STEM CELLS

doi:10.1093/neuonc/not190

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-001. ROLES OF GLIOBLASTOMA STEM CELL-DERIVED VASCULAR PERICYTES

Lin Cheng ¹, Zhi Huang ¹, Wenchao Zhou ¹, Qiulian Wu ¹, Jeremy Rich ¹, Shideng Bao ¹

Abstract

Glioblastomas (GBMs) are highly lethal brain tumors with florid vascularization. GBMs display striking cellular hierarchies containing self-renewing tumorigenic glioma stem cells (GSCs). As GSCs often reside in perivascular niches within a GBM tumor, we interrogated GSC potential to generate vascular pericytes in vivo. We demonstrated that GSCs give rise to pericytes to support vessel structure and function and promote tumor growth (Cheng et al., Cell 2013). In vivo cell lineage tracing with constitutive and lineage specific fluorescent reporters indicated that GSCs generate the majority of vascular pericytes. Selective elimination of GSC-derived pericytes disrupted tumor vasculature and potently inhibited tumor growth. Furthermore, GSCs are recruited toward endothelial cells via the SDF-1/CXCR4 axis and induced to become pericytes predominantly by TGF-β. Thus, GSCs function as pericyte progenitors and contribute to vasculature formation in GBMs. The potent capacity of GSCs to generate vascular pericytes allows active vascularization to support GBM tumor growth. We extended this study and found that GSC-derived pericytes contribute to the resistance to the current anti-angiogenic therapy that targets endothelial cells, indicating that therapeutic targeting of GSC-derived pericytes may improve the anti-angiogenic therapy.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-002. SILENCING BMI1 ELIMINATES TUMOR FORMATION OF PEDIATRIC GLIOMA CD133+ CELLS NOT BY AFFECTING INK4A AND ARF BUT BY DOWN-REGULATING STAU2, ALDH3A, API5, EIF4G2 AND RPS6KA2

Patricia Baxter ¹, Hua Mao ¹, Xiumei Zhao ¹, Zhigang Liu ¹, Yulun Huang ¹, Horatiu Voicu ⁴, Sivashankarappa Gurusiddappa ¹, Jack M Su ¹, Laszlo Perlaky ¹, Robert Dauser ³, Hon-chiu Eastwood Leung ³, Karin M Muraszko ⁵, Jason A Heth ⁵, Xing Fan ⁵, Ching C Lau ¹, Tsz-Kwong Man ¹, Murali Chintagumpala ¹, Xiao-Nan Li ¹

Abstract

Clinical outcomes in children with malignant glioma remain dismal. Although cancer stem cells are shown to play critical roles in tumorigenesis and drug resistance, few therapeutic targets have been identified for pediatric gliomas. BMI1 is a member of the polycomb group gene family and an important regulator of stem cell self-renewal. However, its role in pediatric gliomas has not been established. Here, we show that BMI1 is over-expressed in 29 of 54 (53.7%) pediatric gliomas and in 8 of 8 (100%) patient tumor-derived orthotopic xenograft mouse models. Both CD133+ and CD133- cells expressed BMI1 protein as well. Using two independent orthotopic xenograft models of glioblastoma multiforme (GBM), we further demonstrated that silencing BMI1 with lentivirus mediated shRNAs suppressed GBM cell proliferation in vitro and eliminated the xenograft tumor formation of CD133+ and CD133- GBM cells in vivo in mouse brains. Through whole genome gene expression profiling of paired CD133+ and CD133- cells derived from three orthotopic mouse models, we discovered that lentiviral mediated silencing of BMI1 did not alter the expressions of most of the known target genes that were associated with the over-expressed BMI1, and identified a novel set of core genes, including STAU2, RPS6KA2, ALDH3A2, FMFB, DTL, API5, EIF4G2, KIF5c, LOC650152, C20ORF121, LOC203547, LOC653308, and LOC642489, as potential down-stream targets of the silenced BMI1 in pediatric GBMs. These findings suggested that BMI1 signaling pathway responsible for promoting tumor growth may not be the same as those mediating the inhibition of tumor formation. It highlighted the importance of careful re-analysis of the affected genes following the knocking down of an aberrantly activated oncogenic gene to identify the potential determinants and predicators of efficacy. Our results identified BMI1 as a novel therapeutic target in pediatric gliomas.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-003. IDENTIFICATION OF GLIOBLASTOMA CANCER STEM-LIKE CELL-BINDING HUMAN ANTIBODIES VIA YEAST EXPRESSION LIBRARY BIOPANNING

Paul Clark ¹, Michael Zorniak ¹, Yongku Cho ¹, Xiaobin Zhang ¹, Daniel Walden ¹, Eric Shusta ¹, John Kuo ¹

Abstract

Detection and isolation of cancer stem-like cells (GSC) from the incurable brain cancer, glioblastoma (GBM), require more optimal markers and methods. A naïve yeast expression library of single-chain human antibodies (scFv) was mined for human GSC-specific antibodies via biopanning. Briefly, nine rounds of positive selection enriched against patient-derived GSC cells for GSC-binding scFv, with selected pools also negatively screened against normal human astrocytes, neural stem cells, and serum-cultured GBM. 62 unique scFv clones were identified from ∼600 candidates by differential PCR and restriction analysis. Each clone was characterized for its qualitative binding specificity against 12 distinct lines of normal, GSC, and matched GBM. Clone 1.7 demonstrated specificity against 5 GSC lines representing three different invasiveness classes and multiple neural stem/progenitor states. Secreted and purified scFv-1.7 was validated by flow cytometry for GSC-specificity, and ability to detect highly infiltrative GSCs from tumor xenografts. Ongoing experiments will characterize the tumor initiating ability of scFv-1.7-positive cells from fresh GBM specimens. Rapid screening via yeast antibody library biopanning identified human GSC-specific antibodies for potential development into immunotargeted diagnostics and therapeutics in brain cancer.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-004. THE SMG1 KINASE IS REQUIRED FOR GLIOBLASTOMA STEM CELL GROWTH IN HYPOXIA

Sejuti Sengupta ¹, Surbhi Goel-Bhattacharya ¹, Shreya Kulkarni ¹, Brent Cochran ¹

Abstract

Glioblastomas (GBM) typically have regions of hypoxia. Current evidence suggests that glioblastoma stem cells reside in hypoxic niches and are preferentially resistant to radiation and chemotherapy. In order to identify genes in GBM stem cells that are required for growth and survival of GBM stem cells under hypoxic conditions, we have performed RNAi screens of GBM stem cells under normoxic and hypoxic conditions. The SMG1 kinase was identified in this screen as a gene that is preferentially inhibitory to GBM stem cells grown in 1% oxygen. SMG1 is a member of the phosphoinositide-like family of kinases that includes ATM and ATR. It is been implicated in nonsense mediated decay and the DNA damage response. We have found that multiple GBM stem cell lines are differently sensitive to knockdown of SMG1 by multiple shRNAs under hypoxic as compared with normoxic conditions. In addition, knockdown of SMG1 can sensitize at least one GBM stem cell line to killing by temozolomide. Ongoing studies are investigating the mechanism of action of the SMG1 kinase in GBM stem cells and its possible role in GBM tumorigenesis.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-005. GLIOBLASTOMA STEM- AND PROGENITOR-LIKE CELLS: SIZE MATTERS

Carlo Cusulin ^1,², Artee Luchman ^1,², Sam Weiss ^1,²

Abstract

We isolated and propagated a diverse array of brain tumor stem cell (BTSC) lines with heterogeneous molecular profiles, derived from Glioblastoma multiforme (GBM) patients. These BTSCs grow as spheres and display fundamental cancer stem cell properties. We previously confirmed great heterogeneity amongst BTSC lines in terms of clonogenicity, proliferation, quiescent cell population and marker expression. Hypothesizing that this diversity is also reflected within each line, we set out investigating methods for separating these subpopulations. We reasoned that sphere-forming cells with different proliferative capacity would form spheres of different sizes. We therefore analyzed the variability of sphere size within 20 BTSC lines, revealing two distinct groups. The first had a normal distribution of sphere size, while the second showed a bimodal distribution; we selected the latter for further experiments, separating spheres of diameter <100 µm (small) and >100 µm (big). These two subsets of spheres showed remarkable differences in vitro. Though they had similar clonogenicity (small vs big-derived 107.3 ± 10.7vs110.4 ± 7.6% of control), cells dissociated from small spheres proliferated slower. Spheres grown from these cultures, in turn had different size distributions, with the big sphere-derived cells giving rise to larger spheres and a normal size distribution. Moreover, small sphere-derived cells had more quiescent cells measured by carboxyfluoresceinsuccinimidylester (CFSE) retention (small vs big-derived 213.6 ± 16.9vs78.9 ± 4.7%) and more asymmetric division (small vs big-derived 137.7 ± 20.1vs38.6 ± 18.7%) Together, these results suggest that cells derived from small spheres have more stem cell characteristics, while those derived from big spheres are more similar to progenitors. We are further investigating the behaviour of these subpopulations of spheres, in orthotopic xenografts in NOD-SCID mice. Our results highlight the value of BTSCs as model system to study the intrinsic heterogeneity of GBM, and may provide insights into the great biological complexity of this disease. Funding: Stem Cell Network and Alberta Cancer Foundation.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-006. GROWTH FACTOR-DEPENDENT DIVERGENCE OF STEM CELL IDENTITY IN GLIOBLASTOMA

Megan Wu ², Nestor Fernandez ², Sameer Agnihotri ², Roberto Diaz ², James Rutka ², Markus Bredel ³, Jason Karamchandani ¹, Sunit Das ^1,²

Abstract

Despite therapeutic advances, disease recurrence and tumour progression in patients with glioblastoma remain universal. Tumour resistance is thought to be due in part to cell-intrinsic heterogeneity in these cancers. In particular, glioblastoma is thought to harbour a population of stem-like cells called glioma stem cells (GSCs), that appear to be responsible for tumor recurrence. Given their import in normal stem cell microenvironments, we have examined the role of cell-extrinsic signaling in glioma stem cell biology and resistance. We have focused on two signaling pathways known to be relevant to neural and glioma stem cells, TGF-ß and BMP. The role of TGF-ß and BMP signaling in glioblastoma progression was examined using IHC in patient tumor samples and in vitro studies of glioma stem cells. Our results were then analyzed against clinical and gene expression data from the TCGA and Rembrant datasets. We identified intratumoural heterogeneity in glioma stem cell phenotype driven by cell-extrinsic TGF-ß and BMP signaling. Activation of the TGF-ß pathway gives rise to a GSC that is highly proliferative and motile, but also sensitive to radiation- and chemotherapy-induced cell death. Conversely, BMP-acticated GSCs are relatively quiescent and sedentary, but are resistant to radiation and chemotherapy. These findings correlate with the mesenchymal and proneural glioblastoma subtypes, in which TGF- ß and BMP signaling predominate, respectively. Cell-extrinsic signaling via the TGF-ß and BMP pathways has profound effects on glioma stem cell phenotype and on outcomes in patients with glioblastoma. Modification of GSC phenotype through extrinsic manipulation of these signaling factors could be relevant to the care of patients with glioblastoma.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-007. EphA3 MAINTAINS TUMORIGENICITY AND IS A THERAPEUTIC TARGET IN GLIOBLASTOMA MULTIFORME

Bryan Day ¹, Brett Stringer ¹, Fares Al-Ejeh ¹, Michael Ting ¹, John Wilson ², Kathleen Ensbey ¹, Paul Jamieson ¹, Zara Bruce ¹, Yi Chieh Lim ¹, Carolin Offenhauser ¹, Sara Charmsaz ¹, Leanne Cooper ¹, Jennifer Ellacott ¹, Angus Harding ², Jason Lickliter ⁵, Po Inglis ³, Brent Reynolds ⁶, David Walker ⁴, Martin Lackmann ⁵, Andrew Boyd ^1,⁷

Abstract

INTRODUCTION: Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. This study identifies EphA3 as a functional, targetable protein in GBM. EXPERIMENTAL PROCEDURES: Eph receptor expression analysis was conducted on glioma clinical specimens using QPCR (n = 80). Analysis of EphA3 expression with known glioma tumourigenic markers was conducted using multiplex analysis on dissociated clinical specimens (n = 9). EphA3 tumorigenic potential was assessed by down regulating EphA3 using shRNA, depletion using magnetic beads or acutely sorting patient specimen by FACS followed by both subcutaneous and orthotopic ‘intracranial' xenografts. Tumor formation was assessed using in vivo bioluminescence. EphA3 in vivo targeting was assessed using a Lutetium radiolabelled EphA3-specific mAb. SUMMARY: We show that the receptor tyrosine kinase EphA3 is frequently over expressed in GBM and in particular in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and is critically involved in maintaining tumor cells in an undifferentiated state by modulating MAPK signaling. EphA3 knock down or depletion of EphA3-positive tumor cells dramatically reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific mAb. CONCLUSION: While debate still surrounds the cancer stem cell hypothesis in solid tumors, such as GBM, there is agreement that cells in an undifferentiated, tumorigenic state exist within these highly heterogeneous tumors. These cells are thought to be responsible for tumor recurrence following treatment. Here, we demonstrate that in EphA3-expressing GBM, EphA3 is crucial in maintaining undifferentiated, tumor-initiating cells by modulation of MAPK signaling. EphA3 is lowly expressed in adult tissues and therefore represents a relatively tumor-specific therapeutic target in GBM.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-008. Sox2 KNOCKDOWN IN A PATIENT-DERIVED GLIOSARCOMA MODEL PREVENTS THE FORMATION OF THE ASTROCYTIC BUT NOT OF THE SARCOMATOUS TUMOR COMPARTMENT

Artem Berezovsky ¹, Laila Poisson ¹, Laura Hasselbach ¹, Susan Irtenkauf ¹, Andrea Transou ¹, Tom Mikkelsen ¹, Ana C deCarvalho ¹

Abstract

Remarkable phenotypic plasticity contributing to glioblastoma (GBM) malignancy is manifested multiple instances. Gliosarcoma (GS), a morphological variant of GBM characterized by biphasic glial and sarcomatous compartments is one example. Developmental plasticity is inferred from the observation that GBM cells can exhibit cancer stem cell (CSC) phenotype, operationally defined as the ability to self-renew and differentiate into the cells comprising the bulk of the tumor, phenocopying the parental tumor in mouse xenografts. Sox2 transcription factor is one of the neural stem cell markers highly expressed in GBM CSC-enriched neurosphere cultures. Using immunohistochemistry we observed that 11 to 85% of cells in GBM biopsies expressed Sox2, similar to the pattern observed in patient-derived xenografts, implying that within the tumors Sox2 is expressed not only in CSCs but also in differentiated neoplastic cells. We have previously described a patient-derived GS line which, growing as neurospheres or low passage serum monolayers, undergo mesenchymal metaplasia in vivo, giving rise to biphasic glial and mesenchymal tumors in orthotopic xenografts. Contrary to the norm, this line retains Sox2 expression in low passage serum supplemented cultures. These characteristics rendered this model suitable for Sox2 loss of function studies in differentiated neoplastic cells, while also accessing the distinct roles of Sox2 in the mesenchymal and astrocytic components in GS xenografts. Tumor formation was reduced by 50%, and the tumors that did form in the absence of Sox2 exhibited a sarcomatous phenotype, and did not express astrocytic markers, suggesting that in the context of biphasic gliosarcomas, Sox2 specifically regulates glial tumorigenesis. Transcriptome analysis revealed that Sox2 represses the expression of αSMA and the Wnt pathway and activates the expression of neural stem cell markers in the differentiated cells. Our results suggest that mesenchymal metaplasia could be a mechanism of escape from therapies targeting pathways canonically associated with stem cell maintenance.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-009. EGFRvIII EXPRESSION CONTRIBUTES TO GLIOBLASTOMA STEM CELL PROPERTIES AND ITS EXPRESSION IS REGULATED BY EPIGENETIC MECHANISMS

David Emlet ¹, Catherine Del Vecchio ¹, Puja Gupta ¹, Gordon Li ¹, Steven Skirboll ¹, Albert Wong ¹

Abstract

Amplification and rearrangement of the EGFR gene leading to EGFRvIII expression is frequently found in glioblastoma. Research suggests that EGFRvIII is a marker for glioblastoma stem cells (GSCs). If EGFRvIII genetic alterations are early events then such changes should be preserved throughout the tumor. We found that EGFR amplification and rearrangement is indeed found throughout the tumor yet EGFRvIII expression is focal and sporadic. This restricted expression supports the role of EGFRvIII in GSCs and suggests that EGFRvIII expression can be modulated even in the presence of high gene amplification. In vivo studies showed a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms played a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating a mechanism for re-expression of EGFRvIII. Inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression providing a mechanism for downregulation. Using immunofluorescence to examine cells directly shows EGFRvIII is highly associated with CD133 and CD15 (SSEA-1) but has almost no association with TuJ1 or GFAP. Interestingly, there is also a poor association of EGFRvIII with wt EGFR expression. EGFRvIII partly mediates its effects on CSC properties via the Wnt pathway by increasing b-catenin or Lef1 expression and fos promoter activity which could be downregulated using shRNA or specific inhibitors. This data demonstrates that EGFRvIII is an early event in tumorigenesis, that EGFRvIII and stem cell marker expression follows a hierarchical model, and EGFRvIII activity promotes the CSC phenotype. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs which modulate the epigenome might be used successfully in glioblastoma tumors.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-010. THE GLIOMA-ENHANCING ROLE OF MESENCHYMAL STEM CELL EXOSOMES WITHIN THE TUMOR MICROENVIRONMENT

Javier Figueroa ¹, Tal Shahar ¹, Anwar Hossain ¹, Frederick Lang ¹

Abstract

Cells within the tumor niche use an intricate network of communication not only to establish the tumor microenvironment, but also promote the growth and evolution of the malignancy. In this range of communication systems, the contribution of exosome transfer between tumor forming glioma stem cells (GSCs) and glioma-associated mesenchymal stem cells (GA-MSCs) is not well established. Therefore, we hypothesized that exosomes released by GA-MSCs represent a novel intra-tumoral communication mechanism that promotes aggressive behavior in GBM. Using ultracentrifugation methods we harvested exosomes from four GA-MSC lines, extracted from patient tumors, and characterized the content of these nano-vesicles to include membrane bound proteins and various miRNA. Exosome transfer and uptake was subsequently demonstrated when GA-MSC exosomes labeled with fluorescent membrane dye, were spontaneously absorbed and sorted by four GSC lines (isolated from patient tumors), two of which had matching GA-MSC lines. In vitro experiments then revealed that the tumor-enhancing effects of GA-MSCs on co-cultured GSCs, could not be solely attributed to secreted growth factors, but must also include exosome exchange. We found that the delivery of GA-MSC exosomes increased the proliferation of GSCs between 30% and 145%, as well as enhanced the clonogenicity between 25% and 50%. Furthermore, co-injection of GSC mouse xenografts and GA-MSC exosomes resulted in a greater tumor burden, and a 15% decrease in overall survival. Here we describe for the first time, not only in glioma but for all tumor types, the tumor-promoting capability of exosomes released by MSCs found in the tumor microenvironment. Although exact exosome delivery and GSC altering mechanisms have yet to be established, GA-MSC exosomal miRNA are likely candidates due to their ability to alter recipient GSC gene expression, thus adding a novel pathway by which the tumor microenvironment can impact glioma progression.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-011. IN VITRO TREATMENT RESPONSE AND EPIGENETIC PROFILING REVEAL UNEXPECTED DIFFERENCES IN GLIOMA STEM CELLS COMPARED TO PATIENT-MATCHED NON-STEM CELL CULTURES

Shaun Fouse ¹, Jean Nakamura ¹, C David James ¹, Susan Chang ¹, Joseph Costello ¹

Abstract

Glioblastomas (GBMs) contain a population of cells that exhibit stem cell phenotypes. These cancer stem cells (CSCs) may be a source of therapeutic resistance, although support for this important concept is limited. We established early passage cultures and performed a comprehensive study on the response of primary GBM CSCs and isogenic, patient-matched non-CSCs to clinically relevant single or serial doses of temozolomide (TMZ), radiation (XRT), or alternating TMZ and XRT treatment, which is the standard of care for these patients. The majority of cells in CSC cultures expressed stem cell markers and several cultures gave rise to invasive tumors intracranially, whereas matched non-CSC were negative for these markers and rarely formed tumors. Despite these phenotypic differences, when exposed to clinically relevant doses of TMZ, the majority of CSC and matched non-CSC displayed similar responses, which correlated with MGMT promoter methylation status and MGMT protein levels. In contrast, CSCs were unexpectedly more sensitive to XRT compared to matched non-CSCs from two patients despite equal TMZ-resistance in their CSC and non-CSC cultures. We hypothesized that DNA methylation in addition to MGMT might underlie the differences in response between CSC and their nearly isogenic non-CSC. Genome-wide methylation analyses found that CSC cultures were enriched for hypermethylation in genes involved in neuronal fate determination while metabolic pathway genes are hypomethylated. Taken together, we conclude that response to TMZ is tightly linked to the individual tumor and MGMT status and is independent of the phenotypic differences of the CSC and non-CSC cultures. Additionally, DNA methylation changes between the paired cultures might account for some of the observed differences between the cultures. If the tumor cell response in vitro mirrors therapeutic response across larger patient cohorts, these rapid assays in primary cultures could allow empirical selection of patient-specific therapeutic agents, potentially improving patient outcomes.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-012. INDUCTION OF IMMUNOGENIC CANCER TESTIS ANTIGENS IN TUMOR-INITIATING GLIOMA STEM CELLS

Jason M Frerich ¹, Shervin Rahimpour ¹, Zhengping Zhuang ¹, John D Heiss ¹

Abstract

INTRODUCTION: Tumor heterogeneity impedes immunotherapy medicine strategies in glioma. Glioma stem cells (GSCs) have been identified as tumor initiating and play an important role in recurrence and resistance to chemo- and radiotherapy. Induced expression of immunogenic cancer testis antigens (CTAs) in glioma has been shown to increase T-cell recognition and tumor cell lysis. Immunotherapy strategies capable of inducing expression of CTAs in all glioma cells (GSCs and differentiated tumor cells) may serve as effective adjuvant treatments. METHODS: GSCs and differentiated tumor cell populations were isolated from resected human glioblastoma tumors. DNA hypomethylating agent decitabine (DAC) was utilized over a period of 48-hrs for induction of CTAs. The expressions of NY-ESO-1, LAGE-1, and MAGE-A3 were examined in GSCs and differentiated tumor cells, as well as in U87, U251, and U373 glioma, Mel1300B melanoma, and normal human astrocyte (NHA) cell lines. RNA expressions were determined by conventional and quantitative real-time PCR. Western blot and immunofluorescence analysis were performed to evaluate protein expressions. RESULTS: NY-ESO-1 and LAGE-1 expressions were not detectible prior to treatment with DAC in all cell lines except Mel1300B positive-controls. MAGE-A3 expression was detectible in all cell lines except NHA. Treatment with DAC induced a significant increase in expressions of both NY-ESO-1 and LAGE-1 RNA (p < 0.0001) and protein in GSCs, differentiated cells, and U87, U251, and U373 cell lines. DAC treatment had no significant effect on MAGE-A3 expression, and did not induce the expression of CTAs in NHAs. CONCLUSIONS: Treatment with DAC induced expression of CTAs in both GSCs and differentiated tumor cells. CTA genes were not expressed in NHAs with or without DAC treatment. These results suggest that epigenetic modulation with DAC can overcome tumor heterogeneity and distinguish differentiated and tumor-initiating GSCs from NHAs, serving as an effective means of developing targeted T-cell mediated immunotherapy for glioma.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-013. CLONAL EVOLUTION MEETS CANCER STEM CELLS IN GLIOBLASTOMA

Anna Golebiewska ¹, Daniel Stieber ¹, Lisa Evers ⁴, Elizabeth Lenkiewicz ⁴, Nicolaas HC Brons ², Nathalie Nicot ³, Anaïs Oudin ¹, Sébastien Bougnaud ¹, Frank Hertel ⁵, Rolf Bjerkvig ^1,⁶, Mike Barrett ⁴, Laurent Vallar ³, Simone P Niclou ¹

Abstract

Inter and intratumoral heterogeneity have major therapeutic implications and the presence of genetically distinct clonal populations may underlie multiple phenotypes of cancer cells and influence clinical outcome. By applying ploidy-based flow sorting combined with array CGH in glioblastoma (GBM) patient biopsies we distinguish two tumor types: mono and polygenomic GBMs. Monogenomic tumors were limited to one pseudodiploid tumor clone admixed with normal stromal cells, whereas polygenomic tumors contained multiple clonal populations of different ploidy. Interestingly, polygenomic tumors always contained pseudodiploid tumor cells, carrying identical chromosomal breakpoints than cells from the aneuploid fractions. This suggests an evolutionary process from diploid cells towards aneuploidy after acquisition of the main genomic aberrations, thus aneuploidy appears to be a late event in GBM development. We further show that divergent clones within one patient are tumorigenic and that clonal heterogeneity can be recapitulated in spheroid-based xenografts. Importantly, distinct genetic clones contain different tumorigenic potential and are heterogeneous at the phenotypic level. In particular, putative cancer stem cell populations in polygenomic tumors are heterogeneous and contain genetically distinct clones.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-014. IDENTIFICATION OF BRAIN TUMOUR TARGETING PEPTIDES

Xiaoguang Hao ^1,⁴, Jennifer Rahn ^1,², Eva Ujack ^1,⁴, Xuequing Lun ^1,⁴, Greg Cairncross ^3,⁴, Sam Weiss ⁵, Donna Senger ^1,⁴, Stephen Robbins ^1,⁴

Abstract

Over the last decade there has been an explosion of research focused on the cancer stem cell, a cell believed to be the clonogenic core of tumours. Many efforts have been made to develop therapies designed to target this tumour cell population. Based on numerous studies however it is now evident that the initial iteration of the cancer stem cell hypothesis may be too simplistic, rather recent research supports a more dynamic cancer stem cell hypothesis where both plasticity and heterogeneity of the system is highlighted. To capture the complexity and heterogeneity of high-grade glioma and to identify glioma-specific peptides, we employed a combinatorial phage-display biopanning strategy to isolate peptides that specifically bind and home in vivo to key disease reservoirs within glioma; namely the invasive and stem-like populations of glioma cells. Using this approach we identified a panel of peptides with the ability to detect the heterogeneity of patient gliomas in vivo irrespective of the genetic diversity of each individual patient glioma. Next, we investigated the potential of these peptides as a platform for the development of imaging agents. Synthetic peptides were conjugated to gadolinium or fluorescence tags that could be tracked in vivo. The labelled peptides were administered individually or in combination to animals bearing orthotopic tumours established from human glioma-like-stem cells/brain-tumour-initiating cells and included models of highly infiltrative tumours that are otherwise invisible by conventional imaging technologies. Using a panel of peptides we successfully imaged a range of diffuse infiltrating tumours that are notoriously difficult to image using standard MRI. Additionally, we identified peptides that possessed the ability to inhibit cell invasion, or target subsets of tumour cells that had 'stem-like' properties. Thus, in addition to imaging, these peptides may be useful as therapeutics, as delivery agents for chemotherapeutics, or as diagnostic indicators.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-015. TREATMENT EFFECTS ON STEM AND PROGENITOR SUBPOPULATIONS IN A MODEL OF GLIOBLASTOMA

Julie Harness ¹, Robin Lerner ¹, Yuichiro Ihara ¹, Raquel Santos ¹, Jacqueline De La Torre ¹, Alex Lu ¹, Tomoko Ozawa ¹, Theodore Nicolaides ¹, David James ¹, Claudia Petritsch ¹

Abstract

Glioblastoma multiforme (GBM) is the most deadly CNS malignancy, notoriously resilient despite aggressive treatment. A probable reason for recurrence is the presence of genetically distinct heterogeneous tumor subpopulations (SP) with stem-like properties, exhibiting therapy resistance and heightened malignancy. We aim to identify tumor SPs responsible for therapy resistance and tumor recurrence, thereby ultimately improving treatment outcomes for GBM patients. Whether resistant SPs are present in the treatment-naïve tumor or arise due to treatment is not clear and SP changes are elusive in the complex microenvironment of chemical and radiotherapy. Here we analyze human astrocytoma tumor cells response to inhibition of BRaf and combination therapy. The purpose of the work presented herein is to determine tumor composition before and after in vivo drug administration as well as phenotypic cellular responses to in vitro drug administration. We hope to achieve increased understanding of the origins and dynamics of evolving SPs when challenged with therapy to better inform future treatment strategy. To test if Braf inhibition disrupts key indicators of stemness, we modeled in vitro and in vivo cellular response to drug treatment with a BRAF^VE human astrocytoma cell line (AM38) and PLX4720, a selective BRaf inhibitor. Nude mice were xenografted intracranially with AM38 and treated with PLX4720. Ex vivo tumor analysis revealed division mode disruption, then restoration of normal patterns of at least one subpopulation of progenitor (CD15+) cells with PLX4720 treatment. Cells are also drug-treated in vitro and analyzed on the molecular and phenotypic level for indications of stem-like properties. Preliminary flow cytometry data revealed SP composition changes with treatment. Proliferating NG2+ cells decreased with Braf^VE inhibition and CD133 and CD15 single-positive populations increased in proportion, indicating that stem and progenitor pools are disproportionately affected by treatment. Ongoing work will reveal division mode of multiple SPs and how treatment affects them.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-016. SEMAPHORIN3A REGULATES GBM STEM CELL PROLIFERATION

Dominique Higgins ¹, Mark Schroeder ¹, Bret Ball ¹, Brian Milligan ¹, Fredric Meyer ¹, Jann Sarkaria ¹, John Henley ¹

Abstract

Cancer stem cells present within Glioblastoma multiforme (GBM) are refractory to chemotherapeutics and play a large role in recurrence. Recent studies investigating Semaphorin 3A (Sema3A), classically known for its axon guidance and antiangiogenic properties, have suggested that Sema3A plays an important role in GBM growth. However, the effect of Sema3A on the proliferation of brain tumor stem cells (BTSCs) has yet to be defined. We hypothesized that Sema3A directly inhibits BTSC proliferation via Nrp1 and PlxnA1 receptors. Human GBM xenografts passaged in the flank of nude mice were harvested and grown in defined stem cell media. BTSCs were confirmed by immunofluorescence analysis of known stem cell antigens CD133 and Nestin, and tumorsphere formation. Further, immunostaining, western blotting, flow cytometry and PCR analysis revealed that BTSCs highly express Semaphorin 3A (Sema3A) and its receptors Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1). Treatment with exogenous Sema3A in quantitative BrdU and propidium iodide labeling assays, respectively, demonstrated that Sema3A significantly inhibited BTSC proliferation and did not induce cell death. This finding was corroborated by flow cytometric analysis of DNA content and quantitative immunostaining for the cell cycle protein p27, demonstrating cell cycle arrest in response to Sema3A. We next made shRNA lentiviruses, targeting either Nrp1 or PlxnA1 receptors, which had knockdown (KD) efficiencies greater than 80% as determined by qRT-PCR. KD in BTSCs abrogated Sema3A antiproliferative effects, as determined by BrdU analysis. Interestingly, loss of the receptors alone was sufficient to inhibit BTSC proliferation in vitro. This was confirmed by in vivo studies comparing tumor growth of KD and control infected BTSCs implanted into the flanks of nude mice. Taken together these findings demonstrate that Sema3A inhibits proliferation of BTSCs, potentially via a novel mechanism involving inhibition of pro-proliferative Nrp1 and PlxnA1 signaling, and may thus prove to be an attractive therapeutic target.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-017. BRAIN TUMOR STEM CELLS ADAPT TO RESTRICTED NUTRITION THROUGH PREFERENTIAL GLUCOSE UPTAKE

William Flavahan ¹, Qiulian Wu ¹, Masahiro Hitomi ¹, Nasiha Rahim ¹, Youngmi Kim ¹, Andrew Sloan ², Robert Weil ¹, Ichiro Nakano ³, Jann Sarkaria ⁴, Brett Stringer ⁵, Meizhang Li ¹, Justin Lathia ¹, Jeremy Rich ¹, Anita Hjelmeland ^1,⁶

Abstract

Regions of necrosis are a hallmark of glioblastoma and other solid tumors, and we determined how a critical component of this microenvironment, nutrient restriction, contributes to tumor progression through modulation of the tumor cell hierarchy. We demonstrate that nutrient restriction enriches for brain tumor stem cells (BTICs) due to preferential BTIC survival and adaptation of non-BTICs to low glucose through acquisition of BTIC-like phenotypes. Metabolic adaptation of BTICs includes a unique ability to uptake larger amounts of glucose using the high affinity glucose transporter, type 3 (Glut3). Glut3 is elevated in BTICs and targeting of Glut3 (SLC2A3) diminished BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in multiple cancers, including glioblastoma, suggesting that TICs co-opt this neuronal specific transporter in a broad range of tumor types. As altered metabolism represents a cancer hallmark, metabolic reprogramming may instruct the tumor hierarchy and predict poor prognosis.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-018. TARGETED THERAPY OF GLIOMA STEM CELLS AND TUMOR NON-STEM CELLS USING CETUXIMAB-CONJUGATED IRON-OXIDE NANOPARTICLES

Milota Kaluzova ¹, Simon Platt ², Marc Kent ², Alexandros Bouras ¹, Revaz Machaidze ¹, Costas Hadjipanayis ¹

Abstract

Malignant gliomas remain aggressive and lethal primary brain tumors in adults. The epidermal growth factor receptor (EGFR), is frequently overexpressed in the most common malignant glioma, glioblastoma (GBM), and remains an important therapeutic target. Glioma stem cells (GSCs) present in tumors are felt to be highly tumorigenic and responsible for tumor recurrence due to their resistance to radiation and chemotherapy. Multifunctional magnetic iron-oxide nanoparticles (IONPs) can be directly imaged by magnetic resonance imaging (MRI) and designed to therapeutically target cancer cells. The targeting effects of IONPs conjugated to the EGFR inhibitor, cetuximab (cetuximab-IONPs), were determined with EGFR- and EGFRvIII-expressing human GBM neurospheres and GSCs. Transmission electron microscopy (TEM) revealed cetuximab-IONP GBM cell binding and internalization. Fluorescence microscopy and Prussian blue staining showed increased uptake of cetuximab-IONPs in EGFR- as well as EGFRvIII-expressing GSCs and neurospheres in comparison to cetuximab or free IONPs. Treatment with cetuximab-IONPs resulted in a significant antitumor effect, greater than cetuximab alone due to more efficient cellular targeting, EGFR signaling alterations, and apoptosis induction in EGFR-expressing GSCs and neurospheres, regardless of CD133 status. A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery (CED) treatment of rodents intracranially implanted with human EGFR-expressing GBM xenografts. Translation of the cetuximab-IONP antitumor effect to the larger, more relevant spontaneous canine glioma model was performed. A single cetuximab-IONP CED treatment of two canine patients with spontaneous EGFR-expressing gliomas resulted in a marked antitumor response months after treatment.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-019. INCREASED INVASION OF GLIOMA CANCER STEM CELLS UNDER C5α FROM GLIOMA STROMA MESENCHYMAL STEM-LIKE CELLS BY MEANS OF pp38 NETWORKING

Seok-Gu Kang ¹, Se-Hoon Kim ², Yong-Min Huh ³, Eui-Hyun Kim ¹, Eun-Kyung Park ¹, Jong Hee Chang ¹, Sun Ho Kim ¹, Yong Kil Hong ⁴, Dong Seok Kim ¹, Su-Jae Lee ⁵

Abstract

The presence of glioma stroma mesenchymal stem-like cells (GS-MSLCs) from Korean glioma patients has been recently reported. How these cells function as a part of the glioma microenvironment, however, remains incompletely understood. Here, we investigated the biological effects of GS-MSLCs on glioma cancer stem cells (gCSCs), testing the hypothesis that GS-MSLCs alter the biological characteristics of gCSCs. gCSCs were cultured alone and co-cultured with GS-MSLCs or bone marrow mesenchymal stem cells (BM-MSCs). Glioneural differentiation, stemness, invasion, epithelial mesenchymal transition (EMT), and cytokine assay, were compared between the three groups (gCSCs alone vs. gCSCs with GS-MSLCs vs. gCSCs with BM-MSCs). Next, three groups of orthotopic xenografts in mice were created. Then tumor size, survival, and invasion extent were examined using immunohistochemical analyses. When we co-cultured gCSCs with GS-MSLCs, increased glioneural differentiation (increased Olig2, GFAP, and Tuj1), decreased stemness (decreased CD133, nestin, Sox2, notch2), and increased invasion/EMT (zeb1, β-catenin and N-cadherin). However there were no increased invasion/EMT with gCSCs alone group or gCSCs co-cultured with BM-MSCs group. According to cytokine assay, we found 7 cytokines (C5α, GROα, IL-6, IL-8, MCP1, MIF, PAI-1) from GS-MSLCs. Among them, C5α from GS-MSLCs may be related with increased invasion/EMT of gCSCs through siRNA study. In functional study, we finally found C5 and pp38 were key signal networking molecules. Mice co-injected with gCSCs and GS-MSLCs had significantly larger tumor size, and shorter survival then control groups. Immunohistochemical analysis showed increased glioma invasion/ EMT related expression (zeb1). We have successfully proved the biological effect of human GS-MSLCs on human gCSCs. GS-MSLCs can differentiate gCSCs into neuro-glial cells and enhance invasion by C5α and pp38. Our results indicated that GS-MSLCs may influence the biological properties of gCSCs, shifting them towards a more aggressive status. GS-MSLCs could be a new target cells for the treatment of glioma in the future.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-020. PRESENCE OF MENINGIOMA STROMAL MESENCHYMAL STEM-LIKE CELLS

Eui Hyun Kim ¹, Seok Gu Kang ¹

Abstract

Presence of cells resembling bone marrow-mesenchymal stem cells (BM-MSCs) from glioma was proved, however it has been little known about existence of MSCs from meningioma. We try to investigate whether we are able to isolate cells resembling BM-MSCs. Thus, we hypothesized that cells similar to BM-MSCs exist in meningioma specimen. We cultured fresh meningioma specimen using the same protocols for culturing BM-MSCs. The cultured cells were analyzed by fluorescence-activated cell sorting (FACS) for surface markers associated with BM-MSCs. These cells were exposed to mesenchymal differentiation conditions. To evaluate the tumorigenecity of cells resembling MSCs from meningioma, we injected these cells into the brain of athymic nude mice. To find possible locations, sections of meningioma specimens were analyzed by immunofluorescent labeling. In 5 of 17 meningioma specimens, we isolated cells similar to BM-MSCs which were called meningioma stroma mesenchymal stem-like cells (MS-MSCs). MS-MSCs were spindle shaped and adherent cells. FACS analysis showed that the MS-MSCs had similar surface marker to BM-MSCs. (Cells that were positive for two of the markers among CD105, CD90, CD73 and negative for two of the markers among CD45, CD31, NG2 were chosen to see their differentiation capability.) Chosen cells were observed for their ability to differentiate into adipocytes, osteocytes and chondrocytes. MS-MSCs immnohistological analysis indicated that were closely associated with the endothelial cells and pericytes cells. No tumor was discovered within the brains of mice that received the MS-MSCs orthotopic inoculation. Our results recognized that cells similar to BM-MSCs exist in meningioma specimen for the first time. MS-MSCs could be a source of meningioma microenvironment.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-021. GAP JUNCTION SWITCHING MAINTAINS THE TUMOR HIERARCHY IN GLIOBLASTOMA

Masahiro Hitomi ¹, Loic Deleyrolle ², Maksim Sinyuk ¹, Meizhang Li ¹, Winston Goan ¹, Balint Otvos ¹, Mark Rohaus ², Muna Oli ², Vinata Vedam-Mai ², David Schonberg ¹, Qiulian Wu ¹, Jeremy Rich ¹, Brent Reynolds ², Justin Lathia ¹

Abstract

The orchestration of coordinated processes during tumor progression requires cells to respond to external signals and direct the behavior of adjacent cells. Communication is the mechanism that empowers these relatively simple systems to achieve complexity and stability. As glioblastoma (GBM) has an elevated cellular density, we hypothesized that direct cell-cell communication was important in tumor progression. We interrogated cell junction proteins using bioinformatics interaction datasets, and found that these proteins as a whole could predict patient survival with higher expression correlating with poorer prognosis. Among the top genes to negatively correlate with survival was a gap junction family member, GBM connexin (GBMCx). When we evaluated GBMCx expression in GBM tumors, we found high GBMCx expression in a fraction of cells with the capacity to form spheres, a surrogate of self-renewal and a hallmark of the cancer stem cell (CSC) phenotype. When we compared CSCs and non-CSCs, we observed elevated GBMCx mRNA and protein in CSCs. Cx43, another gap junction family member, showed elevated expression in non-CSCs and when CSCs were differentiated, GBMCx expression was reduced while Cx43 expression increased. RNA interference abrogating GBMCx function resulted in decreased proliferation, self-renewal, and tumor initiation with a concomitant increase in Cx43, suggesting that gap junction switching is associated with tumor progression. As multiple FDA approved gap junction inhibitors are available for immediate use, we determined the impact of targeting gap junctions on GBM progression. We found these inhibitors more severely inhibited proliferation of CSCs as compared to non-CSCs and neural progenitor cells. Gap junction inhibitors decreased self-renewal and when used in established xenograft tumors, decreased tumor growth and had an additive effect when combined with Temozolomide. Taken together, our data demonstrate that gap junctions are important in tumor progression and can be targeted with inhibitors that can be immediately integrated into clinical paradigms.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-022. microRNAs INVOLVED IN ENDOTHELIAL DIFFERENTIATION OF GLIOBLASMA STEM CELLS

Soon-Tae Lee ¹, Kon Chu ¹, So-Hee Kim ¹, Sang Kun Lee ¹, Manho Kim ¹, Jae-Kyu Roh ¹

Abstract

BACKGROUND: Glioblastoma stem cells (GSCs) can contribute to angiogenesis not only by releasing pro-angiogenic factors but also by direct differentiation into endothelial cells. MicroRNAs (miRNAs) are ∼ 22-nucleotide small noncoding RNAs that control a variety of cellular and disease processes. Here, we show the involvement of miRNA in endothelial differentiation of GSCs. METHODS: GSCs were culture from human glioblastoma, maintained in sphere-forming conditions, containing epidermal growth factor and fibroblast growth factor, and differentiated in endothelial medium. Using a microarray, miRNA expression was compared in GSCs and endothelial cells differentiated from GSCs. Regulation of target gene by a selected miRNA was analyzed by in silico prediction, target gene luciferase assay, and miRNA-transfected cells. Up- or down-regulation was defined as a > 1.5-fold change in expression and a P-value < 0.05. RESULTS: By 7-day differentiation, endothelial cells differentiated from GSCs expressed endothelial markers. Seventeen miRNAs were differentially expressed in the endothelial cells (>1.5 fold, P < 0.05). Of these, miR-204 was up-regulated most significantly, and was predicted to target stemness-associated genes. Delivery of miR-204 enhanced the endothelial differentiation and blocking of miR-204 by antagomir reduced the angiogenic process. In addition, treatments with angiogenesis inhibitors blocked the endothelial differentiation of GSCs and also reduced the level of miR-204, which resulted in the maintenance of stemness genes. CONCLUSION: This study suggests that miRNAs participate in the de novo endothelial differentiation of GSCs and the maintenance of stemness after anti-angiogenic therapies.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-023. DIVERGENT EFFECTS OF BRAF^V600E EXPRESSION AND PHARMACOLOGICAL INHIBITION IN Ink4a/Arf DEFICIENT NEURAL STEM AND PROGENITOR CELLS

Robin Lerner ¹, Amelie Griveau ¹, Yuichiro Ihara ¹, Brian Reichholf ¹, Martin McMahon ¹, David Rowitch ¹, David James ¹, Claudia Petritsch ¹

Abstract

The activating BRaf^V600E mutation in combination with loss of CDKN2A has been identified in a significant subset of pediatric astrocytoma (Cancer Res. 2010; 70; 512-519). However as Braf^V600E-targeted therapies enter clinical trials for treatment of glioma, the mechanisms by which BRaf activation leads to tumor formation are still unclear. We have previously shown that disruption of asymmetric divisions in NG2⁺ oligodendrocyte progenitor cells (OPC) causes aberrant self-renewal in an EGFR-driven model of oligodendroglioma (Cancer Cell; 2011; 20; 320-40). Our earlier data further suggested a neural progenitor origin of tumors that form in response to BRaf^V600E expression in combination with Ink4a/Arf loss in young adult mouse brain (PNAS. 2012; 109; 8710-8715). Here, we investigate the molecular changes in asymmetric divisions in response to Braf^V600E expression in CDKN2A deficient stem and progenitor cell populations as they relate to pediatric astrocytoma. BRaf^V600E Ink4a/Arf-deficient neurospheres were generated by transducing neurospheres isolated from adult BRaf^CA (Genes Dev. 2007; 21; 379-84) Ink4a/Arf^loxp (Genes Dev. 2003; 17; 3112-3126) mice with adenovirus-driven Cre recombinase expression. Neurosphere cells exhibit stem and progenitor properties such as cell type specific marker expression and asymmetric cell division. Interestingly, sensitivities to BRaf^V600E expression and inhibition are cell type-specific. OPC marker NG2⁺ but not stem cell marker Prominin-1⁺ BRaf^V600E Ink4a/Arf-deficient cells exhibit decreased rates of asymmetric cell division and increased proliferation capacities. The cell-type specific disruption of asymmetric division is accompanied by differential expression of asymmetry regulating genes following Braf^V600E expression and pharmacological inhibition. Together these data describe a diverse response of stem and progenitor cells to BRaf^V600E expression. Understanding the molecular changes in actionable asymmetry regulators downstream of oncogene expression will allow intelligent design of novel treatment strategies. These novel treatments are expected to complement emerging BRafV600E-targeted therapies and conventional radiation and chemotherapy.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-024. CASEIN KINASE 2 ALPHA REGULATES GBM CANCER STEM CELL GROWTH THROUGH THE BETA-CATENIN PATHWAY

Ryan Nitta ¹, Siddartha Mitra ¹, Maya Agarwal ¹, Timothy Bui ¹, Gordon Li ¹

Abstract

Casein kinase 2 (CK2) is a highly conserved pleiotropic serine/threonine kinase that has been associated with cell survival in many cancers. Elevated levels have been shown to correlate with a worse prognosis in many solid tumors and we have preliminary data demonstrating a worse prognosis in glioblastoma (GBM) patients who exhibit high expression of a CK2 isoform, CK2alpha. Through our own work we discovered that inhibition of CK2alpha using small molecules or short interfering RNA (siRNAs) decreased proliferation and anchorage independent growth in multiple GBM cell lines confirming that CK2alpha plays an important role in GBM tumorigenesis. In this report we demonstrate for the first time that CK2alpha is an important component of GBM cancer stem cell (CSC) through the regulation of pathways necessary for glial stem cell growth and maintenance. We discovered that inhibition of CK2alpha resulted in a 5-fold and 3-fold reduction in Beta-catenin and GLI1 protein expression, respectively, and a 6-fold and 3-fold reduction in transcriptional activity. To verify that CK2alpha plays an important role in GBM CSC growth we extended our study to primary glial tumor neurospheres. We discovered that inhibition of CK2alpha activity using TBB or TBBz reduced GBM tumor neurosphere formation 10-fold, while the self-renewing capacity of the spheres were decreased 20- to 40-fold. To verify that CK2alpha is important for glial tumor sphere growth we reduced CK2alpha expression using an inducible shRNA lentiviral system. We determined that reducing CK2alpha significantly reduced tumor sphere size and decreased expression of Beta-catenin 5-fold indicating that CK2alpha is involved in GBM CSCs. Together our results indicate that CK2alpha plays an important role in glial CSC through the regulation of transcription factors linked to glial stem cell growth and maintenance. In addition, our findings suggest that CK2alpha could potentially be a viable therapeutic target or biomarker for GBM.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-025. EXPRESSION OF AJAP1 IN U87MG CELLS INHIBITS GLIOMA STEM CELL MAINTENANCE

James Lin ^1,³, Cory Adamson ^1,²

Abstract

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults and its dismal prognosis and universal recurrence have been partly attributed to the presence of invasive glioma stem cells (GSCs). The novel adherens junction associated protein 1 (AJAP1) has been shown to associate with β-catenin of the Wnt pathway, a key GSC signaling pathway. Therefore, our study aims to determine the effect of AJAP1 on GSC maintenance. Using U87 cell lines that stably express either GFP (U87-GFP) or the AJAP1-GFP fusion protein (U87-AJAP1-GFP), we quantified tumorsphere formation by growing cells for 72h in the presence of serum, and measuring the number of spheres per field (at 40X) and their average diameters. Serial passaging of tumorspheres was then preformed to verify presence of GSCs in the spheres. Finally, both cell lines were stained for CD133, a GSC marker, and analyzed using flow cytometry. Tumorsphere formation was only seen in U87-GFP cells with an average of 10 ± 2.6 tumorspheres/field and an average diameter of 106.7 ± 19.3 microns. There were no observable sphere formations by U87-AJAP1-GFP cells. Tumorsphere cells from U87-GFP could be serially passaged four times in the presence of serum before losing their ability to form spheres. CD133 staining for GSCs showed a 1.5 fold increase in fluorescence intensity (compared to unstained cells) in the U87-GFP cell line, while the U87-AJAP1-GFP cell line showed no change (P = 0.021). Our results suggest that the presence of AJAP1 in U87 cells is able to reduce GSCs to undetectable levels, evidenced by the inability of U87-AJAP1-GFP cells to form tumorspheres and their lack of CD133 expression. We believe that given AJAP1's association with β-catenin, it could function by disrupting the Wnt pathway and therefore causing GSCs to lose their stemness.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-026. THERAPEUTIC EFFICACY AND FATE OF BIMODAL ENGINEERED STEM CELLS IN MICE MODELS OF MALIGNANT BRAIN TUMORS

Jordi Martinez-Quintanilla ¹, Sung-Hugh Choi ¹, Deepak Bhere ¹, Pedram Heidari ², Derek He ¹, Umar Mahmood ², Khalid Shah ¹

Abstract

Therapeutic Stem cells (SC) engineered to secrete pro-apoptotic, antiangiogenic and anti-proliferative molecules have been used in the past few years as a tumor specific therapeutic approach for different cancer types. However, the assessment of the long-term fate and eradication of therapeutic SC post-tumor treatment is critical in order to translate those new therapies to the clinics. In this study, we have developed a dual stem cell based therapeutic strategy that allows therapeutic SCs to target tumor cell proliferation, angiogenesis or death pathways and simultaneously follow the assessment and eradication of SC post highly malignant glioblastoma multiforme (GBM) brain tumor treatment. Both human and mouse mesenchymal stem cells (MSC) were engineered to co-express the therapeutic agents, secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL), thrombospondin-1 (TSP-1) or interferon-beta, and the prodrug converting enzyme, herpes simplex virus thymidine kinase (HSV-TK). MSC-TK expressing S-TRAIL induced GBM cell death and were selectively sensitized to the prodrug ganciclovir (GCV) in vitro. A significant decrease in tumor growth and a subsequent increase in survival were observed when mice bearing highly aggressive GBMs were treated with the bimodal engineered MSC. Furthermore, their fate was monitored by positron emission-computed tomography (PET) imaging utilizing 18F-FHBG, a substrate for HSV-TK. Finally, the systemic administration of GCV post-tumor treatment selectively eliminated therapeutic MSC expressing HSV-TK. These findings demonstrate the development and validation of a dual therapeutic and safe strategy that has implications in translating stem cell based therapies in cancer patients.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-027. ISOLATING THE PRE-MALIGNANT NEOPLATIC AND NORMAL NEURAL STEM CELLS IN HUMAN GLIOBLASTOMA TO IDENTIFY THE GENOMIC DRIVERS OF TUMOR INITIATION

Siddhartha Mitra ^1,², Sharareh Gholamin ², Abdullah Feroze ¹, Achal Achrol ², Suzana Kahn ², Irving Weissman ¹, Samuel Cheshier ^2,¹

Abstract

One of the major difficulties in identifying the cell of origin for glioblastoma is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. To identify the cell of origin we decided to first dissect out the various stages of CNS stem cell differentiation in fetal and adult human brain. After a functional high throughput screen, we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90) that in various combinations can separate long-term self-renewing multi-potent neural stem cells (NSCs) from multi-potent progenitors with limited self-renewal capacity (NPC) from non-neoplatic human fetal and adult Brain (sub-ventricular zone). FACS sorted cells were analyzed for single cell lineage multi-potentiality, lineage bias and self-renewal to trace and characterize their lineage relationships. In surgical human glioblastoma samples, however we observed in-vivo tumor initiating and in-vitro tumorsphere-forming frequency was predominant in the progenitor cells and not in the neural stem cells. Interestingly we did observe a small frequency of NSC cells forming distinct lesions in the brain, which on further analysis showed the presence of both the NSC and NPC, where as the tumor initiating enriched population did not show the presence of the NSC but only tumor initiating NPCs. Taken together our data suggests the presence of "normal" or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. Using single cell RNAseq and targeted sequencing of known mutations we will identify the genomic events which can distinguish these pre-malignant neural stem cells from the tumor initiating cancer stem cells. These pre-malignant cells could comprise a cellular reservoir that may need to be targeted to prevent tumor relapse.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-028. EVOLUTION OF CANCER STEM CELLS IN GLIOMA TO PROMOTE THERAPY RESISTANT PHENOTYPE

Ichiro Nakano ¹

Abstract

High grade glioma (HGG) is a life-threatening brain tumor. Among heterogeneous tumor cells, glioma stem cells (GSCs) are defined as a subpopulation resistant to chemo- and radiotherapy with prominent tumorigenic ability. Identification of novel therapeutics designed to target the GSC population in HGGs is a challenge. The recent genome-wide transcriptome analysis identified 3 subtypes, proneural (PN), proliferative, and mesenchymal (MES), in HGGs. These HGG subtypes harbor distinct gene signatures and altered signaling pathways with some clinical relevance. During malignant transformation, HGGs appear to gain therapy resistance, at least in part, through their phenotypic drift into mesenchymal tumors. In addition, some of the current therapies, if not all, appear to induce mesenchymal transformation of HGGs resulting in more therapy-resistant tumors. Nonetheless, identification of subtype-specific GSCs remains elusive. My lab recently identified clinical HGG sample-derived two mutually-exclusive GSCs: PN and MES GSCs with striking phenotypic differences with clinical relevance (Mao et al., PNAS 2013). Transcritpome microarray analysis, miRNA profiling, and subsequent functional analysis demonstrated that distinct signaling pathways regulate tumorigenesis and propagation of the individual GSC subtypes. For example, PN GSCs are dependent on the oncogenic transcription factor c-Myb (Miyazaki et al., Clinical Cancer Res. 2012) and MES GSCs are dependent on the oncogene/mitotic kinase MELK and Survivin (Guvenc et al., Clinical Cancer Res. 2012; Gu et al., Stem Cells 2013; Joshi et al., Stem Cells 2013). Intriguingly, upon radiation treatment, PN GSCs gain the phenotypes of MES GSCs (PN-to-MES transformation; PMT), which are much more aggressive and radioresistant than original PN GSCs. Of note, targeting the MES-specific pathways partially blocks PMT of GSCs. These results may patially explain the molecular mechanisms underlying mesenchymal transformation of HGGs to promote therapy resistance. Here I summarize our latest data to elucidate the molecular mechanisms that drive GSC evolution to promote their therapy resistant phenotype.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-029. INTEGRATION OF GENE AND PROTEIN EXPRESSION IN THIRTY-SEVEN GLIOMA STEM CELL LINES

Erik P Sulman ¹, Qianghu Wang ², Ekaterina Mostovenko ³, Huiling Liu ³, Cheryl F Lichti ³, Alexander Shavkunov ³, Roger A Kroes ⁴, Joseph R Moskal ⁴, Charles A Conrad ⁶, Frederick F Lang ⁷, Mark R Emmett ⁵, Carol L Nilsson ³

Abstract

Glioma-derived cancer stem-like cells (GSCs) are hypothesized to provide a repository of cells in tumor cell populations that can self-replicate and be refractory to radiation and chemotherapeutic agents developed for the treatment of these tumors. The potential resistance GSCs to traditional cytotoxic and radiation therapies has significant implications for tumor biology and therapeutics. Improved understanding of biomolecular expression correlated to patient responses to therapy and clinical outcomes could improve the future standard of care for glioma patients. We assembled a panel of GSCs that reflect the biological heterogeneity of human malignant gliomas. Thirty-seven GSC lines isolated at The University of Texas M.D. Anderson Cancer Center from fresh surgical specimens and representing several molecular sub-types of malignant gliomas were isolated and characterized with respect to gene expression by RNAseq. Targeted transcriptomic analyses were performed at the Falk Center for Molecular Therapeutics (Evanston, IL). The systems biology team at The University of Texas Medical Branch performed quantitative measurements of protein expression in the cell lines, relative to a global proteomic standard comprised of a defined mixture of proteins derived from all 37 cell lines. The use of an external global proteomic standard allows for unlimited quantitative comparisons between any two cell lines. Transcriptomic signature was correlated to proteomic expression to develop a comprehensive, functional landscape of the GSCs. Here, we present our first insights into differences in GSC biology based on integrated proteomic and transcriptomic data. Molecular annotation of these GSCs will facilitate their use as models for novel treatment identification. Support from the Cancer Prevention Research Institute of Texas, The University of Texas Medical Branch, the Falk Foundation (Chicago), and The University of Texas M.D. Anderson Cancer Center is gratefully acknowledged.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-030. THE ROLE OF IGF1 RECEPTOR SIGNALING IN ADAPTIVE RADIOPROTECTION OF GLIOMA STEM CELLS

Satoru Osuka ¹, Oltea Sampetrean ¹, Takatsune Shimizu ^1,², Isako Saga ¹, Nobuyuki Onishi ¹, Eiji Sugihara ¹, Jun Okubo ^1,³, Satoshi Fujita ^1,⁴, Shingo Takano ⁵, Akira Matsumura ⁵, Hideyuki Saya ¹

Abstract

BACKGROUND: Cancer stem cells play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as quiescence and high DNA repair capability. It is unclear, however, how cancer stem cells further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes. METHODS: Initial tumors were formed by implantation of Ink4a/Arf null neural stem cells overexpressing H-RAS^V12 into the forebrain of wild-type mice. GSCs purified from the tumors were then grown as tumorspheres (TS), with a subgroup, TS-RR, surviving repeated radiation (12x 5Gy). The two types of cells and allografts were compared to identify differentially expressed factors that underlie acquired radioresistance. RESULTS: Analysis of the molecular changes induced in TS during fractionated radiation revealed an increase in IGF1 secretion and a gradual up-regulation of the IGF type 1 receptor (IGF1R). Interestingly, IGF1R up-regulation exerted a dual radioprotective effect: in the resting state, continuous IGF1 stimulation ultimately induced down-regulation of Akt/ERK and FoxO3a activation, which resulted in slower proliferation and enhanced self-renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promoted a rapid shift from a latent state towards activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity. CONCLUSION: Our results show that GSCs can evade the damage of repeated radiation not only through innate properties, but also by establishing an IGF1-IGF1R autocrine trophic loop. Elucidation of stem-cell-specific adaptive radioprotection mechanisms and identification of targetable key factors are crucial to the refinement of radiosensitizing strategies and prevention of tumor relapse.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-031. PTEN REGULATES GBM'S SENSITIVITY TO γ-SECRETASE INHIBITORS: IMPLICATING THE NEED FOR SIMULTANEOUS INHIBITION OF PI3K AND NOTCH PATHWAYS IN PTEN MUTANT GBMS

Norihiko Saito ¹, Jun Fu ¹, Shuzhen Wang ¹, W K Alfred Yung ¹, Dimpy Koul ¹

Abstract

NOTCH pathway regulates normal stem cells in the brain and glioma initiating cells (GICs) with high NOTCH activity. Blocking the proteolytic activation of NOTCH by γ-secretase inhibitors (GSIs) seems to be active in only a fraction of GICs (responders) with constitutive NOTCH activity. We further observed that within the NOTCH activated group there is a selective response of PTEN-WT cells to GSIs. Therefore an understanding of the mechanisms for sensitivity to GSIs would be critical for future drug development. Here we report that loss of PTEN function as a critical event leading to decreased response to GSIs, which causes the transfer of "oncogene addiction" from the NOTCH to the PI3K pathway. We studied the effects of NOTCH pathway inhibition in GICs, by treating a panel of high NOTCH pathway, PTEN-mutant and PTEN-WT GICs with GSIs. PTEN-mutant GICs seems to be less sensitive to GSIs treatment as compared to PTEN-WT GICs. Further investigation showed that in PTEN-WT GICs, GSIs increase PTEN expression and thereby downregulate the activity of the PI3K/AKT pathway. We showed that GSI treatment attenuated NICD, Hes-1, Hes-3, and Hes-5 and increases PTEN expression. Further, NOTCH seems to regulate PTEN expression via Hes-1, since knockdown of either NOTCH or Hes-1 led to increased PTEN expression. We further showed that PTEN reconstitution in PTEN-mutant cells increases the sensitivity to GSIs confirming that PTEN is complimentary to GSIs mediated cellular effects. In contrast, PTEN knockdown decreases the response of GICs to GSIs in PTEN-WT GICs. Our preliminary data reveal the synergistic attenuation of cell growth using combination of GSIs and PI3K inhibitors in PTEN-mutant GICs. These results indicate that PTEN is an important mediator of GSIs induced attenuation of cell growth through a regulatory circuit linking NOTCH signaling with PTEN expression, providing the basis for combination therapeutic strategies in GBMs.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-032. THE ROLE OF REGIONAL ASTROCYTE IDENTITY IN ASTROCYTOMA GENOMIC HETEROGENEITY

Ralf S Schmid ¹, David M Irvin ¹, Mark Vitucci ¹, Ryan E Bash ¹, Andrea M Werneke ¹, C Ryan Miller ¹

Abstract

The cells from which astrocytomas originate have remained elusive. We previously showed that human GFAP-CreER-driven inactivation of Rb, Kras activation, and Pten deletion induces low-grade astrocytoma (LGA) tumorigenesis throughout the adult brain. To define the cellular origin of these LGA and map the fates of transformed cells, we used genetic lineage tracing with a floxed TdTomato reporter and labeled proliferating cells with EdU. In the absence of oncogenic mutations, hGFAP-CreER induced recombination in ∼50% of GFAP⁺/BLBP⁺ astrocytes in the cortex, diencephalon, brainstem, and olfactory bulb (4 to 6% of all DAPI⁺ cells) and 0.01-0.05% of these cells were actively proliferating after 7 days. Astrocyte proliferation increased 70 to 340-fold 3 weeks after induction of oncogenic mutations and 39-46% of cells in these regions were TdTomato⁺ by 8 weeks. Similar results were obtained when these oncogenic mutations were induced by Glast-CreER. In both GFAP- and Glast-CreER mice, transformed astrocytes maintained their astrocytic identity (GFAP/BLBP expression) and formed increasingly perineuronal satellites over time. Expression profiling of 8 week LGA showed that olfactory bulb and forebrain LGA had distinct transcriptomes. EdU and Ki-67 labeling demonstrated clonal expansion of transformed astrocytes, as hypercellular foci with a 3 to4-fold increase in proliferation relative to less-cellular areas of tumor developed by 8 weeks. aCGH of ≥ 2 spatially distinct high-grade astrocytomas (HGA) masses in 8 individual mice showed divergent landscapes of DNA copy number abnormalities after malignant progression. Transcriptomal analysis of terminal HGA showed that their transcriptome profiles correlated with brain region, suggesting that astrocytes within these four brain regions can be transformed by identical oncogenic mutations to produce genomically-distinct HGA. These findings support the notion that cell of origin is reflected in the genomic profiles of astrocytomas and that malignant progression occurs through clonal expansion of transformed astrocytes upon the stochastic acquisition of additional mutations.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-033. TGF-β MEDIATES HOMING OF BONE MARROW-DERIVED HUMAN MESENCHYMAL STEM CELLS TO GLIOMA STEM CELLS

Naoki Shinojima ^4,¹, Anwar Hossain ^1,³, Tatsuya Takezaki ^4,¹, Juan Fueyo ^2,³, Joy Gumin ^1,³, Feng Gao ^1,³, Felix Nwajei ^1,³, Frank C Marini ⁵, Michael Andreeff ⁵, Jun-Ichi Kuratsu ⁴, Frederick F Lang ^1,³

Abstract

Although studies have suggested that bone-marrow human mesenchymal stem cells (BM-hMSCs) may be used as delivery vehicles for cancer therapy, it remains unclear whether BM-hMSCs are capable of targeting cancer stem cells, including glioma stem cells (GSCs), which are the tumor-initiating cells responsible for treatment failures. Using standard glioma models, we identify TGF-β as a tumor-factor that attracts BM-hMSCs via TGF-β receptors (TGFβR) on BM-hMSCs. Using human and rat GSCs, we then show for the first time that intravascularly administered BM-hMSCs home to GSC-xenografts that express TGF-β. In therapeutic studies, we show that BM-hMSCs carrying the oncolytic adenovirus Delta-24-RGD prolonged the survival of TGF-β-secreting GSC-xenografts and that the efficacy of this strategy can be abrogated by inhibition of TGFβR on BM-hMSCs. These findings reveal the TGF-β/TGFβR-axis as a mediator of the tropism of BM-hMSCs for GSCs, and suggest that TGF-β predicts patients in whom BM-hMSC delivery will be effective.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-034. GLIOMA STEM CELL SPECIFIC microRNA-mRNA INTERACTION NETWORK

Sanjay Singh ¹, Kelly Burrell ¹, Elizabeth Koch ², Sameer Agnihotri ¹, Shahrzad Jalali ¹, Alenoush Vartanian ¹, Joy Gumin ³, Erik Sulman ⁴, Frederick Lang ³, Bradley Wouters ², Gelareh Zadeh ¹

Abstract

The role of cancer stem cells in tumor formation and tumor heterogeneity is currently one of the most researched topics in cancer biology. A better understanding of molecular mechanisms regulating the biology of cancer stem cells may ultimately help provide a better management of cancer patients. Various individual or families of microRNAs have been shown to have oncogenic or tumor suppressor function in glioblastoma (GBM). It is postulated that there exists an extensive microRNA mediated RNA-RNA interaction network in GBMs utilizing systems biology approach supporting a competitive endogenous RNA (ce-RNA). MicroRNAs have functional relevance in regulation of critical genes and pathways implicated in maintenance of glioma stem cell (GSC) properties. To avoid inclusion of inherent bias of miRNA-target prediction algorithms, we have applied biochemical methods to establish direct miRNA-mRNA interaction network relevant and specific to GSCs. We have generated an unbiased global miRNA mediated RNA-RNA interactome by performing RNA-sequencing all RNA species (small and large RNAs) isolated from AGO2-miRISC (microRNA-induced silencing complex) of GSCs and normal human neural stem cells (hNSCs). Additionally, we have also established this interactome after exposure of GSCs and normal hNSCs to hypoxia, a key tumor micro-environmental factor that is known to be pivotal in generating GBM heterogeneity. The rank order list of miRNA-mRNA interaction nodes generated from RNA sequence reads reveals that enrichment of specific RNAs in functional AGO2-miRISC is not a direct function of their relative abundance in cells, thus this biochemically generated interactome is distinct from that generated by bioinformatics tools. We demonstrate that scope and influence of GSC specific miRNA-mRNA network and specific nodes of this interactome varies with hypoxia and tumor region in GBMs.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-035. TARGETING THE THERAPEUTIC RESISTANCE AND PRONEURAL –MESENCHYMAL SHIFT IN GBM USING NON-TOXIC MODULATORS OF ROS

Renza Spelat ¹, Eric Singer ¹, Lisa Matlaf ¹, Sean McAllister ¹, Liliana Soroceanu ¹

Abstract

Our group has recently shown that treatment with a non-toxic ID1 inhibitor, cannabidiol (CBD), could inhibit glioma dispersion and progression in vivo and downregulate self-renewal and expression of stemness markers in primary glioma stem cells (GSCs) grown as neurospheres. Our recent data demonstrate that the effects of CBD are directly related to the induction of ROS in glioma cells. Using analyses at the mRNA and protein level as well as functional studies, we recently found that CBD treatment induced a compensatory cell survival program in GSC, characterized by upregulation of the XC system (which is critical in maintaining the redox status in GBM cells) and induction of a proneural- mesenchymal shift. These results suggest that ID1 inhibition alone may not be sufficient to produce sustained inhibition of glioma growth and that combining CBD with additional compounds targeting the XC system may improve antitumor efficacy. To date, we show that CBD and the XCT inhibitor Sulfasalazine (SAS) can act additively to inhibit GBM aggressiveness as measured by tumor cell survival, neurosphere growth, and invasion in culture. In vivo, CBD alone demonstrated anti-tumor efficacy in two primary GSC models of glioma. Studies testing the combination of CBD with SAS in vivo are currently underway. Our data also suggest that response to therapy (i.e., CBD) correlates with specific GBM molecular signatures, which can have implications on patient stratification for testing novel combinatorial therapies. Taken together, our results demonstrate that non-toxic compound CBD can be used alone or in combination with other FDA approved therapies (SAS) to inhibit GBM growth and prevent GSC reprogramming underlying therapeutic resistance.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-036. STEM CELL-LIKE GBM SUBPOPULATIONS EXHIBIT ALTERED TELOMERE STABILIZATION EFFICACY, MGMT PROMOTER METHYLATION AND DRUG RESPONSE PATTERN

Sabine Spiegl-Kreinecker ¹, Daniela Loetsch ^2,³, Magdalena Laaber ¹, Christoph Schrangl ^2,³, Adelheid Wöhrer ^4,³, Johannes Hainfellner ^4,³, Christine Marosi ^5,³, Josef Pichler ⁶, Serge Weis ⁷, Gabriele Wurm ¹, Georg Widhalm ^8,³, Engelbert Knosp ^8,³, Walter Berger ^2,³

Abstract

Glioblastoma multiforme (GBM), the most common malignant brain tumor, is characterized by high invasiveness and treatment resistance. Stem-cell like GBM subpopulations (GSCLS) are believed to cause tumor initiation and treatment resistance. Aim of this study was to analyse GSCLS derived from glioma cell lines and surgical specimen regarding gene expression pattern, telomere stabilization mechanisms and response to anticancer drugs. Therefore neurosphere forming GSCLS were compared to their adherently growing cell counterparts. Freshly resected GBM tissues (n = 36) and glioblastoma cell lines (N = 7) were in parallel cultured both serum-free as neurospheres and as adherent monolayers. Whole genome mRNA expression pattern were established on the Agilent platform. Telomerase activity and hTERT gene expression were investigated using the TRAP assay and RT-PCR, respectively. MGMT promoter methylation and gene expression was verified using pyrosequencing and Western blot. Thirty three percent (12/36) and 3/7 (40%) of the GBM tissues and cell lines, respectively, grew as neurospheres correlating with enhanced expression of stem cell markers (CD133, nestin, SOX2) in the tumor tissue and with lower patient age (t-test; p < 0.05). Moreover telomerase activity was shown to be enhanced in GSCLS. Surprisingly, hTERT mRNA expression was lower in GSCLS than in their adherent monolayer counterparts. Additionally, MGMT promoter methylation was upregulated in GSCLS and accompanied by lack of MGMT mRNA expression. In contrast, GSCLS tended to by slightly resistant towards temozolomide. This might possibly be attributed to the 3-dimensional compact neurosphere morphology. In contrast, however, GSCLS were hypersensitive against the multi-kinase inhibitor sorafenib and the epidermal growth factor inhibitor erlotinib. Summarizing we demonstrate that cancer stem cell subpopulations capable of spheroid formation exhibit altered telomerase activity regulation, enhanced MGMT promoter methylation and hypersensitivity against kinase inhibitors targeting oncogenic receptor tyrosine kinases.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-037. BONE MARROW DERIVED HUMAN MESENCHYMAL STEM CELLS (hMSCs) LOCALIZE SPECIFICALLY IN THE HUMAN PRIMARY GLIOBLASTOMA XENOGRAFTS BY INTRAVASCULAR INJECTION AND IRRADIATION DRIVES THE HOMING OF hMSCs

Tatsuya Takezaki ^1,², Naoki Shinojima ^1,², Jun-ichi Kuratsu ¹, Frederick Lang ²

Abstract

We show for the first time that bone marrow derived human mesenchymal stem cells localize specifically in most primary glioblastoma xenografts by intravascular injection and radiation drives the hMSC homing to the xenografts. Until now, the clinical applicability of hMSCs, like other cellular delivery systems, is still uncertain because true capacity of hMSCs to localize to glioblastoma in patients is unknown. We exploit a clinically pertinent in vivo model of human glioblastoma, which faithfully recapitulate phenotype and genotype of patient glioblastomas by serially maintained in vivo. Significantly, 5 of 5 intracranial xenografts newly established from surgical specimens at MD Anderson Cancer Center and 18 of 20 samples given from Mayo Clinic attracted hMSCs injected intravascularly. There is still heterogeneity of capability of hMSCs localization. To enhance the tropism of hMSCs, perturbation of xenografts by irradiation was performed. The efficacy of hMSCs homing was enhanced, furthermore, even non-attracting xenografts attracted hMSCs to some extent after irradiation. These facts indicate that hMSCs have great chances for their clinical application as a noble treatment for glioblastoma patients.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-038. ENHANCING RETROVIRAL REPLICATING VECTOR-MEDIATED PRODRUG ACTIVATOR GENE THERAPY WITH ENGINEERED HUMAN MESENCHYMAL STEM CELLS IN ANIMAL MODELS OF GLIOMA

Quincy Tam ¹

Abstract

Retroviral replicating vectors (RRVs) achieve efficient tumor transduction through selective viral replication in cancer cells. Toca 511, an improved RRV expressing an optimized version of the yeast cytosine deaminase (CD) prodrug activator enzyme, is currently being evaluated in multicenter first-in-human Phase I/II clinical trials for patients with recurrent high grade glioma, using either stereotactic intratumoral injections of virus (www.clinicaltrials.gov, NCT01156584), or multiple virus injections into the post-resection tumor bed (NCT01470794). To enhance the kinetics of intratumoral RRV dissemination, we have engineered human mesenchymal stem cells (MSC) as tumor-homing cellular carriers that produce and release RRV. Multiple human MSC isolates from different commercial sources and tissues of origin (bone marrow, adipose, cord blood) or from clinical specimens (fetal liver) were engineered to produce RRV (MSC-RRV). Cytoxicity assays confirmed efficient prodrug activator function in U87 glioma cells transduced with vectors from MSC-RRV. To evaluate intratumoral migration activity and tumor-homing migration activity in vivo, each MSC isolate was labeled with luciferase or DiI, and injected either directly into U87 human glioma xenografts, or contralateral to the intracerebral U87 tumors. MSC-RRV isolates showing the highest levels of intratumoral migration activity were selected, and the efficiency of intratumoral dissemination and tumoricidal activity achieved by these isolates was compared in subcutaneous glioma models against intratumoral bolus injection of RRV virus alone. Compared to RRV alone, MSC-RRV achieved more rapid virus spread and enhanced tumor growth inhibition at early time points. Notably, no difference in systemic biodistribution and no evidence of hematopoietic genotoxicity was observed with either delivery method, indicating an acceptable safety profile for MSC-RRV. Studies are currently on-going to further investigate these findings in orthotopic intracranial glioma models, to evaluate the potential of MSC-RRV for human studies.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-039. STRONG THERAPEUTIC POTENTIAL OF γ-SECRETASE INHIBITOR MRK003 FOR GBM STEM-LIKE CELLS WITH HIGH CD44 AND LOW CD133 EXPRESSION

Shingo Tanaka ^1,², Mitsutoshi Nakada ¹, Daisuke Yamada ², Ichiro Nakano ³, Tomoki Todo ⁴, Yutaka Hayashi ¹, Jun-ichiro Hamada ¹, Atsushi Hirao ²

Abstract

BACKGROUND: Glioblastoma (GBM) is the most malignant brain tumor with worst clinical outcome. Notch signal is important for cancer stem/initiating cells to maintain stemness, induce cell proliferation and regulate apoptosis. Notch signal inhibition by γ-secretase inhibitor may be effective strategy for the treatment of cancer stem/initiating cells. MATERIALS AND METHODS: We analyzed 9 patient's derived GBM stem-like cells with treatment by MRK003, a novel clinically available γ-secretase inhibitor. The effects of MRK003 were assessed by viability assay, apoptosis assay and sphere forming assay. Akt dependent effect of MRK003 was evaluated by rescue assay with myristoylated Akt vector. The expression ratio of CD44 and CD133 in 9 GBM stem-like cells were quantified by flow cytometric analysis. RESULTS: MRK003 suppressed proliferation and stemness maintenance, and induced apoptosis in all species of GBM stem-like cells. Based on the sensitivity for MRK003, 9 GBM stem-like cells could be clearly divided into two types; high-sensitive and low-sensitive cells. Sensitivity for MRK003 corresponded to the inhibitory ability of Akt pathway which is associated with notch downstream signal. Transfection of myristoylated Akt vector into high-sensitive cells incompletely abrogated the effect of MRK003, suggesting that the effect of MRK003 partially depended on the inhibition of Akt pathway. High-sensitive cells expressed CD44 with high percentage (87.9-100%) and CD133 with low percentage (0-33.4%), whereas low-sensitive cells expressed CD44 with broad percentage (0.24-87.7%) and CD133 with high percentage (84.2-99.4%). IC50 of MRK003 in GBM stem-like cells was significantly correlated with the expression percentages of CD44 and CD133 expression (r = -0.865, r = 0.712, respectively). CONCLUSIONS: Our data suggested that MRK003 might be effective for the GBM stem-like cells with high CD44 and low CD133 expression (mesenchymal-like subtype).

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-040. REGULATION OF GLIOMA STEM CELL GENE EXPRESSION AND PHENOTYPE BY KRÜPPEL-LIKE FACTOR 9 TRANSCRIPTION FACTOR

Jessica Tilghman ¹, Mingyao Ying ², John Laterra ²

Abstract

Tumor-initiating stem-like cells (alternatively called cancer stem cells, CSCs) are a subpopulation of tumor cells that play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is becoming increasingly important to understand the molecular signaling that regulates the self-renewal and differentiation of CSCs. Transcription factors are critical for the regulation of normal and neopolastic stem cells. The Krüppel-like family of transcription factors (KLFs) consists of 17 evolutionarily conserved zinc finger containing proteins with diverse regulatory functions. KLFs bind to GC-GT rich sites (basic transcription element, BTE) in gene promoter and enhancer regions. One KLF family member, KLF4, is known for its capacity to induce Notch1 expression and to drive differentiated cells to a pluripotent stem-like state . We recently identified KLF9 as a relatively unique KLF family member that induces glioma neurosphere cell differentiation and inhibits the growth of tumor xenografts derived from human glioblastoma neurospheres. How KLF9 contributes to cell differentiation is still unknown. We performed ChIP-seq and RNA-seq analyses to identify KLF9 gene targets in GBM-derived neurosphere cells. Over 3,000 candidate KLF9 target genes coding for proteins with diverse functions including regulators of neural development, Notch signaling, and cell-cell/cell-matrix interactions were identified. The expression of one candidate KLF9 target gene ITGA6 coding for integrin α6, a cell surface receptor associate with neoplastic cell stemness, was verified to be down-regulated by KLF9 in human GBM stem cells. ChIP-PCR confirmed KLF9-binding to ITGA6 BTE promoter sites. ITGA6 transcription repression by KLF9 altered GBM neurosphere cell behavior as evidenced by reduced cell adhesion to and migration through the integrin α6 ligand laminin. Forced expression of integrin α6 partially rescued GBM neurosphere cells from the differentiating and adhesion/migration-inhibiting effects of KLF9. These results suggest that KLF9 regulates cancer stem cell phenotype in part by altering integrin α6-dependent functions.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-041. TARGETING GLIOBLASTOMA CANCER STEM CELLS VIA MITOTIC KINESINS

Monica Venere ¹, Cathleen Chang ¹, Qiulian Wu ¹, Matthew Summers ², Steven Rosenfeld ², Jeremy Rich ^1,³

Abstract

Glioblastoma stem cells (GSCs), the tumor-propagating subpopulation within the neoplastic cellular hierarchy, contribute to treatment resistance and ultimately tumor recurrence. Consequently, there is a need for new therapies that target not only the bulk of the tumor but also this treatment-resistant subpopulation. We find that the process of mitosis in GSCs is dysregulated. In particular, the mitotic kinesin Eg5, essential for bipolar spindle formation and progression through mitosis, is consistently overexpressed in GSCs, compared to non-GSCs. Furthermore, unlike normal cells or non-GSCs, Eg5 levels in GSCs do not fluctuate through the course of the cell cycle, implying that GSCs have a fundamental defect in regulating their mitotic machinery. Conventional anti-mitotics, such as the microtubule inhibitors paclitaxel and vincristine, have failed to demonstrate efficacy for gliomas. However, newly developed targeted anti-mitotic therapies warrant a reevaluation of adjuvant anti-mitotic chemotherapy for GBM. Highly specific inhibitors of Eg5 are available and several have moved to phase I and II clinical trials in patients with solid malignancies. We find that GSCs exhibit a greater sensitivity to the Eg5 inhibitor ispinesib compared to non-GSCs from the same tumor. Importantly, Eg5 inhibition compromises the tumor initiation capability of the GSCs as well as tumor progression in an orthotopic xenograft model. We therefore propose that the mitotic machinery may provide a novel therapeutic target for GSCs, and that Eg5 inhibitors, specifically, may hold promise in preventing treatment resistance and recurrence in GBM.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-042. ESTABLISHMENT OF GENETICALLY DISTINCT BRAIN TUMOR STEM CELLS FROM GLIOBLASTOMA BEFORE AND AFTER MOLECULAR TARGETED THERAPY

Shota Tanaka ¹, Samantha Luk ¹, Clarice Chang ¹, John Iafrate ¹, Daniel Cahill ¹, Robert Martuza ¹, Samuel Rabkin ¹, Andrew Chi ¹, Hiroaki Wakimoto ¹

Abstract

Cellular heterogeneity within a tumor represents one of the mechanisms by which tumors escape molecularly targeted therapy. Establishing a cell-based model that preserves the unique molecular characteristics of the tumor from which the cells were derived will provide an opportunity to better understand resistance and develop strategies for overcoming recurrence. In this study, we isolated and established a matched pair of stem-like cell cultures from a patient glioblastoma before and after EGFR inhibitor treatment, and analyzed the phenotypic and molecular characteristics of the stem-like cells and compared them with the patient tumors. The patient presented with recurrent glioblastoma (MGG70R), which was resected and found to have focal, high-level EGFR gene amplification. After treatment with 2 cycles of an irreversible EGFR inhibitor, PF-00299804, the tumor recurred again (MGG70RR). The surgical specimen now showed diploid EGFR carried by all tumor cells, suggesting inhibitor-mediated elimination of EGFR-amplified tumor cells and the survival and propagation of EGFR non-amplified cell subsets. Neurosphere-forming tumorigenic stem-like lines MGG70R-GSC and MGG70RR-GSC were established from MGG70R and MGG70RR, respectively. MGG70R-GSC retained high level EGFR amplification, and overexpressed EGFR and phosphorylated EGFR, while MGG70RR-GSC lacked EGFR amplification and expressed lower levels of EGFR and phosphorylated EGFR both in vitro and in vivo. MGG70R-GSC proliferated faster in vitro, and generated larger and more proliferative intracerebral tumors in SCID mice than MGG70RR-GSC, mirroring the observation that patient MGG70R is more proliferative than MGG70RR. Furthermore, in vitro MGG70R-GSC was significantly more sensitive to EGFR inhibitors than MGG70RR-GSC, while PI3Kinase inhibitors were equally cytotoxic to MGG70R-GSC and MGG70RR-GSC. Thus, we established molecularly distinct GSC lines over the clinical course of glioblastoma evasion of molecular targeting, and demonstrated GSC recapitulation of a “subpopulation switch” seen in a patient who received EGFR inhibitor. Our findings provide insights into targeting glioblastomas that progress after EGFR inhibitors.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-043. THYMOSIN β4 GENE SILENCING DECREASES STEMNESS AND INVASIVENESS IN GLIOBLASTOMA

Hans-Georg Wirsching ^1,⁴, Shanmugarajan Krishnan ^1,⁴, Karl Frei ^2,⁴, Niklaus Krayenbühl ², Guido Reifenberger ³, Michael Weller ^1,⁴, Ghazaleh Tabatabai ^1,⁴

Abstract

Thymosin β4 (TB4) is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. In the developing brain, TB4 gene silencing promotes differentiation of neural progenitor cells and TB4 overexpression inhibits neuroglial apoptosis and promotes an expansion of the proliferating cell pool. In colon cancer cells, TB4 overexpression promotes malignant transformation and inhibits apoptosis. However, published data on the role of TB4 in malignant gliomas is not yet available to date. TB4 expression increased with the grade of malignancy in gliomas and was correlated with patient survival in both, a tissue microarray and the TCGA glioma dataset. In vitro, lentiviral TB4 gene silencing decreased migration, invasion and growth, and promoted apoptotic cell death in LNT-229 and U87MG cells. In glioma initiating cells, TB4 gene silencing inhibited self-renewal and promoted differentiation. In vivo, survival of nude mice bearing tumors derived from TB4-depleted glioma cells was improved and the tumorigenicity of glioma-initiating cells was decreased. An Affymetrix^™ genome-wide mRNA expression array was performed to unravel shifts in the gene expression pattern upon TB4 gene silencing. TB4 gene silencing inhibited the expression of mesenchymal signature genes and promoted expression of pro-neural signature genes. A STRING interactome analysis of differentially regulated transcription factors yielded two major clusters involving TGF-β and p53 signalling networks. We conclude that TB4 is a regulator of malignancy and may serve as a therapeutic molecular target in glioblastoma.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii206–iii216.

SC-044. IMPROVING RADIOSENSITIZATION OF GLIOBLASTOMA INITIATING CELLS WITH HYPERTHERMIA BY DOWNREGULATION OF PI3K-AKT

Jianghong Man ¹, Jocelyn Shoemake ¹, Monica Venere ¹, Jeremy Rich ¹, Jennifer Yu ¹

Abstract

INTRODUCTION: Glioblastoma is the most common and lethal primary brain tumor. Glioblastoma initiating cells (GICs) are a subpopulation of cells that activate pro-survival pathways to resist chemoradiation. The PI3K-AKT pathway is frequently activated in GICs. We hypothesized that hyperthermia improves radiosensitivity of GICs through downregulation of AKT signaling. METHODS: GICs were isolated from patient specimens and functionally validated. GICs were sham-treated, or treated with hyperthermia (42.5C, 1 hr), radiation (2 Gy/1fraction), or thermoradiotherapy (42.5C, 1 hr followed by 2 Gy/1 fraction). We interrogated cell proliferation, apoptosis, DNA repair, PI3K-AKT and RAS-MAPK pathway components. RESULTS: Radioresistance of GICs correlated with increased PI3K-AKT signaling. The addition of hyperthermia improved radiosensitivity and correlated with downregulation of AKT signaling. pERK levels were similar between all treatment groups. Introduction of constitutively activated AKT (myristoylated AKT) rescued radiosensitization by hyperthermia. Pharmacologic inhibition of PI3K further improved thermoradiosensitivity. CONCLUSIONS: Hyperthermia improved radiosensitivity through downregulation of AKT signaling. These studies suggest that combined thermoradiotherapy combined with pharmacologic PI3K-AKT signaling may further improve glioblastoma therapy.

PERMALINK

STEM CELLS

SC-001. ROLES OF GLIOBLASTOMA STEM CELL-DERIVED VASCULAR PERICYTES

Lin Cheng

Zhi Huang

Wenchao Zhou

Qiulian Wu

Jeremy Rich

Shideng Bao

Abstract

SC-002. SILENCING BMI1 ELIMINATES TUMOR FORMATION OF PEDIATRIC GLIOMA CD133+ CELLS NOT BY AFFECTING INK4A AND ARF BUT BY DOWN-REGULATING STAU2, ALDH3A, API5, EIF4G2 AND RPS6KA2

Patricia Baxter

Hua Mao

Xiumei Zhao

Zhigang Liu

Yulun Huang

Horatiu Voicu

Sivashankarappa Gurusiddappa

Jack M Su

Laszlo Perlaky

Robert Dauser

Hon-chiu Eastwood Leung

Karin M Muraszko

Jason A Heth

Xing Fan

Ching C Lau

Tsz-Kwong Man

Murali Chintagumpala

Xiao-Nan Li

Abstract

SC-003. IDENTIFICATION OF GLIOBLASTOMA CANCER STEM-LIKE CELL-BINDING HUMAN ANTIBODIES VIA YEAST EXPRESSION LIBRARY BIOPANNING

Paul Clark

Michael Zorniak

Yongku Cho

Xiaobin Zhang

Daniel Walden

Eric Shusta

John Kuo

Abstract

SC-004. THE SMG1 KINASE IS REQUIRED FOR GLIOBLASTOMA STEM CELL GROWTH IN HYPOXIA

Sejuti Sengupta

Surbhi Goel-Bhattacharya

Shreya Kulkarni

Brent Cochran

Abstract

SC-005. GLIOBLASTOMA STEM- AND PROGENITOR-LIKE CELLS: SIZE MATTERS

Carlo Cusulin

Artee Luchman

Sam Weiss

Abstract

SC-006. GROWTH FACTOR-DEPENDENT DIVERGENCE OF STEM CELL IDENTITY IN GLIOBLASTOMA

Megan Wu

Nestor Fernandez

Sameer Agnihotri

Roberto Diaz

James Rutka

Markus Bredel

Jason Karamchandani

Sunit Das

Abstract

SC-007. EphA3 MAINTAINS TUMORIGENICITY AND IS A THERAPEUTIC TARGET IN GLIOBLASTOMA MULTIFORME

Bryan Day

Brett Stringer

Fares Al-Ejeh

Michael Ting

John Wilson

Kathleen Ensbey

Paul Jamieson

Zara Bruce

Yi Chieh Lim

Carolin Offenhauser

Sara Charmsaz

Leanne Cooper

Jennifer Ellacott

Angus Harding

Jason Lickliter

Po Inglis

Brent Reynolds

David Walker

Martin Lackmann