Neurosurgical FOCUS - 24(3-4):E27

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March 2008 Volume 24, Number = 3-4

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Brain tumor stem cells and the = tumor=20 microenvironment

Rahul=20 Jandial, M.D.¹², Hoisang U, M.D.¹, = Michael L. Levy, M.D.,=20 Ph.D.¹, and Evan = Y. Snyder,=20 M.D., Ph.D.²

¹Division of Neurosurgery, = University of California, San Diego; and ²Center for Neuroscience, = Aging and=20 Stem Cell Research, Burnham Institute for Medical = Research, La=20 Jolla, California

Abbreviations used in=20 this paper: BTSC =3D brain tumor stem cell; = CNS =3D=20 central nervous system; CSC =3D cancer stem cell; GBM = =3D=20 glioblastoma multiforme; NSC =3D neural stem cell.

Address correspondence = to: Evan=20 Y. Snyder, M.D., Ph.D., Burnham Institute for Medical=20 Research, 10901 North Torrey Pines Road-Building 7, La = Jolla,=20 CA 92037. email: esnyder@burnham.org<= /NOBR>.

DOI: 10.3171/FOC/2008/24/3-4/E26

Figures = and Tables=20

Abstract	IntroductionPrevailing Theory of = Tumo...Cancer Stem Cells: = Discov...Caveats to BTSCsTumor = MicroenvironmentPotential Clinical = Releva...References

3D""=20 Recent advances in stem cell research = and=20 developmental neurobiology have uncovered new = perspectives=20 from which to investigate various forms of cancer.=20 Specifically, the hypothesis that tumors consist of a=20 subpopulation of malignant cells similar to stem cells = is of=20 great interest to scientists and clinicians and has = been=20 dubbed the =93cancer stem cell hypothesis.=94 The = region in which=20 this assertion is most relevant is within the brain. = Cancer=20 stem cells have been isolated from brain tumors that = exhibit=20 characteristics of differentiation and proliferation = normally=20 seen only in neural stem cells. These cancer stem = cells may be=20 responsible for tumor origin, survival, and = proliferation.=20 Furthermore, these cells must be considered within = their=20 immediate microenvironment when investigating = mechanisms of=20 tumorigenesis. Evidence of brain tumor stem cells is = reviewed=20 along with the role of tumor environment as the = context within=20 which these cells should be understood.

Keywords:=20 brain tumor; brain tumor stem cell; cancer stem cell; = stem=20 cell therapy; tumor microenvironment.

Introduction	AbstractIntroduction = <<Prevailing Theory of = Tumo...Cancer Stem Cells: = Discov...Caveats to BTSCsTumor = MicroenvironmentPotential Clinical = Releva...References		=

The intersection of = stem cell=20 biology with oncology has offered new insight into the = cellular and molecular mechanisms underlying both = normal and=20 abnormal development. Of particular interest to = clinicians and=20 scientists is the hypothesis that solid tumors harbor = a=20 sub-population of malignant cells that display stem = cell=96like=20 properties=97CSCs.

A normal stem cell can both self-renew and = differentiate=20 into multiple lineages. In the CNS, somatic (adult) = NSCs can=20 undergo asymmetric division to create 2 daughter = cells=97one an=20 NSC and the other a more committed progenitor able to=20 differentiate and proliferate but not self-renew. = Similarly,=20 the putative CSC can sustain and spread the tumor, as = well as=20 populate the tumor with the various types of = differentiated=20 cells.

Brain tumor stem cells are the CSCs of the CNS and = exhibit=20 both self-renewal and multilineage differentiation. = This=20 subpopulation of cells within brain tumors may be = responsible=20 for lesion origin, survival, and proliferation.

The process of carcinogenesis may be = more=20 accurately viewed as the aberrant response of the = entire=20 tissue to genetic and epigenetic stress. The cancerous = cell=20 defining the tumor and constituting the CSC = subpopulation must=20 be considered within its immediate tumor = microenvironment, and=20 elucidating the role of this microenvironment may be = as=20 important as investigating the transformed glial or = neuronal=20 cells within brain tumor. In this paper the prevailing = evidence in support of BTSCs will be reviewed along = with the=20 evolving and integral role of the tumor environment as = the=20 context within which these stem cells must be=20 understood.

Prevailing Theory of Tumor=20 Initiation	AbstractIntroductionPrevailing Theory of Tumo...=20 <<References

It has been widely accepted that = cancer occurs=20 as a consequence of genetic and epigenetic alterations = in a=20 differentiated cell. These alterations provide a = proliferative=20 advantage and ultimately lead to uncontrolled growth = and=20 spread of the malignant cells. This theory suggests = that=20 tumors, such as gliomas, result from mutations to = terminally=20 differentiated astrocytes and oligodendrocytes that=20 dedifferentiate into a less differentiated = phenotype.^7,18,21

Cancer Stem Cells: Discovery = and=20 Evidence of BTSCs	AbstractIntroductionPrevailing Theory of = Tumo...Cancer Stem Cells: Discov...=20 <<References

Approximately 150 years ago pathologists Rudolph = Virchow=20 and Julius Cohnheim suggested that there were = histological=20 similarities between the developing fetus and certain = cancers=20 (such as teratocarcinomas) and that both tissues have = the=20 capacity to differentiate and proliferate. This = embryonal-rest=20 hypothesis is the historical version of today's CSC=20 hypothesis.¹¹ As=20 defined at an American Association for Cancer Research = workshop, CSCs are cells that: 1) self-renew and 2) = resupply=20 the tumor with the various cell lineages of which it = is=20 composed. Self-renewal can only be defined = experimentally as=20 the ability to recapitulate the generation of a = continuously=20 growing tumor or tumor initiation cell.^6,17

Original work establishing the CSC model was based = on the=20 hematopoietic system. Evidence of leukemia CSCs was = first=20 reported in 1994 when Lapidot et al.¹⁶ isolated=20 a rare population of CD34+CD38=96 cells from patients = with acute=20 myeloid leukemia. Infusion of these CD34+CD38=96 cells = into=20 severe combined immunodeficient mice resulted in = leukemic=20 blast generation; however, more differentiated cells=20 (CD34+CD38+) did not generate leukemia.^4,16 This CSC=20 model was later extended to solid organ tumors = (breast) by=20 Al-Hajj et al.² = in a 2003=20 report of tumor recapitulation with the = transplantation of as=20 little as 100 CD44+ CD24=96 cells from the primary = lesion;=20 whereas tumorigenesis failed to occur with = transplantation of=20 ≥ 100,000 CD44+CD24+ cells. On a molecular = level, CSCs share=20 properties with normal stem cells. They have similar = markers=20 and signaling pathways and respond to environmental = cues,=20 telomerase activity, apoptosis clearance, and = increased=20 membrane transporter activity.²¹

Cells with stem cell=96like properties in brain = tumors were=20 first described by Ignatova et al.¹² in 2002;=20 surgical specimens of GBMs were shown to have = clonogenic=20 neurosphere-forming cells that expressed both neuronal = and=20 glial markers on differentiation (Fig. = 1).²⁵=20 Subsequently, another group demonstrated CSCs in brain = tumors=20 through the intracerebral transplantation of CD133+ or = CD133=96=20 populations into immunodeficient mice. With as few as = 100=20 CD133+ cells from the primary tumor, a new phenocopy = of the=20 lesion could be created in the recipient mice. = Furthermore,=20 unsorted or CD133=96 primary tumor cells did not cause = de novo=20 tumor generation. Self-renewal capacity, a trait that = has come=20 to define CSCs, was also shown by confirming the = ability of=20 serially transplanted CD133+ cells to recapitulate the = original tumor.^4,22 These=20 findings established the presence of BTSCs, cells that = can=20 differentiate into the neural lineages and exhibit=20 self-renewal, as demonstrated by the recapitulation of = primary=20 tumors with serial transplantation. These = characteristics of=20 self-renewal, differentiation, and the ability to = recapitulate=20 tumors have been verified for other types of brain = tumors as=20 well.^19,24

View=20 larger version = (58K)

Fig. 1. = Schematic=20 demonstrating isolation and perpetuation = of BTSCs=20 in culture. The neurosphere assay is a = defined=20 serum-free culture system that allows = isolation=20 and propagation of CNS-derived stem cells. = Adult=20 precursor cells are dissociated and plated = in a=20 liquid growth medium that contains the = stem cell=20 mitogens, epidermal growth factor, and/or=20 fibroblast growth factor=962. Given the = lack of=20 serum and the low plating density, most = cells die,=20 except those that divide in response to = the stem=20 cell mitogens. The growth = factor=96responsive cells=20 proliferate to form floating clusters of = cells=20 that are referred to as =93neurospheres, = =93 which can=20 be further dissociated into a single-cell=20 suspension and then replated in fresh = medium to=20 produce secondary neurospheres. The = process can be=20 repeated, resulting in a geometric = expansion in=20 the number of cells that are generated at = each=20 passage. On mitogen removal, the progeny = of the=20 proliferating precursor cells can be=20 differentiated into neurons, astrocytes, = and=20 oligodendrocytes, which are the 3 primary = cell=20 types found in the adult mammalian CNS. = Under=20 these conditions, the growth = factor=96responsive=20 precursor cells can be expanded = indefinitely with=20 little change in their growth or = differentiation=20 characteristics. These findings indicate = the=20 existence of an adult NSC, as these cells = possess=20 the fundamental stem cell features of = extensive=20 self-renewal, generation of many progeny, = and=20 production of the primary cell types of = the tissue=20 from which they were obtained. Reprinted = by=20 permission from Macmillan Publishers Ltd: = Vescovi=20 AL, Galli R, Reynolds B: Brain tumor stem = cells.=20 Nat Rev Cancer 6:425=96436,=20 2006.

Bao and colleagues³ = further=20 defined the molecular mechanisms by which BTSCs may = escape the=20 lethal damage of ionizing radiation and provided a = reason for=20 revisiting clinical radiation therapy. The CD133+ = cells=20 isolated from fresh tumor specimens were shown to=20 preferentially activate DNA repair checkpoints, = including=20 phosphorylation of the checkpoint proteins Chk1 and=20 Chk2.²³

Caveats to BTSCs	AbstractIntroductionPrevailing Theory of = Tumo...Cancer Stem Cells: = Discov...Caveats to BTSCs = <<Tumor = MicroenvironmentPotential Clinical = Releva...References

With the surge in interest in CSCs and BTSCs, some=20 confusion regarding methods and definitions has = arisen,=20 risking the inappropriate assignment of properties to = a=20 heterogeneous population of cells and leading to = erroneous=20 characterization. Some concerns about the current CSC=20 hypothesis remain (Fig. = 2).¹⁰

View=20 larger version = (101K)

Fig. 2. = Schematic=20 illustrating the potential difficulties in = sorting=20 and identifying CSCs. Upper portion = of the=20 figure outlines the theoretical concerns; = the=20 lower portion, the technical = concerns.=20 Reprinted with permission from Hill RP: = Cancer=20 Res 66:1891=961895, = 2006.

Brain tumor stem cells, shown to be tumor = initiating, are=20 not independent of their site of primary culture and = ultimate=20 site of transplantation. In fact, their ability to = proliferate=20 is significantly dependent on the tumor = microenvironment, as=20 discussed later. Furthermore, the current in vivo = serial=20 transplantation assay for self-renewing BTSCs utilizes = immunodeficient mice, and in this nonphysiological = scenario=20 one would expect higher engraftment of transplanted = tumor=20 cells. The use of proteolytic enzymes to disassociate = primary=20 brain tumor specimens, as used in all the BTSC = studies, could=20 also change the cell surface proteins by which these = BTSCs are=20 sorted. Although it is clear that CD133=96cells are = not=20 tumorigenic, it remains unclear which subpopulation of = CD133+=20 cells is the bona fide BTSC conferring = tumor-initiating=20 capacity.

The identification of reliable markers = will=20 allow prospective isolation and characterization of a = pure=20 population of BTSCs=97not just a population of cells = containing=20 BTSCs. Fundamental questions about these cells then = can be=20 investigated, such as the underlying signaling = mechanisms for=20 self-renewal and whether the BTSC is also the tumor = cell of=20 origin.

Tumor = Microenvironment	AbstractIntroductionPrevailing Theory of = Tumo...Cancer Stem Cells: = Discov...Caveats to BTSCsTumor Microenvironment=20 <<References

The microenvironment of stem cells is known to help = maintain the cells' quiescent state and preserve their = potential to proliferate and differentiate.^1,9 = This=20 assertion extends to tumors as well. The tumor=20 microenvironment, composed of nonepithelial stromal = cells=20 (inflammatory cells, endothelial cells, and = fibroblasts),=20 increasingly is shown to be essential for lesion = growth (Fig.=20 3).^8,14 Other=20 solid-organ tumors (such as breast cancer) demonstrate = that=20 genetic changes occur in the stroma at the earliest = stages of=20 tumori-genesis, and in murine models genetic lesions = confined=20 to the stroma are sufficient to induce epithelial=20 tumors.^13,15,26 In fact,=20 this concept is an extension of the =93seed and = soil=94 hypothesis=20 presented by Paget many years ago, and now authors are = underscoring the role of the tumor microenvironment as = the=20 lesion =93soil.=94

View=20 larger version = (94K)

Fig. 3. = Schematic=20 depicting a model of CSCs in solid tumors. = The=20 CSCs are associated with the stromal = components of=20 the tumor, including fibroblasts and/or = blood=20 vessels, which make up the CSC niche. The = niche=20 cells secrete factors that support CSC=20 self-renewal. The CSCs retain = differentiation=20 potential, giving rise to = non=96self-renewing tumor=20 cells that make up the bulk of the tumor. = The CSCs=20 can be drug and/or radiation resistant and = can=20 express CSC-specific antigens. Current = research=20 focuses on the identification of the = molecular=20 mechanisms regulating these properties of = CSCs=20 (highlighted in green[AU Q: Note = that I see=20 no green in your submitted figure. Please=20 advise.]), as they represent potential = targets for=20 therapy. Reprinted from Curr Opin=20 Biotechnol, 18, Ailles LE, Weissman = IL, Cancer=20 stem cells in solid tumors, 460=96466, = 2007, with=20 permission from Elsevier.

Brain tumors and BTSCs must be considered as = dysfunctional=20 phenomena that occur not only in individual cells, but = also in=20 the tissue that harbors the malignant lesion. This = tissue and=20 the microenvironment it creates are integral parts of = the=20 cancer. Neural stem cells have been shown to lie = within a=20 vascular microenvironment, and Calabrese et al.⁵ = have=20 demonstrated that CD133 + nestin + tumor cells = (containing the=20 BTSCs) were similarly associated with the vasculature. = Furthermore, increasing the number of endothelial = cells and=20 blood vessels in xenografts augmented the CSC = population and=20 the rate of tumor growth. Accordingly, antiangiogenic=20 treatment decreased both the CSC population and tumor = growth=20 rate, demonstrating that the secreted factors or cell = contact=20 with the tumor microenvironment helped maintain the = functional=20 characteristics of the CSCs.¹ = A CSC is=20 not subject to the same internal and external = regulation as a=20 normal stem cell and cannot be identified without = considering=20 the influential microenvironment within which it = resides.

The tumor microenvironment offers a = novel=20 approach to treatment through the targeting of cells = of the=20 tumor niche as well as the potential means of = chemoprevention.=20 Characterizing this niche could allow the development = of in=20 vitro assays that recreate the in vivo environment and = obviate=20 the current time-consuming dependence on serial=20 transplantation as the standard for defining = CSCs.

Potential Clinical = Relevance	AbstractIntroductionPrevailing Theory of = Tumo...Cancer Stem Cells: = Discov...Caveats to BTSCsTumor = MicroenvironmentPotential Clinical Releva...=20 <<References

The BTSC, as it is investigated and characterized, = could=20 become the ideal target in brain tumor treatment. One = approach=20 would be to force the differentiation of BTSCs; by = depleting=20 this CSC compartment, tumor proliferative and = initiating=20 capacity would decrease. Piccirillo and = colleagues²⁰=20 demonstrated this concept with (physiologically mature = neural=20 progenitor cells) bone morphogenetic = protein=96mediated=20 differentiation of the CD133+ BTSCs and reduction in = tumor=20 initiating capacity (Fig. = 4).²³ Brain=20 tumor stem cell resistance to chemotherapy and = radiotherapy=20 could be targeted with inhibitors. As the molecular = signature=20 of these cells becomes defined, each tumor could be = better=20 targeted through the development of patient-specific = therapies=20 based on molecular characteristics. The stromal = compartment=20 and tumor microenvironment could also be targeted to = modulate=20 and inhibit tumor growth. The potential for = therapeutic=20 progress by targeting the BTSC is considerable, and = realizing=20 this potential depends on continued molecular and = cellular=20 investigations into the character of these cells and = the=20 underlying molecular mechanisms.

View=20 larger version = (77K)

Fig. 4. = Schematic=20 showing GBM treatment targeted to the = tumor stem=20 cell compartment. A GBM is a heterogeneous = tumor=20 initiated and maintained by a minority of = CD133+=20 stem cells, which have a high tumorigenic=20 potential and low proliferation rate. New=20 therapeutic strategies include: 1) forcing = tumor=20 stem cells into a more differentiated = phenotype=20 characteristic of the CD133=96 tumor bulk, = for=20 example, with bone morphogenetic proteins = (BMPs);=20 2) overcoming the inherent resistance of = tumor=20 stem cells to radio- or chemotherapy by = using=20 suitable inhibitors, such as inhibitors of = DNA=20 repair checkpoints; 3) selecting therapies = based=20 on the tumor's molecular profile; and 4) = targeting=20 the tumor stroma, which provides the = specialized=20 milieu for tumor stem cells, angiogenesis, = and=20 immune response. Reprinted by permission = from=20 Macmillan Publishers Ltd: Stupp R, Hegi = ME:=20 Targeting brain-tumor stem cells. Nat=20 Biotechnol 25:193=96194, = 2007.

References	AbstractIntroductionPrevailing Theory of = Tumo...Cancer Stem Cells: = Discov...Caveats to BTSCsTumor = MicroenvironmentPotential Clinical = Releva...References = <<

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