Seeing is = Believing: Are Cancer Stem Cells the Loch Ness Monster of Tumor = Biology?

Justin D. Lathia^{1, 3}, Monica Venere¹,=20 Mahendra S. Rao² and = Jeremy N. Rich^1,=20
3

Keywords Brain tumor stem=20 cell - Review - Cell of origin - Cancer = stem cell=20 hypothesis

Once Upon a Time=97Introduction

In general, as our understanding of a disease = improves, so does=20 our ability to design effective therapies. For some highly lethal = tumors, this=20 has not been the case. Since President Nixon declared the war on cancer = in 1971,=20 tumors such as the most common primary brain tumor, Glioblastoma = Multiforme=20 (GBM), have not seen a major change in survival [1].=20 For GBMs in particular, the 5 year survival rate for patients = treated with=20 radiation alone remains at 2% [2]=20 and current therapies offer mere palliation. Overall, cancer deaths = account for=20 the second most common disease related cause in the US and the change in = survival over the last 20 years remains minimal [1].=20 The recent characterization of a fraction of tumor cells in many solid = cancers=20 responsible for their maintenance and propagation has brought much = excitement=20 and promise to cancer research. This fraction of cells, termed cancer = stem=20 cells, stem cell-like cancer cells, tumor initiating cells, or tumor = propagating=20 cells have been identified in a variety of tumors including those in the = blood=20 [3],=20 breast [4],=20 brain [5=969],=20 and colon [10,=20 11].=20 These cancer stem cells appear to be resistant to many current therapies = including radiation [12]=20 and chemotherapy [13]=20 as a result of potentially primed DNA damage responses. In addition, = cancer stem=20 cells appear to have unique regulatory pathways to promote their = maintenance=20 (reviewed recently by Li et al. [14]),=20 some of which are shared by stem cells during development and adult = homeostasis.=20 As we being to fully appreciate the role of cancer stem cells in tumor=20 maintenance, we=92re likely to find cancer stem cell specific targets = that can be=20 incorporated into the development of multimodal therapeutic strategies.=20

The Legend of the Loch Ness=97Cancer Stem Cells in = the=20 Brain

Despite recent attention being focused on cancer stem = cells, the=20 general concept can first be attributed to Virchow=92s embryonal rest = theory which=20 suggested that cancers arose from embryo-like cells which remained = remnant in a=20 tissue. Interestingly, this has been shown to be the case at the = transcription=20 level as poorly differentiated cancers have an embryonic stem cell-like=20 signature [15].=20 This link between normal and cancer stem cells is a common theme and has = provided much insight into the mechanism of regulation and contribution = of=20 cancer stem cells to the biology of the tumor. In the nervous system, = cancer=20 stem cells have been identified in GBMs [5,=20 7,=20 9],=20 and pediatric brain tumors [8]=20 including medulloblastomas [6].=20 Interestingly, GBM stem cells have been proposed to be highly localized = to blood=20 vessels [16]=20 which has been shown to be the case in neural stem/progenitor cells = [17,=20 18]=20 as well as in regions of hypoxia [19].=20 Additionally, GBM stem cells have been shown to have unique properties = which=20 define them from the bulk tumors cells including radiation resistance = [12],=20 chemoresistance [20],=20 and elevated expression of developmental pathways such as Sonic Hedgehog = [21],=20 Notch [22],=20 and signal transducer and activator of transcription-3 (stat3) [23].=20 Despite the identification of this cell population, there appears to be=20 controversy over the existence of cancer stem cells in the brain and = throughout=20 the body. There are several reasons for the controversy, many of which = originate=20 from inappropriate definitions and expectations of what a cancer stem = cell=20 should be or must be able to do. In the following sections, we will use = GBM stem=20 cells to examine some of these controversies and suggest functional = requirements=20 for cancer stem cells.

Lurking in the Fog=97Functional Definitions

While there are many caveats to the work described for = GBM stem=20 cells, and for that matter other cancer stem cell types, what is clear = is that=20 it is possible to distinguish fractions of a tumor which have different=20 capabilities with respect to tumor initiation. This ability to = recapitulate a=20 phenotypically similar tumor in itself is the single most important = feature of a=20 cancer stem cell. As discussed previously, many somatic stem cells are = solely=20 defined by functional aspects and with regards to GBM and other cancer = stem=20 cells, the most logical functional definition would be the ability to = generate=20 tumors upon secondary transplantation. Another important feature of = cancer stem=20 cells is the ability to self-renew. However, conclusively showing = self-renewal=20 using current methodologies is not a trivial task. The first hurdle is = to=20 establish what is self? Currently, it is possible to measure = non-invasive=20 aspects of an individual cell (presence/absence of a marker, growth) and = use=20 subsequent analysis to determine if these properties are maintained. A = recent=20 demonstration of this has been shown nicely at the single cell level of = neural=20 stem/progenitor cells (NSPCs) [24].=20 However, establishing self-renewal in the most literal sense in vivo is = not=20 currently possible at the single cell level. The neurosphere/tumorsphere = assay=20 has been used as a measure of self-renewal in vitro but it must be = carefully=20 interpreted. First, it measures more than one aspect of cell behavior = taking=20 into account survival, proliferation, and self-renewal. Second, careful=20 interpretations are required to link sphere formation to the in vivo = setting as=20 these in vitro studies are often done in growth permissive conditions = and many=20 microenvironmental cues present in tissue are lacking.

The controversy over cancer stem cells has resulted, = in part,=20 from assigning additional features to cancer stem cells that are present = in=20 somatic stem cell such as low frequency and marker expression. A recent = report=20 has called into question both the transplantation model as well as = frequency of=20 cancer stem cells [25].=20 However, in the developing embryo, many organs have a large fraction of=20 stem/progenitor cells during histogenesis. In addition, the growth = requirements=20 of a tumor are far different from that of a homeostatically stable = organ. For=20 many highly aggressive tumors (such as GBMs), it is not unreasonable to = consider=20 that there may be many cancer stem cells driving tumor growth. Hence, = while=20 there is a low frequency of stem cells present in many tissues, this may = not the=20 case in all tumors and should not be a consideration in defining a = cancer stem=20 cell. The additional requirement for cancer stem cells posed by some=20 investigators is marker expression. While informative, marker expression = alone=20 cannot be a singular determining factor of a cancer stem cell just as it = should=20 not for a somatic stem cell. For tumors which display a great = heterogeneity,=20 such as GBMs, marker expression may serve as a good stating point but = functional=20 analysis is a critical determining factor for the presence of a cancer = stem=20 cell. The single most critical factor in defining a cancer stem cell is = the=20 ability to recapitulate the parental tumor upon translation into a = secondary=20 site. With such a definition in place, much of the controversy and = confusion=20 with regards to cancer stem cells can be avoided.

Tools for Sighting the Monster=97How to Identify a = GBM Stem=20 Cell

One of the major controversies surrounding the = existence of=20 cancer stem cells is their identification and enrichment from bulk tumor = tissue.=20 For the brain, examining stem/progenitor cells present in the normal = embryonic=20 and adult brains is likely to provide insight into what will be = successful in=20 defining GBM stem cells. Despite being located in distinct anatomical = regions=20 and possessing somewhat unique marker expression, NSPCs are defined = strictly by=20 function which appears to be the case with stem/progenitor cells in = various=20 systems. In the nervous system, the functional definition(s) a NSPC must = fulfill=20 remains relatively unclear. Ideally, NSPCs would be able to self-renew = and=20 generate all lineages within the brain but the assays used to examine = these=20 cells are unable to provide appropriate insight. To date, there are = several=20 neural stem cell (embryonic ventricular zone, adult subependymal zone, = adult=20 dentate gyrus) and progenitor cell (embryonic subventricular zone, = embryonic=20 glial progenitors, adult white matter/glial progenitors) populations = which have=20 been described for the developing and adult brain (see review by = Kriegstein and=20 Alvarez-Buylla [26]).=20 The ability to culture mammalian NSPCs in vitro by the neurosphere assay = [27]=20 provided a substantial advance in our ability to understand their = biology and=20 allowed for the examination of which populations of cells are putative = NSPCs.=20 However, it has become clear that the neurosphere assay measures more = than=20 self-renewal (also adhesion independence, proliferation, and survival) = and has=20 forced us to be careful with our evaluation of results generated from = the assay=20 [28].=20 While culturing NSPCs as neurospheres has been useful in expanding these = populations, the anatomical complexity (cell to cell, cell to = extracellular=20 matrix interactions) is lost as is the appropriate mitogenic balance. = The in=20 vivo examination of regeneration first shown by Doetsch et al. [29]=20 is another way to examine NSPCs. While this assay is complex, it allows = for the=20 examination of quisencent NPSCs repopulation in vivo, which is not = possible in=20 vitro. While both these assays have provided a greater understanding of = NSPCs,=20 they both do not adequately measure self-renewal, which given our = current=20 technology, appears to remain a difficult endeavor.

It is logical to draw parallels between NSPCs in the = brain to=20 GBM stem cells and in principle, both appear to have similar functions = (maintain=20 a normal or aberrant organ). As such, our understanding of GBM stem = cells has=20 greatly benefited from assays used for NSPCs. Using identical culture = media, it=20 is possible to expand GBM stem cells as spheres similar to neurospheres, = also=20 called tumorspheres. In addition, the majority of cancer stem cell = models,=20 including those for GBM stem cells, have relied on mouse transplantation = models.=20 With in vitro and in vivo evaluation assays, it would theoretically be = possible=20 to evaluate which marker(s) are best for enrichment of GBM stem cells. = While=20 this has been an area of major effort for those working in GBM and other = cancer=20 stem cell systems (Table 1),=20 there are several caveats which must be considered in marker evaluation. = First=20 off, in the normal brain (both in the embryo and adult), unlike in other = systems, there is no single defining NSPC marker. Second, given the=20 heterogeneity of GBMs, it is unlikely that a single defining marker will = be=20 capable of enriching for GBM stem cells in all cases. As such, = approaches=20 integrating several markers, either by positive or negative selection = are likely=20 to prove successful. Against this backdrop, several cell surface markers = have=20 been proposed for GBM stem cell enrichment:

Table 1 Summary of=20 markers used for NSPC, GBM, and other cancer stem cell enrichment

Cell surface marker(s)	NSPC	GBM stem cell	Other cancer stem cell = systems
ALDH	Activity unknown	Activity unknown	Colon [82,=20 83],=20 Leukemia [84,=20 85],=20 AML [86],=20 Liver [87],=20 Lung [88],=20 Mammary [89]=20
Cadherins	N-cad is critical in NSPC adhesion in embryonic = brain but=20 utility for sorting unknown [90]=20	Present but utility for sorting unknown	Prostate=97absence of Cadherin-10 [91],=20 Leukemia=97VE Cadherin [92,=20 93]=20
CD15	Embryonic [38]=20 and adult [39]=20 mouse NSPCs	Human patient specimens [40]=20	Medulloblastoma [94,=20 95]=20
CD24	Present but utility for sorting unknown	Present but utility for sorting unknown	Pancreas=97in combination with CD44 and ESA = [96,=20 97],=20 Colon=97in combination with CD133 [97]=20
CD44	Present but utility for sorting unknown	Present but utility for sorting unknown	Bladder=97in combination with CK5+/CK20- = [98],=20 Breast [4],=20 Colon [99],=20 Gastric [100],=20 Head and Neck [101],=20 Ovarian=97in combination with CD117 [102],=20 Prostate [103]=20
CD133	Fetal human NSPCs [31],=20 mouse postnatal cerebellum [104]=20	Human patient specimens [6,=20 9]=20	Colon [10,=20 11],=20 Other CNS tumors (Medulloblastomas, Pilocytic astrocytoma, = Anaplastic=20 ependymoma) [6]=20
Chemokine receptors	Present but utility for sorting unknown	Present but utility for sorting unknown	Present but utility for sorting = unknown
Integrins	Integrin α6β1 in human fetal neural = stem cells [46],=20 integrin β1 in rodent NSPCs [105,=20 106]=20	Human patient specimens (integrin = α6β1) [47]=20	Breast=97integrin β3 [107]=20 and integrin β1 [108]=20 in mouse models, Colon=97integrin β1 [97],Prostate=97integrin=20 α2β1 [109],=20 and integrin α6 in mouse model in combination with Lin-Sac1+ = [110]=20
Syndecans	Syndecan-1 in mouse NSPCs [106]=20	Present but utility for sorting unknown	Syndecan-1 overexpressed in multiple myeloma = [111]=20

Fig. 1 Schematic=20 depicting potential cell(s) of origin for GBMs. Proliferating cell = populations=20 are present in several regions of the CNS, including the=20 subventricular/subependymal zone (SEZ/SVZ) adjacent to the lateral = ventricle=20 (LV), the corpus callosum (CC), and the subgranzular zone of the = hippocampus=20 (not shown). Based on heterogeneity present in cell surface marker = expression,=20 molecular subclasses, and GBM models, it is plausible that multiple = cells of=20 origin exist for GBM, consisting of various proliferating cell = populations that=20 acquire genetic lesions. Adapted from Mouse Atlas [112]=20 and Kazanis et al. [113]=20

CD133

CD133 (also known as Prominin-1) is a pentaspan = membrane protein=20 with relatively unknown function where the mutant mice have a = photoreceptor=20 defect phenotype [30].=20 CD133 has previously been shown to be a marker of normal embryonic = neural=20 [31]=20 and hematopoietic [32]=20 stem cells and also been shown to be a useful marker of GBM stem cells = [9]=20 and other cancer stem cells [10,=20 11].=20 However, CD133 is not consistently expressed in all GBMs and recent = reports have=20 demonstrated that CD133-negative cells are capable of giving rise to = tumors in=20 transplant assays [33].=20 Confounding the use of CD133 for fluorescence activated cell sorting = (FACS) are=20 recent reports that its expression is cell cycle dependent [34]=20 so it is possible to miss a population of CD133-positive cells during = sorting.=20 When present, CD133 expression can be a useful enrichment method for GBM = stem=20 cells, however the low expression in some tumors suggests additional = markers=20 need to be explored. In addition, as we begin to understand the = biological=20 function of CD133 in greater detail, we are likely to fully appreciate = its=20 contribution to GBM stem cell and cancer stem cell biology.

A2B5

A2B5, an antibody against a surface glycoside = [35]=20 which is a commonly used glial/white matter progenitor cells marker has = also=20 been explored as a potential GBM stem cell marker. While expressed in a = large=20 percentage of brain tumor and GBM cells, transplantation studies showed = both=20 A2B5-positive/CD133-positive and A2B5-positive/CD133-negative cell = populations=20 were capable of generating tumors in transplantation models [36]=20 suggesting that A2B5 may be used to enrich for CD133-negative GBM stem = cells. In=20 addition, this suggests that there may be a link between glial/white = matter=20 progenitor cells and GBM stem cells. Further work will be required to = fully=20 understand the role of A2B5 as a GBM stem cell marker and suggests that=20 progenitor cell markers should be evaluated in other cancer stem cell = system.=20

L1CAM

L1CAM, a neural cell adhesion molecule found to be = overexpressed=20 in GBMs has been shown to also enrich for GBM stem cells [37].=20 Interestingly, while the biology in the normal brain is yet to be fully=20 elucidated, targeting L1CAM in GBM stem cells showed therapeutic = benefits in=20 vitro (tumorsphere assays) and in vivo (increased tumor latency in=20 transplantation models). Furthermore, it is worth noting the L1CAM is = also=20 overexpressed in other solid cancers so it use as a cancer stem cell = marker=20 would be worth exploring.

CD15

A recently proposed cell surface marker which has been = explored=20 for GBM stem cell enrichment is CD15 (also known as SSEA-1 or LewisX = (LeX)).=20 This cell surface carbohydrate is expressed in embryonic [38]=20 and adult [39]=20 NSPCs and has been linked to bFGF responses. Recently, it has been shown = to be=20 useful to enrich for GBM stem cells in scenarios where CD133 is either = not=20 present or present in a small fraction of cells [40].=20 However, like CD133, little is known about the biology of CD15 and it is = thought=20 that it may be cell cycle dependent [34].=20 In addition, it may be the case that CD15 is marking proliferating tumor = cells=20 rather than actual GBM stem cells (DM Park, R Ravin, DJ Hoeppner, = unpublished=20 observation) and a recent study showed that CD15 in NSPCs was not an = informative=20 marker for in vitro cultures [24].=20 Clearly, further studies to define the role of CD15 in GBMs will be = required but=20 the preliminary data which exists suggest that it can be an informative = GBM stem=20 cell enrichment marker.

Integrin α6

Integrin α6 is a member of the heterodimeric = integrin family of=20 extracellular matrix (ECM) receptors and interacts with the laminin = family of=20 ECM proteins. Integrin α6 was identified as a core stemness marker = by=20 meta-analysis of microarray studies from multiple labs profiling = embryonic,=20 hematopoietic, and neural stem cells [41].=20 In addition, integrin α6 has been shown to be critical in embryonic = NSPCs during=20 development [42,=20 43],=20 expressed by several progenitor cell types in the adult brain [44],=20 and used by adult NSPCs to interact with blood vessels in the niche = [18,=20 45].=20 Integrin α6 can also be used as a NSPC enrichment marker [46].=20 Recently, an examination of the perivascular microenvironment, a region = enriched=20 in GBM stem cells identified integrin α6 as an additional GBM stem = cell marker=20 with potential therapeutic usage [47].=20 Integrin a6 targeting, either by RNA interference or blocking antibodies = resulted in decreased growth in vitro (tumorsphere formation assays) and = in vivo=20 (increased tumor latency in transplantation models). The use of integrin = α6 in=20 the context of GBM therapy will need to be carefully assessed in the = context of=20 normal tissue. The use of integrin α6 as a GBM stem cell enrichment = marker=20 highlights the importance of evaluating the microenvironment in order to = identify additional GBM enrichment markers and potential therapeutic = pathways.=20

While all of these enrichment markers have shown = success in=20 selecting for GBM stem cells from bulk tumors, several issues have = become=20 apparent. First, selection using fluorescence activated cell sorting = (FACS) has=20 been a critical tool but it should be understood that the phenotype of = the=20 selected cells are only known at the time of selection. Enzymatic = treatment,=20 additional culture, or transplantation has the potential to influence = the=20 phenotype in addition to the possibility that certain markers are cell = cycle=20 dependent and could be missed at the time of sorting. Second, no single = marker=20 has been demonstrated to enrich for all GBM stem cells, which is likely = due in=20 part to the heterogeneity of the disease. Third, the time in culture and = culture=20 conditions must be considered as if not controlled properly, will result = in=20 phenotypes similar to highly passages GBM cells lines [48].=20 Finally, and possibly most interesting, all tumor initiating populations = do not=20 appear to share the same marker profile suggesting that there could be = more than=20 one cell of origin or original clone driving the tumor maintenance. As = progress=20 moves forward, it will be critical to develop additional markers for GBM = stem=20 cell enrichment, such as those which have been shown to be successful = for neural=20 stem cell as well as other cancer stem cell system, and understand how = they=20 relate to other markers (Table 1).=20 A systematic screen of cell surface markers with combined functional = assessments=20 will likely yield additional markers for GBM stem cell enrichment. = Future=20 multi-marker sorting schemes may allow for the selection of multiple = tumor=20 initiating cell populations and assist in the development of putative = GBM and=20 cancer stem cell hierarchies.

Birth of the Legend=97Cell(s) of Origin for = GBMs

Another point of contention for the cancer stem cell = hypothesis=20 is that the cell of origin for the tumor must be a stem cell. For GBMs, = like=20 other solid tumors, there could be a variety of cells that could be = responsible=20 for generating the tumor and thus the term =93cancer stem cell=94 = implies the=20 phenotype of the tumor cells, not the cell of origin. With our ability = to=20 genetically engineer mouse disease models, attempts have been made to = create an=20 accurate model which fully recapitulates GBMs and could be used, at = least in=20 part, to infer the cell type(s) of origin. However, GBMs are a complex=20 heterogeneous disease and to date, no single mouse model has been able = to=20 accurately recapitulate the disease. In addition, genetic models rely on = the=20 principle that tumor initiation is a cell intrinsic process and excludes = cell=20 extrinsic influences which are likely to also be critical in the initial = steps=20 of tumor development [49].=20 With that said, several models exist which have been shown to be useful = in the=20 understanding of the disease as well as potential cell type(s) of = origin. The=20 earliest animal model of brain tumors was based on mutagenesis using DNA = alkylating agents such as N-ethyl-N-nitrosourea (ENU). The = ENU=20 model induces sporadic brain tumors of various grades by injection of = ENU into=20 gestating rat embryos [50].=20 This experimental approach has resulted in a cohort of rat brain tumor = models=20 that have yielded insight into the biology of the disease as well as = allowed for=20 therapeutic testing [51].=20 However, this model fails to allow for interrogation of the cell of = origin or=20 the tumor-initiating mutations.

Genetically engineered mice (GEMs), on the other hand, = provide=20 an experimental system that allows researchers to manipulate the = genetics of a=20 specific cell type within the adult animal and evaluate for tumor = formation.=20 This is accomplished by a germline modification that is conditionally=20 manipulated in the adult or by somatic cell gene transfer in a targeted = cell=20 population. Conditional transgenic models take advantage of drug = administration=20 (e.g.; 4-hydroxytamoxifen or tetracycline) to temporally activate a gene = (such=20 as an oncogene) or recombination by cell type specific expression of=20 Cre/Flp to delete a gene (such as a tumor suppressor). Somatic = cell gene=20 transfer, on the other hand, involves the stereotactic injection of a = virus=20 engineered to express a gene of interest to a defined location within = the brain=20 and at a specific time. Furthermore, these systems can be used together = to=20 provide for the multiple genetic alterations required for cancer = progression.=20 Utilizing the aforementioned murine systems, numerous groups have = attempted to=20 evaluate whether NSPCs are the source of malignant gliomas. For the = purpose of=20 the following discussion, we will focus on those studies aimed at = modeling GBM.=20 The link between glioma formation and NSPCs was highlighted by reports = from=20 knockout studies that described alterations in pathways commonly mutated = in GBM=20 (namely p53, Neurofibromatosis type 1 (NF1), and PTEN) led to = deregulation of=20 NSPCs within the subependymal/subventricular zone (SEZ/SVZ), a germinal = region=20 know to harbor adult stem cells [52=9655].=20 To more specifically target this population, the glial fibrillary acidic = protein=20 (GFAP) or nestin promoters have been used as they have = been shown=20 to be active in adult NSPCs [56].=20 Of note, both of these genes are selective rather than specific for this = population, underscored by the expression of GFAP in differentiated = astrocytes=20 and nestin in additional non-NSPC populations. Nonetheless, deletion of = both the=20 p53 and PTEN tumor suppressor genes in GFAP positive cells = (using=20 a hGFAP-Cre transgenic line) led to GBM-like tumors and revealed = a role=20 for these proteins in regulating differentiation and self-renewal of = NSPCs=20 [57].=20 However, this system failed to allow for temporal regulation and = therefore=20 carries the caveat of loss of gene expression at an early developmental = stage.=20 Use of the recently generated Nestin-creER or GFAP-creER = mice=20 overcomes this limitation by allowing for cre activation upon = 4-hydroxytamoxfen=20 injection in the adult animal [58=9660].=20 Using such an approach, it was demonstrated that tumor suppressor = inactivation=20 (p53, NF1, and PTEN) under the control of the Nestin-creER = promoter led=20 to tumors in adult mice and results were confirmed using viral-mediated = cre=20 recombinase injections into the adult SEZ/SVZ [61].=20

Even with these improved GEMs, validating the cell of = origin=20 using models based on conditional induction via the GFAP or = nestin=20 promoter is problematic due their being selective rather than specific = for=20 NSPCs, therefore allowing for potential transformation of more = differentiated=20 cell types. Somatic cell gene transfer using systems such as the = RCAS/tv-a=20 retroviral model circumvents this issue by allowing for both spatial and = temporal control, targeting the NSPC niche in adult animals only. RCAS = vectors=20 stereotactically injected into a defined brain region are engineered to = express=20 a transforming element (oncogene or tumor suppressor) only upon = infection in=20 cells expressing the TVA receptor. Mouse lines driving tv-a by the = GFAP=20 or nestin promoters have been generated and have indeed generated = gliomas=20 from NSPCs in adult mice in the context of various genetic disruptions = [62=9666].=20 In a similar vein, Cre-loxP-controlled lentiviral vectors have = been used=20 to transform GFAP cells in the neurogenic zones via activated H-Ras and = AKT=20 overexpression in a p53 heterozygous background, resulting in malignant = gliomas=20 [67].=20 Moving beyond Nestin and GFAP, the orphan nuclear reseptor tailless = (Tlx) has=20 recently been reported to be neural stem cell specific and tumorigenic = when=20 overexpressed, yielding invasive GBMs when combined with the loss of p53 = [68,=20 69].=20

Lending support to the idea that NSPCs may be a = potential cell=20 of origin is a recent study in which mutations were introduced into = NSPCs and=20 astrocytes ex-vivo and then transplanted to assess tumor formation = [70].=20 The study demonstrated that mature astrocytes were not capable of = forming tumors=20 upon the introduction of mutations whereas deletion of PTEN and p53 in = NSPCs=20 resulted in transformed cells that gave rise to tumors, suggesting in = part that=20 mature astrocytes are not likely to be the cell or origin in this mouse=20 transplant model. In addition to traditional GEMs, several other model = systems=20 have been developed which are likely to be useful in understanding = cellular=20 contributions to GBMs. A recent report has demonstrated that by mutating = epidermal growth factor receptor (EGFR) and phosphoinositide 3-kinase = (PI3K)=20 signaling in drosophila glia and glial precursors, GBM-like tumors can = be=20 generated [71].=20 This model may be a good candidate to screen drugs or identify = additional=20 pathways contributing to the formation and maintenance of GBMs. Although = powerful, many of the systems discussed above rely on the mutation of a = defined=20 cell type to produce tumors. These model systems have not ruled out = other=20 potential cells or origin and assuming the multiple-hit hypothesis holds = true,=20 it is likely that any cycling cell population in the brain subject to = the=20 appropriate set of mutations could generate a tumor. In support of this, = recent=20 studies have reported the generation of gliomas by cells outside of the=20 neurogenic niches [72=9674].=20

Another potential way of evaluating the cell(s) or = origin for=20 GBMs is by using microarray analysis. Attempts to generate distinct gene = signatures have not produced a unified profile but rather three main = classes of=20 tumors: proneural, proliferative, and mesenchymal [75].=20 The proneural gene signature is associated with neuronal marker = expression and=20 longer survival while the proliferative and mesenchymal signatures have = higher=20 neural stem cell marker expression and poorer prognosis [75].=20 Additional interpretation of these tumor signatures suggests that the = proneural=20 tumors resembled neurogenic processed and specifically neuroblast cells, = the=20 proliferative tumors resembled proliferation processed and neural stem = and/or=20 transit amplifying progenitors, and mesenchymal tumors resembles = angiogenic=20 processes and NSPCs. No uniform mutation pattern exists for each tumor = type but=20 there are some associations: loss of PTEN, gain of EGFR, and Akt = activation in=20 mesenchymal and proliferative groups, elevated presence of notch pathway = members=20 in the proneural group. A recent follow up study by the Cancer Genome = Atlas=20 Network (TCGA) delineated four GBM molecular subclasses (proneural, = neural,=20 classical, and mesenchymal), expanding on the previous three groups = [76].=20 In their analysis, the TCGA found that the following mutation patterns: = the=20 proneural group has frequent mutations in p53 and isocitrate = dehydrogenase 1=20 (IDH1), the classical group had increased mutations in EGFR and the = presence of=20 EGFRvIII, and the mesenchymal group had increased mutations in NF1. In = addition,=20 an evaluation of survival after therapy (which included radiation and=20 chemotherapy) demonstrated that the proneural group was the worst to = respond as=20 assessed by patient survival. Additional classification of GBMs based on = CD133=20 status has suggested that CD133-negative tumors have a proneural gene = signature=20 and resemble fetal NSPCs while CD133-positive tumors have a mesenchymal = gene=20 signature and are associated with adult NSPCs [77].=20 The studies discussed above demonstrate the heterogeneity present with = GBMs and=20 a simplistic interpretation would be that these data suggest that more = than one=20 cell type is capable of generating a GBM. Lending support to this idea = would be=20 data which demonstrates that GBMs contain populations of cells that = share=20 markers present in embryonic and adult stem/progenitor cell populations = such as=20 CD133 [9],=20 NG2 [78],=20 Sox2 [79],=20 and Olig2 [80].=20 Additional profiling and phenotypic assessment of the GBM stem cells = from each=20 tumor type may yield insight into differences between tumor classes and=20 potential molecular subclass-specific therapies. Furthermore, a = systematic=20 comparison of GBM stem cells to that of normal stem and progenitor cells = present=20 from the embryo and adult are likely to yield core pathways that are = conserved=20 between populations. Such efforts will also will aid in the = identification of=20 additional GBM stem cell pathways and provide conserved pathways which = will need=20 to be considered when designing targeted therapies. Overall, these = microarray=20 studies have provided a good starting point for additional genomic = analysis=20 linking tumors classes to survival and will be critical as will = attempting to=20 assess which therapies are most effective to which tumor classes. =

Rising from the Water=97Final Thoughts

In this review, we have used GBM as an example to = explore the=20 cancer stem cell hypothesis. GBM, like many other solid tumors, is a=20 heterogeneous disease and as such, requires the appreciation of the = diversity=20 (both cellular and genetic) present within the tumor. To improve our=20 understanding of cancer stem cells and their contribution to cancer, = additional=20 enrichment paradigms must be developed that are likely to include = multiple=20 markers. In addition, current models will need to be improved to address = the=20 possibility that multiple cells of origin may exist (Fig. 1)=20 and previous classified solid tumors may represent several distinct = tumor types=20 with variant genetic abnormalities, such as is the case with GBM. = Finally,=20 additional genetic profiling will be critical to understand the = diversity=20 present within a tumor and if combined with animal models, such as = demonstrated=20 recently with ependymoma [81],=20 will be a powerful tool to understand how different classes of cancer = stem cells=20 contribute to different subclasses of tumors. Amidst the controversy = surrounding=20 the cancer stem cell hypothesis, what is clear is that the existence of = cancer=20 stem cells has helped explain certain aspects of tumor behavior and = demonstrates=20 the complexity of cancer. Moving forward, efforts to detect the monster = at a=20 distance (i.e. diagnostic studies) and kill the monster (multi-modal = tumor=20 therapies) will likely result in improved patient outcomes.

Acknowledgements We sincerely apologize to those whose work we were unable = to=20 discuss due to space limitations. We would like to thank members of the = Rich lab=20 for stimulating discussion and critical review of this manuscript. Work = in the=20 Rich laboratory is supported by the Childhood Brain Tumor Foundation, = the=20 Pediatric Brain Tumor Foundation of the United States, Accelerate Brain = Cancer=20 Cure, Alexander and Margaret Stewart Trust, Brain Tumor Society, = Goldhirsh=20 Foundation, Duke Comprehensive Cancer Center Stem Cell Initiative Grant, = and NIH=20 grants NS047409, NS054276, CA112958, and CA116659. J.N.R. is a Damon=20 Runyon-Lilly Clinical Investigator supported by the Damon Runyon Cancer = Research=20 Foundation. M.V. is supported by an American Brain Tumor Association = Basic=20 Research Fellowship and a National Service Research Award (NINDS F32 = NS058042).=20 J.D.L. is supported by an American Brain Tumor Association Basic = Research=20 Fellowship (sponsored by the Joelle Syverson Fund) and a National = Service=20 Research Award (NCI F32 CA142159).

Conflict of=20 Interest =20

Dr. Mahendra S. Rao is an employee of Invitrogen = Corporation,=20 co-founder of Q Therapeutics, and currently serves at its Chief = Scientific=20 Consultant.

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