, 166], at the same time

sustain mTOR activation, which in turn suppresses autop-

hagy and apoptosis of GSCs [167]. Both, endothelial cells

and GSCs, express nestin and the cell surface antigen

CD133 [168]. Almost 75% of nestinC cells co-expressCD133 while less than 0.1% of nestinC cells coexpress theendothelial marker CD34 [116]. In addition, GBM SP

cells, characterized by their efflux properties using flow

cytometry were identified as brain endothelial cells [55].

Co-culture of GBM and endothelial cells results in a

GSC population with higher proliferation rates and

subsequently formation of bigger spheres and more

aggressive tumors than observed with GSCs in mono-

culture [116]. On the other hand, co-culture of GSCs and

endothelial cells also augments number and prolifera-

tion of endothelial cells [126]. The majority of GSC-

enrichment studies employing co-culture conditions

were done with immortalized human brain endothelial

cells (hCMEC) [167, 169], umbilical vein endothelial

cells (HUVEC) [164], microvascular endothelial cells

(HMVEC) [126], primary endothelial cells [116] and

primary bovine endothelial cells [117]. In general, trans-

well inserts are used to co-culture endothelial and GSCs

ensuring free diffusion of signaling molecules without

direct cell contact [116, 117, 126, 164]. Alternatively,

conditioned media from 72 h endothelial cell culture in

serum-free medium might also be employed [167].

Endothelial cells cultivated in three-dimensional system

enhanced secretion of IL-8, upregulating IL-8 cognate

receptor CXCR1 and CXCR2 activities with the conse-

quence of enhanced GSC migration, growth and stem-

ness properties [169].

3.4 Effects of pericyte cells

Pericytes are perivascular contractile stromal cells that

surround the wall of endothelial cells regulating blood

flow [170]. In GBM, it was demonstrated that pericytes

mediate co-option of modified pre-existing blood vessels

supporting the expansion of the tumor margin [161].

Besides, pericytes present mesenchymal stem cell (MSC)

phenotypes, exhibiting neural multi potential activity

[171, 172], also mediating immunosuppression effects

on GBM. [173]. Interestingly, although GSCs have been

suggested to differentiate into endothelial cells, recent

evidence indicates that GSC preferentially differentiate

into pericytes, attempting to support vasculature function

and tumor growth [174, 175]. Besides, endothelial cells

have been proposed to promote paracrine stimulation of

pericytes, specifically in the context of hypoxia. In this

context, hypoxic exosomes secreted by GBM cells

enhance pericyte vessel coverage and induce endothelial

cells to secrete factors stimulating pericyte PI3K/Akt

pathway activation and migration [163]. Endosialin

(CD248), not expressed by endothelial cells, is a marker

of closely associated pericytes in GBM [176].

3.5 Effects of immunosuppressed animals

One of the initially described characteristics of GSCs is

tumorigenicity in vivo [26]. Considering that GSCs

present a distinct capacity for tumor growth and prop-

agation, these cells have been enriched by serial

passages in immune-compromised animals. The enrich-

ment of human GSCs in vivo is based on tumor xeno-

engrafting into immunosupressed animals (generally

mice and rats). The congenitally athymic and hairless

nude mouse immunodeficient in functional T cells has

been routinely used in tumorigenesis assays and shown

to be suitable for GSC transplantation due to its

impaired capability of rejecting human xenografts

tumors. On the other hand, the severe combined immu-

nodeficient (SCID) mouse carries a genetic defect pre-

venting functional development of T and B cells due to

dysfunction in the TCR and BCR recombination

machinery. However, the SCID mouse retains normal

numbers of natural killer (NK) cells [177], which might

interfere in the rejection of xenografts, as NK cells

promote selective lysis of particular tumor targets

[178]. An appropriate model for analysis of GSCs has

been created by the inclusion of the non-obese diabetic

background (NOD-SCID) with reduced NK cell func-

tion. Properly, injection of anti-NK cell antibodies

before transplantation enhances the engraftment of

human xenografts [179]. Followed by the NOD/

SCID/g cnull (NOG) strain, with the above-noted defi-ciencies including cytokine production incapability, the

NOG strain has been considered a more appropriate

animal recipients and especially suitable for GSC trans-

plantation [180].

Intracranial GSC grafting has been widely performed

using stereotactic approach, whereas few studies have

explored subcutaneous or intraperitoneal engrafts.

Engrafting cells into a mouse brain is a critical step as

the site of injection determines the location of the tumor

bulk and requires special care for in fact reaching the

brain tissue [181]. Alternatively to stereotactic proce-

dures, an implantable guide screw was developed to

establish intracranial xenografts in mice, allowing a large

number of animals to be engrafted [182]. Later, the guide

screw method was modified incorporating an infusion

pump that allows up to 10 animals to be simultaneously

intracranial injected with tumor cells [181]. In general

the number of intracranial injected cells was 105 cells per

animal, although tumor formation was also observed with

injection of 2 102 to 103 cells after 12 weeks [183].Injection of 5 103 to 104 cells resulted in tumorestablishment after 8 weeks. Tumor formation in vivo,

a functional method for defining GSCs, is widely used

since for enrichment of these cells. Cells are replicated by

serial passages re-injecting them into secondary mice

[26, 159], which also serve as a validation assay. Xeno-

graft tumor excision later allows further histological and

molecular characterization.

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4. VALIDATION OF THE GSC PHENOTYPE4.1 Sphere-formation assay

Tumor cells have less requirements for proliferation, in

general being less independent from growth factor stim-

ulation and anchorage when compared to normal cells.

However, not every cell within the tumor present the same

independence in order to survive and propagate. On the

other hand, GSCs are considered to be capable of clono-

genic proliferation as anchorage independent spheres

under serum withdrawal, as similarly observed for NSCs.

Also similar to NSCs, the frequency of GSCs has been

predominantly assessed by primary spheres assays. The

studies on GSCs usually refer to GBM primary tumors

cultured with defined medium supplemented with EGF

and FGF. Ignatova et al. [106] cultured tumor cells using

non-adhesive dishes previously treated with poly-Hema

and detected after 18 days a yield of GSC frequency from

0.05% to 1.26% (n 10) in the GBM population. More-over, the authors of this paper reported that the size of

formed tumorspheres is heterogeneous (

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primary endothelial cells, xenograftswere generatedwithin

7 days of injection of 1,000,000 cells [116]. Co-culturewith

endothelial cells increases tumor formation by a factor of

six compared to the monoculture. After 11 weeks, animals

intracranially injected with 100,000 cells formed tumors

with a size of 110 mm3, whereas previous co-cultures with

endothelial cells resulted in 600mm3 tumors [169]. Inmice,

co-injection of GL261glioma and b.END3 endothelial cells

resulted in larger allografts when those observed following

transplantation of glioma cells alone [164].

Alternative methods for animal experimentation

include GBM growth in semi-solid medium employing

agar or agarose suspension [189, 190]. Agarose-suspen-

sion culture is for long known to be one of the best and low

cost in vitro assays correlating with tumorigenicity

[108, 189, 190], still used for GSC validation [191

193]. Alternatively, GSC enrichment has also been per-

formed using agarose as non-adhesive surface [194].

4.3. Expression of biomarkers

GSCs can be validated based on expression patterns of

classical NSCmarkers, correlating with the progression of

WHO grades astrocytomas [195]. Among them, the tran-

scription factor Octamer 4 (OCT-4), a marker of stem cell

pluripotency [196], has been employed. OCT-3/4 pro-

motes migration and invasion of GBM [197], whose

knockdown was described to result in CSC apoptosis

[114]. In stem cells, OCT-4 expression was shown to be

upregulated by HIF-2a during hypoxia, increasing itsmRNA level four times [150].However, in another studied

OCT-4 expression could not be detected in GSC, although

performed with GSC enriched in adhesive cultures pre-

viously treated with laminin [126]. On the other hand,

SRY (sex determining region Y)-box 2, also known as

SOX-2, which is a encoding group of transcription factors,

is also found in NPCs and are also used as GSC validation

marker [83]. Musashi encodes for various RNA-binding

proteins which contribute to the maintenance of stemness

during CNS development and are also applied in the

validation of the GSC phenotype [83, 150]. Nestin is an

intermediate filament protein specific for glia, usually

applied as a multipotent marker in NSC characterization

and also in the validation of the GSC phenotype [198].

Nestin-knockout embryos revealed reduced self-renewal

with no overt defects in cell proliferation or differentia-

tion, surprisingly uncoupled from nestins structural

involvement in the cytoskeleton [199]. Nestin has been

found to be highly expressed in invasive GBM xenografts

delineating tumor infiltration [200]. However, as neuro-

genesis precedes gliogenesis during development, the

more undifferentiated stem cell-like phenotypes are thus

negative for nestin expression and found nestinC later[112]. In line with this observation, nestin mouse NPCsgave raise to neurons and glia [201]. In addition, 75% ofnestinC cells in GBM coexpressed CD133 [116]. How-ever, in GSCs after 5 days of differentiation with serum,

nestin expression levels were still at a high level, whereas

CD133 was much lower expressed [105]. CD133 is the

most used marker for GSC phenotype validation. How-

ever, a controversial discussion remains about the suit-

ability of CD133 expression for validating and targeting

GSCs. Beier and co-workers supported evidences that

CD133C GSCs maintain only a subset of primary GBMs[104]. At the same time, CD133 derived GBM cellsformed tumors in nude rats beyond the efficiency of

CD133C cells [61]. In addition, CD133 has been presentedas marker of bioenergetic stress in GBM rather than for

being a marker protein of their stem-like properties [202].

However, considering that surface phenotype of stem cells

may remain intact despite decay in functional activity, a

more functional assay would be appropriate, as those

employing ALDH expression. According to Choi and

co-workers ALDHC cells range from 0.328.9% in GBMprimary cultures. Furthermore, near 40% of ALDHC cellsoverlapped with CD133 expression whereas only near

0.3% of CD133C cells exhibit ALDH activity [82].

4.4 Chemoresistance assay

GSCs are proposed to be chemoresistant, a feature expli-

cating GBM recurrence after conventional therapy [38].

Attempting to predict this chemoresistant phenotype, in

vitro chemotherapeutic drug assays assess the chemosen-

sitivity of CSCs [194]. Temozolomide (TMZ) is a che-

motherapeutic alkylating agent included in the standard

therapy against GBM. Although experimental data dem-

onstrate that TMZ preferentially depletes GSCs [203], and

that primary GBM cell response to therapy occurs in

patient-specific fashion and independent of GSC pheno-

type [204], the majority of studies regarding the suscep-

tibility of GSCs points at a chemoresistant phenotype of

GSCs towards alkylating agents [205]. TMZ induces

apoptosis and senescence in GBM cells cultured as tumor-

spheres, a phenotype also used for GSC validation [206].

In fact, conversion of differentiated GBM cells into GSCs

could take place upon treatment with TMZ [207].

Recently, TMZ was described to down-regulate P-gly-

coprotein expression, subsequently promoting survival of

GSCs by theWnt3a/glycogen synthase-3 kinase/b-cateninsignaling pathway [208]. In line with this observation,

BMP2-induced differentiation sensitized GSC to TMZ

therapy [141]. However, GSCs are not uniformly resistant

to TMZ. In fact, CSCs are neither resistant nor susceptible

to chemotherapy per se. DNA repair mechanisms restore

the integrity of alkylated DNA bases thus contributing to

drug resistance and subsequent tumor reoccurrence.

O6-methylguanine-DNA-methyltransferase (MGMT),

a DNA repair enzyme, possesses detoxifying functions

during TMZ treatment, when the MGMT gene is non-

methylated, conferring a strong intrinsic resistance toGSC

[209]. However, MGMT methylatio stauts alone sill does

not predict the TMZ response with high precision as

atypical TMZ-resistant GSC presented both methylated

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and unmethylated forms [210]. TMZ resistance of GSCs

via regulation of MGMT expression is promoted by c-Jun

N-terminal kinase (JNK) and Ras-Raf-MEK-ERK signal-

ing pathways, whose inhibition enhances cytotoxicity of

TMZ on GBM [211213]. In addition to MGMT, another

DNA repair protein, ALKBH2, was shown to mediate

TMZ resistance in humanGBM[214], however its specific

effects on GSCs remain unknown. Recently, microRNA-

125b has been shown to play a role in the resistance of

GSCs against TMZ through down regulation of Bak1 and

PIAS3 (protein inhibitor of activated STAT3) expression

[215, 216]. In view of that, detection of microRNA-125b

expression levels may be useful for validation of the GSC

phenotype.

4.5. Differentiation assay

GSCs are postulated to recapitulate the heterogeneity of

the parental tumor in immunosupressed animals due to

their multipotency capacity. As the differentiation prop-

erty is a key feature of stemness, differentiation assays

attempt to evaluate the functional GSC phenotype.

Similar to assessing stemness in NSCs, the assay is

done by cultivating GSC under pro-differentiation con-

ditions. Thus, upon induction of differentiation, cells are

going to express specific genes and proteins of the three

cell types of neural lineage (neurons, oligodendrocytes

and astrocytes). These results suggest that initial cells

have stem cell-like characteristics in vitro. Differentia-

tion progress is usually evaluated based on expression

determination of cytoskeleton-associated proteins such

as neuron-specific b3-tubulin (TuJ1) and MAP2, or glialcell-specific GFAP [217]. Moreover, glycoproteins, pos-

tulated to determine stem cell fates, are differentially

expressed during differentiation, and used to determine

the differentiation status of GSCs [218]. The loss of the

CD133 epitope, ABC transporters, and ALDH-1 also are

evidence for CSC differentiation [56, 60, 73].

Theglial differentiationofGSCs, evidencedby increased

expression of GFAP, can be induced by all-trans retinoic

acid (RA) GSC cultures were cultured in growth factor-free

medium [70]. Similarly, BMPs promote also astro-glial

differentiation of GSCs [219]. Interestingly, BMPs were

already demonstrated to sensitize GSC for TMZ by affect-

ingHIF-1a stability andMGMTexpression [141]. Lee et al.[217] showed that GBM-derived GSCs cultured with N2

supplement, RA or 10% fetal bovine serum alone, within amonth progressively differentiate losing their NSCmarkers

nestin, SSEA-1 and Sox2 and developingmorphologies and

immunohistochemical staining patterns (GFAP, TuJ1 and

MAP2), consistent with phenotypes of glial and neuronal

lineages. Finally, in vivo experiments with immunosu-

pressed animals provide a proper environment for differ-

entiation of GSCs, recapitulating GBMmass with a variety

of cell types [26]. Therefore, since differentiation property

is a key feature of stemness, this processmight be applied in

the validation of GSC phenotypes.

5. CONCLUSIONSA number of methods have been used to identify GSCs.

Emerging evidence suggests that GSCs do not comprise

a fixed entity, but a state-dependent phenotype of the

micro-environmental niche. In this sense, diverging strat-

egies to obtain GSC hinders inter-laboratory comparisons

when not giving rise to conflicting results. Enrichment and

isolation methods proposed to obtain GSC are evolving,

allowing mimicking in vivo niches to enrich GSC cultur-

ing in vitro and introducing novel markers for cytometry

isolation. Hypoxic and co-cultivation with endothelial

cells enriches the cell population with the expected GSC

phenotype. In linewith this, the isolation ofGSCs based on

expression of CD133 remains a valuable approach, while

ALDH expression has turned into a promising strategy for

GSC isolation, contemplating functional and viable

assessment of the GSC phenotype (see Figure 1 for a

comprehensive scheme). On the other hand, validation

assays are usually performed bymeasuring the capacity of

forming tumorspheres, stemness marker expression and

tumorigenesis in vivo. However, these strategies for val-

idation might overlap with those used to enrich and/or

isolate GSCs, such as in the endless mobius ribbon par-adigm. Similar to this paradigm, the overlap of the meth-

ods of enrichment/isolation with the ones to validate the

obtained GSC is presented in an endless cycle potentially

introducing bias into the process to obtain bona fide GSC.

In this sense, differentiation assays, together with chemo-

resistance assays, assess more interesting predictive func-

tional features of GSC, although precautions regarding the

expression of DNA repair genes should be taken in

account in order to interprete the latter test. Remarkably,

little knowledge exists on quiescent GSCs, the subset of

GBM cells responsible for the production of transient

populations of highly proliferative cells. Notably, the

hierarchy of markers during NSC versus NPC stage tran-

sition remains obscure, especially considering the high

plasticity of this process, with more committed NPCs

reverting back to a more primitive NSC state. Interest-

ingly, ALDH expression has been suggested as marker for

distinguishing between NSCs and NPCs. On the other

hand, the sphere frequency assay is more suitable for

progenitor cell than for stem cell activity. In summary,

the scheme presented in Figure 2 puts together how

methods used for GSC enrichment and isolation might

interfere with the parameters used for GSC phenotype

validation. Investigation ofGSC as a relatively recent field

relies on the establishment and optimization of universal

methods focusing at isolation of GSC and robust multi-

parameter characterization

ACKNOWLEDGMENTSHUacknowledges grant support fromFAPESP andCNPq,

Brazil. TLS acknowledges Slovenian Agency for

Research (ARRS) for supporting the work by granting

the Program P1-0105-0245 to TTL. TLS is grateful for a

12 E. Molina et al.

J Cancer Stem Cell Res http://cancerstemcellsresearch.com

1007; 8/12/2014; 15:21:44

visiting professor fellowship granted by the Sciences

without Frontiers Program of the CNPq. ESM and MPP

are grateful for fellowship support by the Brazilian fund-

ing agencies FAPESP and CNPq, respectively.

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