Volume=20 6, Issue=20 3, Pages 458-464 (July 2009)

7 of 27

ABSTRACT

FULL=20 TEXT

FULL-TEXT PDF (739=20 KB)

Novel Treatment Strategies for = Malignant=20 Gliomas Using Neural Stem Cells

Michael=20 C. Oh

, Daniel=20 A. Lim =E2=80=A0 3D"Corresponding

Summary

Recent studies in stem cell = biology=20 have refined our understanding of the origin and progression = of=20 cancer. Identification and characterization of endogenous = neural=20 stem cells (NSCs), especially those in the adult human = brain, have=20 inspired new ideas for selectively targeting and destroying=20 malignant gliomas. Gliomas consist of a heterogeneous = population of=20 cells, and some of these cells have characteristics of = cancer stem=20 cells. These brain tumor stem cells (BTSCs) share certain=20 characteristics with normal NSCs. It is still unclear, = however,=20 whether malignant gliomas in human patients originate from = these=20 aberrant BTSCs. Nonetheless, the cellular and molecular = similarities=20 between BTSCs and normal NSCs suggest a common research = landscape=20 underlying both normal and cancer stem cell biology, wherein = findings of one field are relevant to the other. = Furthermore, the=20 natural tropism of NSCs to gliomas has generated the idea = that=20 modified NSCs can deliver modified genes to selectively = destroy=20 malignant brain tumor cells, and even BTSCs, while leaving = healthy=20 surrounding neurons intact. These studies and others on the = basic=20 biology of both BTSCs and NSCs will be crucial to expanding = our=20 treatment strategies for malignant gliomas.

Key=20 Words: Neural=20 stem cells, brain=20 tumor stem cells, glioma,=20 gene=20 therapy

Article Outline

=E2=80=A2 Summary

=E2=80=A2 Introduction

=E2=80=A2 Molecular=20 and Cellular Similarities Between NSCS and BTSCS

=E2=80=A2 NSCS=20 are Attracted to Gliomas

=E2=80=A2 Novel=20 Treatment Strategies Using NSCS

=E2=80=A2 Conclusion

=E2=80=A2 Acknowledgment

=E2=80=A2 References

=E2=80=A2 Copyright

Introduction

Identification and characterization = of=20 endogenous neural stem cells (NSCs), especially those in the = adult=20 human brain, have inspired new ideas about the cellular = origin of=20 malignant brain tumors,1, 2, 3, 4 as = well as novel=20 approaches for selectively targeting and destroying = malignant=20 gliomas. Malignant gliomas remain one of the most = devastating=20 cancers, despite recent advancements in surgical resection = and=20 adjuvant therapies.5, 6 For = glioblastoma=20 multiforme, the most common and malignant subtype of glioma, = the=20 median survival time remains approximately 14 months.7 The = resistance=20 of gliomas to current therapeutic measures is in part due to = their=20 highly infiltrative nature. Given the invasiveness of glioma = cells,=20 with tumorigenic cells often spreading widely into = relatively normal=20 brain parenchyma,8, 9 = total surgical=20 resection is essentially impossible. Furthermore, adjuvant = treatment=20 strategies (e.g., external beam radiation and chemotherapy) = have not=20 been able to completely treat disease remaining after = surgical=20 resection because these treatment modalities cannot = specifically=20 target the widely disseminated tumor cells. The toxicity of = adjuvant=20 treatments can also result in both systemic illness and = cognitive=20 impairment.10 = New approaches=20 are therefore critical to advancing our ability to treat = this=20 devastating disease.

The identification of neural stem = cells (NSCs)=20 in the adult human brain and their characterization at the = cellular=20 and molecular levels have led to new strategies using such = cells to=20 selectively target and destroy malignant gliomas. NSCs can = be=20 defined as a population of cells that have the ability to = self-renew=20 and produce differentiated neural cell types.

In the adult brain, NSCs have=20 immunocytochemical and ultrastructural characteristics of=20 astrocytes,11 = and there is a=20 large pool of NSCs found in a region of the brain called the = subventricular zone (SVZ), a layer of cells found under the=20 ventricle ependymal cell layer. In rodents, SVZ NSCs give = rise to a=20 large number of interneurons for the olfactory bulb and also = can=20 generate oligodendrocyte and astrocytes.12 In = the human=20 brain, SVZ NSCs have been also reported to produce neurons = for the=20 olfactory bulb,13 = but this=20 finding remains controversial.14 = In vivo=20 NSCs, however, are likely quite different from NSCs = propagated in=20 vitro for therapeutic purposes. For example, culture = conditions=20 that include high concentrations of growth factors can alter = differentiation characteristics of NSCs.11, = 15, = 16 = Nonetheless,=20 cultured NSCs derived from endogenous NSCs as well as=20 transit-amplifying populations that have dedifferentiated17 = still satisfy=20 the defining characteristics of NSCs, including self-renewal = and=20 production of differentiated neural progeny, and are likely = to be=20 useful for novel glioma treatment strategies and cell = replacement=20 therapies.

Molecular and = Cellular=20 Similarities Between NSCS and BTSCS

The cellular origin of malignant = gliomas is=20 still poorly understood. Some recent studies suggest that = gliomas=20 originate from aberrant NSCs,18, = 19, = 20, = 21, = 22, = 23 = but it is also=20 possible to generate malignant gliomas from dedifferentiated = astrocytes.24, = 25 = Both NSCs and=20 glioma cells have the ability to self-renew, express cell = surface=20 antigen prominin-1 (CD133)21, = 26, = 27, = 28 = and the NSC=20 marker nestin, and produce both neurons and glia. = Furthermore, both=20 cell types appear to be attracted to the same molecular = niche,=20 comprised of molecular signals released by either the glioma = cells=20 themselves or by surrounding supportive cells. Thus, = deciphering the=20 niche and molecular properties of NSCs in the adult brain = (reviewed=20 elsewhere; see references29 = and30) = may lead to=20 novel approaches of treatment that are more selective and = potent=20 than current methods.

The adult human brain harbors NSCs = in the=20 subventricular zone (SVZ) of the brain ventricles31, = 32 = and the=20 subgranular zone (SGZ) of the hippocampal dentate gyrus.33 = Human brain=20 specimens collected during neurosurgical procedures = demonstrate the=20 presence of NSCs in the form of ribbons that line the SVZ of = the=20 lateral ventricles.32, = 34 = Similar to=20 NSCs found in the rodent brain,31, = 35 = these human=20 SVZ NSCs correspond to a subpopulation of glial fibrillary = acidic=20 protein (GFAP) expressing astrocytes and are capable of=20 differentiating into neurons, oligodendrocytes, and neurons = in=20 vitro.32 In = the rodent,=20 SVZ NSCs produce a large number of young neuroblasts that = migrate to=20 the olfactory bulb, where they differentiate into several = different=20 types of interneurons.36 = Curtis et=20 al.13 = have proposed=20 that the human SVZ NSCs similarly produce olfactory bulb = neurons=20 throughout life; however, this finding has not been observed = by=20 others.14 = Future studies=20 of the human SVZ should shed more light on the fate of = normal human=20 SVZ NSCs.

Normal NSCs share molecular and = cellular=20 characteristics with BTSCs found in gliomas, and these = parallels may=20 provide important therapeutic targets and novel strategies = for brain=20 tumor treatment. For instance, it has been shown in studies = of SVZ=20 NSCs that the bone morphogenetic protein (BMP) family of = signaling=20 molecules causes NSCs to exit the cell cycle and terminally=20 differentiate into astrocytes at the expense of generating = other=20 lineages.37, = 38 = Given the=20 parallels between NSCs and glioma BTSCs, it was reasonable = to=20 believe that BMP signaling would have a similar effect on = the=20 malignant phenotype of gliomas in vivo. Intriguingly, = Vescovi's group demonstrated that treatment of glioma BTSCs = with BMP=20 dramatically reduces the oncogenic potential of specific = types of=20 cancer stem cells in a mouse transplantation model.39 = Clinical=20 trials evaluating BMP in the treatment of brain tumors are = currently=20 being planned.

Despite the many similarities = between BTSCs=20 and NSCs, there are important differences that are likely to = influence treatment strategies. First, unlike normal NSCs, = BTSCs=20 likely harbor the oncogenic mutations found in the tumor = mass (e.g.,=20 activation of oncogenes and loss of tumor suppressor genes). = These=20 genetic alterations at the level of the DNA sequence = undoubtedly=20 underlie differences between BTSCs and NSCs. Second, there = are=20 epigenetic differences in BTSCs that account for variability = in the=20 biological behavior of specific subsets of BTSCs in = comparison to=20 other BTSCs or normal NSCs. For instance, certain subsets of = BTSCs=20 cannot differentiate in response to BMP, and this difference = is due=20 to epigenetic silencing (by DNA methylation and the polycomb = factor=20 EZH2) of the BMP receptor promoter.40 = Thus, defining=20 such differences between NSCs and specific BTSCs subtypes = will be=20 very important to the design and implementation of glioma = treatments=20 that draw upon parallels between NSCs and BTSCs.

NSCS are Attracted = to=20 Gliomas

Neural stem cells are known to have = high=20 migratory capacity and can travel great distances throughout = the=20 brain. This highly migratory property is reminiscent of the = invasive=20 quality of malignant gliomas. Although a large = conglomeration of=20 tumor cells may be found by radiographic imaging in one = location,=20 small numbers of tumor cells can form microsatellites that = may be=20 located far away from the main tumor, sometimes even in the=20 contralateral hemisphere.8, 41, = 42 = The presence=20 of these microsatellite tumors makes complete surgical = resection=20 very difficult and is thought to contribute to high = recurrence of=20 glioblastomas.

Neural stem cells have extensive = tropism to=20 gliomas.43, = 44, = 45 = Endogenous=20 NSCs are naturally attracted to gliomas44 = and have the=20 ability to migrate into the tumor mass, even contributing to = the=20 tumor bulk. When NSCs are coinjected into rodent brain with = glioma=20 cells, NSCs rapidly migrate great distances to track and = surround=20 glioma tumors. NSCs injected into the side contralateral to=20 malignant tumors have been found to cross the midline and = migrate to=20 the tumor. NSCs injected into systemic circulation can also = migrate=20 and target intracranial gliomas. Furthermore, glioblastoma = cells=20 attract endogenous precursor cells from the SVZ.44 = These findings=20 could be of high clinical value if translated into human = glioma=20 therapy (FIG. 1). NSCs may be used to deliver = drugs and/or=20 genes targeted to destroy microsatellite tumor cells that = could not=20 be detected intraoperatively or by radiographic imaging and = thus=20 escaped surgical resection.

FIG.=20 1. Glioma proliferation may be supported = by brain=20 tumor stem cells (BTSCs). A: Although BTSCs usually = comprise a=20 small portion of total tumor bulk, tumor growth and=20 progression (and, more importantly, recurrence) may be = dependent on BTSCs. These cells have a high migratory = capacity=20 and may form microsatellites of tumor cells far from = the=20 primary tumor. Despite attempts to completely resect = the=20 primary tumor, disseminated microsatellites of BTSCs = may form=20 recurrent tumors. B: Neural stem cells (NSCs) are=20 intrinsically attracted to gliomas and can be used to = deliver=20 therapeutic gene products selectively to gliomas. This = property could be most useful when modified NSCs = carrying=20 genes toxic to tumor cells are injected into the tumor = resection cavity to track and kill residual tumor=20 cells.

Although there is ample evidence of = NSC=20 tropism to gliomas in rodent tumor models, it remains to be = verified=20 whether NSCs can track and target glioma cells in the human = brain.=20 Furthermore, it will be important to identify the signals = involved=20 in attracting NSCs to the gliomas. Once these signaling = pathways are=20 better understood, one can apply this molecular = understanding to=20 track down gliomas, not only for the purpose of targeting=20 therapeutic agents to the tumor, but also to identify and = locate=20 residual tumors for further therapy. It will also be = important to=20 elucidate the mechanisms involved in regulating the cellular = mechanisms of NSC and BTSC migration, as this understanding = may be=20 used to identify drugs capable of inhibiting the migratory = capacity=20 of BTSCs, thus decreasing their malignant invasiveness. For = example,=20 cell surface chemokine receptors, such as CXCR346 = and CXCR4,47 = are potent=20 mediators involved in attracting NSCs to gliomas. NSCs = express high=20 concentrations of CXCR4 and blocking these surface receptors = block=20 NSC migration towards gliomas.47 = Furthermore,=20 numerous other cytokines have been implicated in NSC tropism = to=20 gliomas, including human growth hormone (HGH), stem cell = factor=20 (SCF), urokinase plasminogen activator (uPA), and vascular=20 endothelial growth factor (VEGF).48, = 49, = 50, = 51

Although it is unclear why NSCs are = attracted=20 to gliomas, one possible reason for the recruitment of cells = is to=20 repair damaged tissues surrounding the tumor. Demonstrating = that=20 NSCs can replace damaged cells and understanding the = molecular=20 regulation of this process may lead also to enhanced = neurological=20 recovery.

Novel Treatment = Strategies=20 Using NSCS

Because of the attraction of NSCs to = gliomas,=20 NSCs can be used as cellular vehicles for the delivery of=20 therapeutic agents. Recent studies have shown that NSCs can = be=20 engineered to deliver a variety of genes that use different=20 mechanisms to selectively destroy gliomas.52 = These genes,=20 for example, may encode for enzymes that convert prodrugs to = toxic=20 metabolites that kill gliomas, or may produce proteins that = induce=20 apoptosis or enhance immunity against tumor cells (FIG. 2). Coinjection of unmodified NSCs = with=20 glioblastoma cells into the brains of mice results in = increased=20 survival and decreased tumor progression, suggesting that = NSCs have=20 intrinsic antitumorigenic properties and may release factors = that=20 inhibit glioblastoma proliferation.44, = 45 It = is critical=20 to further understand the molecular nature of this = NSC-mediated=20 antitumor effect for the identification of new therapies for = glioblastomas.

FIG.=20 2. Neural stem cells (NSCs) are = genetically=20 modified to selectively target glioma cells. Modified = NSCs=20 have been designed to carry various genes that encode = protein=20 products toxic to gliomas. In suicide gene therapy = (A),=20 modified NSCs carry genes for an enzyme that converts=20 systemically delivered prodrug into a toxic metabolite = that=20 selectively targets glioma cells. Other approaches = have used=20 genes that (B) induce specific immunity against = gliomas or (C)=20 induce apoptotic pathways selectively in dividing = glioma cells=20 to promote glioma cell death.

One potential mechanism for gene = therapy using=20 NSCs is what is called suicide gene therapy.53 = This is also=20 known as the enzyme=E2=80=93prodrug system, wherein = NSCs can be used=20 to deliver genes for an enzyme that can convert a prodrug,=20 administered systemically, into toxic metabolites. These = toxic=20 metabolites then selectively target the actively dividing = glioma=20 cells while leaving nondividing neurons intact (FIG. 2A).

Two such systems have been tested = using NSCs.=20 One system uses cytosine deaminase (CD), which converts = nontoxic=20 5-fluorocytosine (5-FC) into highly toxic 5-fluorouracil = (5-FU).=20 This system has been tested for malignant gliomas using an=20 immortalized neural progenitor cell line ST14A that = expresses CD.54 = Although 5-FC=20 has no effect on tumor mass by itself, coinjection of = CD-expressing=20 ST14A cells with glioma cells followed by systemic treatment = with=20 5-FC results in approximately a 50% reduction in tumor mass. = Another=20 technique uses the HSV-thymidine kinase=E2=80=93ganciclovir = system, wherein=20 HSV-thymidine kinase (HSV-tk)=E2=80=94but not the mammalian = thymidine kinase=20 found in the patient=E2=80=94phosphorylates systemically = administered=20 ganciclovir to form nucleotide-like precursors that = terminate DNA=20 replications in dividing neoplastic cells. Because a = retroviral=20 vector is used to transduce the HSV-tk gene, gene transfer = occurs=20 only in actively dividing tumor cells, leaving healthy = nondividing=20 brain cells intact.

The HSV-tk/ganciclovir system was = tested in a=20 phase III clinical trial, using vector-producing cells = derived from=20 murine fibroblasts that produced replication-incompetent = retrovirus=20 vectors.55 = However,=20 injecting these vector-producing cells into surgical = resection=20 cavities had no significant effects on progression-free = survival or=20 median overall survival. This lack of effect was thought to = be due=20 to low delivery of HSV-tk gene to the tumor cells. More = importantly,=20 the vector-producing cells used in this trial were murine=20 fibroblasts; these cells do not migrate toward glioma cells, = which=20 severely limits the delivery of HSV-tk to tumor cells. In = more=20 recent studies, therefore, NSCs that have intrinsic tropism = to=20 gliomas have been used to carry HSV-tk gene.56, = 57, = 58 = Coinjection of=20 these cells with malignant glioma cells followed by systemic = ganciclovir treatment results in prolonged survival and = significant=20 inhibition of tumor growth.56

This newer system may be more = effective in=20 inhibiting tumor progression and growth than the earlier = suicide=20 gene systems; however, such a NSC-based treatment strategy = has yet=20 to be tested in a clinical trial. Notably, both the = CD=E2=80=935-FC and the=20 HSV-tk=E2=80=93ganciclovir systems show a bystander effect, = wherein tumor=20 cells not carrying the suicide genes are also killed, which=20 amplifies the antitumor activity in the suicide gene therapy = systems. Although the exact mechanism for the bystander = effect is=20 unclear, phosphorylated ganciclovir is thought to be able to = pass=20 through gap junctions and kill nontransduced tumor cells = nearby. The=20 5-FU released by cytosine deaminase-expressing cells is also = capable=20 of diffusing and killing local tumor cells.59 = This is an=20 important component to the suicide gene therapy, given that = the=20 number of modified NSCs injected into the surgical resection = cavity=20 is likely to be small, relative to the residual tumor cells = left=20 behind. Thus, the antitumor activity of NSCs carrying the = suicide=20 gene is amplified by two different mechanisms. The tropism = of NSCs=20 to tumor microsatellites (which may be great distances away = from the=20 tumor resection cavity) allows selective homing of suicide = genes to=20 targeted areas, and the potent bystander effect can kill=20 nontransduced tumor cells that are near the tumor cells = carrying the=20 suicide gene.

NSCs can be also used to deliver = target=20 proteins that induce apoptosis of glioma cells (FIG. 2C), such as tumor necrosis = factor=E2=80=93related=20 apoptosis-inducing ligand (TRAIL). This is a member of the = TNF=20 protein family that has been shown to induce apoptosis in = neoplastic=20 cells.60=20 Transplantations of NSCs infected with adenovirus-expressing = TRAIL=20 induce apoptosis in established intracranial gliomas, = leading to=20 decreases in tumor size.61 = Furthermore,=20 apoptosis of neoplastic cells is found in pockets of tumors = distant=20 from the main tumor, again suggesting that NSCs can track to = microsatellites of glioma cells. Thus, using NSCs may allow = more=20 direct and selective secretion of this proapoptotic protein = near=20 distant microsatellites of gliomas that may be missed during = a=20 surgical resection. Another potential target protein is PEX, = which=20 is a fragment of metalloproteinase-2 that inhibits glioma=20 angiogenesis. Injection of NSCs transfected with the PEX = gene=20 significantly inhibits proliferation and growth of gliomas = by=20 inhibiting angiogenesis in the tumor.62 = Although none=20 of these target proteins have been used in combination, it = would be=20 useful to learn whether NSCs that can secrete more than one = target=20 protein have additive effects of inhibiting tumor = growth.

Immunomodulators have also been = shown to be=20 effective in controlling tumor progression (FIG. 2B), and recent studies indicate = that=20 interleukin-4 (IL-4),45 = interleukin-12=20 (IL-12),63, = 64 = and=20 interleukin-23 (IL-23)65 = are such=20 candidates. Injecting glioblastoma cells (GL261) into either = mouse=20 or rat brain results in rapid growth and expansion of = tumors,=20 followed by death. Coinjection of IL-4 secreting NSCs = prevents tumor=20 growth and progression and significantly lengthens = survival.45 In = fact,=20 coinjection of NSCs not expressing IL-4 also lengthens = survival,=20 although less so than IL-4-secreting NSCs, again suggesting = that=20 NSCs have intrinsic properties to prevent tumor progression. = Thus,=20 the intrinsic capacity to inhibit tumor growth, tropism to = gliomas,=20 and capacity to carry genes that are toxic to dividing = neoplastic=20 cells are all important characteristics of NSCs that can be = applied=20 for development of more effective glioma = therapies.

Conclusion

Despite the recent advancements in = surgical=20 resections of malignant gliomas and adjuvant therapies, = there has=20 been minimal improvement in overall survival. Identification = and=20 characterization of NSCs in the adult brain has inspired new = approaches that use modified NSCs to target and destroy = malignant=20 gliomas. A few important molecular strategies have been = discussed=20 here. Future studies of both NSCs and glioma BTSCs will = likely lead=20 to identification of new therapeutic targets and strategies, = and we=20 envision a synergy of research between these two stem cell=20 types.

The ideal NSC-based therapy for = adjuvant=20 therapy of malignant gliomas will have certain = properties.

First, the molecular product of the = engineered=20 NSC should be a strong inhibitor of tumor growth at low=20 concentrations, because the local concentration of this = antitumor=20 activity may be low, due to limited numbers of transplanted = NSCs=20 and/or inefficient production of the inhibitors from the NSC = itself.=20 Amplification of antitumor effects, such as that provided by = the=20 bystander effect, would therefore greatly aid in the success = of this=20 treatment strategy.

Second, the NSC-based therapy should = be=20 selective for proliferating neoplastic cells, sparing normal = neurons=20 and glial cells from harmful side-effects.

Third, the natural tropism of NSCs = for gliomas=20 should be maintained or perhaps even enhanced. There may be = many=20 tumor satellites located great distances from the resection = cavity,=20 away from the implantation site of the engineered NSCs. = Furthermore,=20 the number of grafted NSCs will likely be small compared to = the=20 patient's tumor burden even after a resection. Thus, = efficient NSC=20 homing activity will likely be a key feature of a successful = cell-based therapy for malignant gliomas.

Finally, we must exercise caution = when using=20 NSCs for human therapy, because there is always the = potential for=20 tumor growth from grafts of undifferentiated NSC = populations.66 It = may be=20 prudent to have some method of eliminating the transplanted = cells=20 (or causing their terminal differentiation) after they have = served=20 their function.

If all these criteria are met, then = the simple=20 implantation of engineered NSCs to the tumor cavity walls = after=20 surgical resection of the primary tumor might be sufficient = to allow=20 rapid migration of these NSCs toward the residual tumor=20 microsatellites. Thus, even residual disease located in = eloquent=20 areas (e.g., language and motor centers) might be = eliminated, while=20 sparing the normal neurons and glia from the collateral = damage of=20 direct surgical manipulation. Clearly, continued studies of=20 malignant gliomas, BTSCs, and the molecular and cellular=20 characteristics of NSCs will be vital for the development of = new=20 adjuvant therapies for malignant gliomas.

Acknowledgments

We thank the reviewers for their = helpful=20 comments and Benjamin Sherman for his editorial assistance = and=20 intellectual input.

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Department = of=20 Neurological Surgery, University of California, San = Francisco, San=20 Francisco, California 94143-0112

=E2=80=A0 Neurosurgical Section, Surgical = Service, San=20 Francisco Veterans Affairs Medical Center, Department of = Veterans=20 Affairs, San Francisco, California 94121

Address = correspondence=20 and reprint requests to: Michael C. Oh, M.D., Ph.D., = Department of=20 Neurological Surgery, University of California, San = Francisco, 505=20 Parnassus Avenue, San Francisco, CA 94143-0112

Daniel A. = Lim, M.D.,=20 Ph.D., Neurosurgical Section, Surgical Service, San = Francisco=20 Veterans Affairs Medical Center, Department of Veterans = Affairs,=20 4150 Clement Street, San Francisco, CA 94121

PII: S1933-7213(09)00099-3

doi:10.1016/j.nurt.2009.05.003

=C2=A9 2009 = The American=20 Society for Experimental NeuroTherapeutics, Inc. All = rights=20 reserved.

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