技术资料

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

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

Pilocytic astrocytoma is commonly viewed as a benign lesion. However,disease onset is most prevalent in the first two decades of life,and children are often left with residual or recurrent disease and significant morbidity. The Hedgehog (Hh) pathway regulates the growth of higher WHO grade gliomas,and in this study,we have evaluated the activation and operational status of this regulatory pathway in pilocytic astrocytomas. Expression levels of the Hh pathway transcriptional target PTCH were elevated in 45% of tumor specimens analyzed (ages 1-22 years) and correlated inversely with patient age. Evaluation of a tissue array revealed oligodendroglioma-like features,pilomyxoid features,infiltration,and necrosis more commonly in specimens from younger patients (below the median patient age of 10 years). Immunohistochemical staining for the Hh pathway components PTCH and GLI1 and the proliferation marker Ki67 demonstrated that patients diagnosed before the age of 10 had higher staining indices than those diagnosed after the age of 10. A significant correlation between Ki67 and PTCH and GLI1 staining indices was measured,and 86% of Ki67-positive cells also expressed PTCH. The operational status of the Hh pathway was confirmed in primary cell culture and could be modulated in a manner consistent with a ligand-dependent mechanism. Taken together,these findings suggest that Hh pathway activation is common in pediatric pilocytic astrocytomas and may be associated with younger age at diagnosis and tumor growth. View Publication

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs,we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently,the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated,stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast,ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer,and despite treatment advances,patient prognosis remains poor. During routine animal studies,we serendipitously observed that fenbendazole,a benzimidazole antihelminthic used to treat pinworm infection,inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines,mebendazole displayed cytotoxicity,with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells,and in vitro activity was correlated with reduced tubulin polymerization. Subsequently,we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections,has a long track-record of safe human use,and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials. View Publication

The recent identification of cancer stem cells (CSCs) in multiple human cancers provides a new inroad to understanding tumorigenesis at the cellular level. CSCs are defined by their characteristics of self-renewal,multipotentiality,and tumor initiation upon transplantation. By testing for these defining characteristics,we provide evidence for the existence of CSCs in a transgenic mouse model of glioma,S100beta-verbB;Trp53. In this glioma model,CSCs are enriched in the side population (SP) cells. These SP cells have enhanced tumor-initiating capacity,self-renewal,and multipotentiality compared with non-SP cells from the same tumors. Furthermore,gene expression analysis comparing fluorescence-activated cell sorting-sorted cancer SP cells to non-SP cancer cells and normal neural SP cells identified 45 candidate genes that are differentially expressed in glioma stem cells. We validated the expression of two genes from this list (S100a4 and S100a6) in primary mouse gliomas and human glioma samples. Analyses of xenografted human glioblastoma multiforme cell lines and primary human glioma tissues show that S100A4 and S100A6 are expressed in a small subset of cancer cells and that their abundance is positively correlated to tumor grade. In conclusion,this study shows that CSCs exist in a mouse glioma model,suggesting that this model can be used to study the molecular and cellular characteristics of CSCs in vivo and to further test the CSC hypothesis. 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

Glioblastoma multiforme is the most common glioma variant in adults and is highly malignant. Tumors are thought to harbor a subpopulation of stem-like cancer cells,with the bulk resembling neural progenitor-like cells that are unable to fully differentiate. Although multiple pathways are known to be involved in glioma tumorigenesis,the role of Wnt signaling has been poorly described. Here,we show that Dishevelled 2 (Dvl2),a key component of the Wnt signaling pathway,is overexpressed in human gliomas. RNA interference-mediated depletion of Dvl2 blocked proliferation and promoted the differentiation of cultured human glioma cell lines and primary,patient-derived glioma cells. In addition,Dvl2 depletion inhibited tumor formation after intracranial injection of glioblastoma cells in immunodeficient mice. Inhibition of canonical Wnt/β-catenin signaling also blocked proliferation,but unlike Dvl2 depletion,did not induce differentiation. Finally,Wnt5a,a noncanonical Wnt ligand,was also required for glioma cell proliferation. The data therefore suggest that both canonical and noncanonical Wnt signaling pathways downstream of Dvl2 cooperate to maintain the proliferative capacity of human glioblastomas. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease,and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly,as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv,and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together,these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library,which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. View Publication

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity,which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications,clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however,the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs,most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification,revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall,43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication