References

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

BACKGROUND Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery,whole brain and spine irradiation,and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. METHODS To test the importance of NFκB to medulloblastoma cell growth,the effects of multiple drugs that inhibit NFκB,pyrrolidine dithiocarbamate,diethyldithiocarbamate,sulfasalazine,curcumin and bortezomib,were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells,xenograft flank tumors,and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB,IκB,was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition. RESULTS We report high constitutive activity of the canonical NFκB pathway,as seen by Western analysis of the NFκB subunit p65,in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though,conversely,the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally,expression of a dominant negative form of the endogenous inhibitor of NFκB,dnIκB,resulted in poor xenograft tumor growth,with average tumor volumes 40% smaller than controls. CONCLUSIONS These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth,and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes. View Publication

The demonstration of the brain's ability to initiate repair in response to disease or injury has sparked considerable interest in therapeutic strategies to stimulate adult neurogenesis. In this study we examined the effect of a progressive neurodegenerative condition on neural precursor activity in the subventricular zone (SVZ) and hippocampus of the R6/1 transgenic mouse model of Huntington's disease (HD). Our results revealed an age-related decline in SVZ precursor numbers in both wild-type (WT) and HD mice. Interestingly,hippocampal precursor numbers declined with age in WT mice,although we observed maintenance in hippocampal precursor number in the HD animals in response to advancement of the disease. This maintenance was consistent with activation of a recently identified latent hippocampal precursor population. We found that the small latent stem cell population was also maintained in the HD hippocampus at 33 weeks,whereas it was not present in the WT. Our findings demonstrate that,despite a loss of neurogenesis in the HD hippocampus in vivo,there is a unique maintenance of the precursor and stem cells,which may potentially be activated to ameliorate disease symptoms. View Publication

The neurosphere assay (NSA) is one of the most frequently used methods to isolate,expand and also calculate the frequency of neural stem cells (NSCs). Furthermore,this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied,overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay,the neural-colony forming cell assay (N-CFCA),which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential,and thus provides a method to enumerate NSC frequency. In the N-CFCA,colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC,while colonies textless 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are textless 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated. View Publication

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises,human iPSCs will ideally be free of exogenous DNA (footprint-free),and be derived and cultured in chemically defined media free of feeder cells. Currently,methods are available to enable efficient derivation of footprint-free human iPSCs. However,each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery,but the process was inefficient and required feeder cells. Here,we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901,GSK3β inhibitor CHIR99021,TGF-β/Activin/Nodal receptor inhibitor A-83-01,ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover,we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts,adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs. View Publication

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs),thought to account for tumorigenesis and therapeutic resistance,have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples,regardless of their histopathologies and grades of malignancy (57% of embryonal tumors,57% of low-grade gliomas and neuro-glial tumors,70% of ependymomas,91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (textgreater7 self-renewals),whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities,the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05,chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013,log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers,the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients,TSCs have been isolated only from high-grade gliomas. In contrast,our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors. View Publication

Maintenance of genomic integrity is essential for adult tissue homeostasis and defects in the DNA-damage response (DDR) machinery are linked to numerous pathologies including cancer. Here,we present evidence that the DDR exerts tumor suppressor activity in gliomas. We show that genes encoding components of the DDR pathway are frequently altered in human gliomas and that loss of elements of the ATM/Chk2/p53 cascade accelerates tumor formation in a glioma mouse model. We demonstrate that Chk2 is required for glioma response to ionizing radiation in vivo and is necessary for DNA-damage checkpoints in the neuronal stem cell compartment. Finally,we observed that the DDR is constitutively activated in a subset of human GBMs,and such activation correlates with regions of hypoxia. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this study,we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor,PI-103,in established glioma lines and primary CD133(+) glioma-initiating cells and explored the potential of combining PI-103 with stem cell-delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion,causes G(0)-G(1) arrest in cell cycle,and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural stem cells (NSC) and glioma cells,we show that PI-103 augments the response of glioma cells to stem cell-delivered S-TRAIL. Using bimodal optical imaging,we show that when different regimens of systemic PI-103 delivery are combined with NSC-derived S-TRAIL,a significant reduction in tumor volumes is observed compared with PI-103 treatment alone. To our knowledge,this is the first study that reveals the antitumor effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanism-based systemically delivered antiproliferative agents and novel stem cell-based proapoptotic therapies to improve treatment of malignant gliomas. View Publication

p73,a member of the p53 family,is a transcription factor that plays a key role in many biological processes. In the present study,we show that TAp73 is expressed in neural stem cells (NSC) and its expression increases following their differentiation. NSC from p73 null mice have a reduced proliferative potential,together with reduced expression of members of the Sox-2 and Notch gene families known to be important for NSC proliferation. In parallel with this in vitro data,the width of the neurogenic areas was reduced in the brains of embryonic and adult p73-/- mice. These data suggest that p73,and in particular TAp73,is important for maintenance of the NSC pool. View Publication

Current data suggests an association between elevations in interleukin 1 (IL-1)α,IL-1β,and IL-6 and the proliferation of neural progenitor cells (NPCs) following brain injury. A limited amount of work implicates changes in these pro-inflammatory responses with diminished NPC proliferation observed as a function of aging. In the current study,adolescent (21day-old) and 1year-old CD-1 male mice were injected with trimethyltin (TMT,2.3mg/kg,i.p.) to produce acute apoptosis of hippocampal dentate granule cells. In this model,fewer 5-bromo-2'-deoxyuridine (BrdU)+ NPC were observed in both naive and injured adult hippocampus as compared to the corresponding number seen in adolescent mice. At 48h post-TMT,a similar level of neuronal death was observed across ages,yet activated ameboid microglia were observed in the adolescent and hypertrophic process-bearing microglia in the adult. IL-1α mRNA levels were elevated in the adolescent hippocampus; IL-6 mRNA levels were elevated in the adult. In subgranular zone (SGZ) isolated by laser-capture microdissection,IL-1β was detected but not elevated by TMT,IL-1a was elevated at both ages,while IL-6 was elevated only in the adult. Naïve NPCs isolated from the hippocampus expressed transcripts for IL-1R1,IL-6Rα,and gp130 with significantly higher levels of IL-6Rα mRNA in the adult. In vitro,IL-1α (150pg/ml) stimulated proliferation of adolescent NPCs; IL-6 (10ng/ml) inhibited proliferation of adolescent and adult NPCs. Microarray analysis of SGZ post-TMT indicated a prominence of IL-1a/IL-1R1 signaling in the adolescent and IL-6/gp130 signaling in the adult. View Publication

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological,genetic,and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells,much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM),epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly,despite its greater biological activity than wild-type EGFR (wtEGFR),individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population,and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes,we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues,glioma cell lines,glioma stem cells,and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130,which in turn activates wtEGFR in neighboring cells,leading to enhanced rates of tumor growth. Ablating IL-6,LIF,or gp130 uncouples this cellular cross-talk,and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass,and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention. View Publication