Primary Neurospheres Research Articles

Glucose-Based Regulation of miR-451/AMPK Signaling Depends on the OCT1 Transcription Factor

In aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5' AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment.

Regulation of the p19(Arf)/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice.

Brain aging is associated with increased neurodegeneration and reduced neurogenesis. B1/neural stem cells (B1-NSCs) of the mouse subependymal zone (SEZ) support the ongoing production of olfactory bulb interneurons, but their neurogenic potential is progressively reduced as mice age. Although age-related changes in B1-NSCs may result from increased expression of tumor suppressor proteins, accumulation of DNA damage, metabolic alterations, and microenvironmental or systemic changes, the ultimate causes remain unclear. Senescence-accelerated-prone mice (SAMP8) relative to senescence-accelerated-resistant mice (SAMR1) exhibit signs of hastened senescence and can be used as a model for the study of aging. We have found that the B1-NSC compartment is transiently expanded in young SAMP8 relative to SAMR1 mice, resulting in disturbed cytoarchitecture of the SEZ, B1-NSC hyperproliferation, and higher yields of primary neurospheres. These unusual features are, however, accompanied by premature loss of B1-NSCs. Moreover, SAMP8 neurospheres lack self-renewal and enter p53-dependent senescence after only two passages. Interestingly, in vitro senescence of SAMP8 cells could be prevented by inhibition of histone acetyltransferases and mimicked in SAMR1 cells by inhibition of histone deacetylases (HDAC). Our data indicate that expression of the tumor suppressor p19, but not of p16, is increased in SAMP8 neurospheres, as well as in SAMR1 neurospheres upon HDAC inhibition, and suggest that the SAMP8 phenotype may, at least in part, be due to changes in chromatin status. Interestingly, acute HDAC inhibition in vivo resulted in changes in the SEZ of SAMR1 mice that resembled those found in young SAMP8 mice.

AI-03 * TARGETING ANGIOGENESIS WITHOUT INCREASING THE STROMAL CELL RESPONSE OR INVASION USING ABT-898, A THROMBOSPONDIN TYPE 1 REPEAT PEPTIDE

Anti-angiogenic therapy is a promising therapeutic approach for highly vascularized tumors including glioblastoma (GBM). Nonetheless, the efficacy of Bevacizumab - a monoclonal antibody to VEGF-A - is limited only to progression-free survival but not overall survival, due at least in part to the induction of a more invasive phenotype. Here, we investigated the anti-tumor effect of a thrombospondin-1&2 mimetic peptide, ABT-898, on intracerebral xenografts derived from primary patient GBM neurospheres in the nude mouse. ABT-898 treatment of xenograft tumors (80 mg/kg/day i.p.) prolonged mouse survival. Unlike Bevacizumab-treated mouse brain tumors, ABT-898-treated tumors did not exhibit a detectable increase in the number of invasive foci or phospho-Met expression, indicating no induction of an invasive phenotype. As stromal cells are thought to promote tumor cell invasion, we quantitated infiltration of Iba1+ activated microglial/macrophages into the tumor. Although Bevacizumab therapy significantly increased the number of recruited Iba1+ cells into the tumors, the number of Iba1+ cells in the ABT-898-treated tumors was not increased as compared to the vehicle-treated xenograft tumors. Supporting this finding, the migration of microglia cells (BV2), was significantly inhibited by ABT898 in a dose-dependent manner, and the inhibition was reversed by CD36-neutralizing antibody. This suggests that recruitment of tumor invasion-promoting microglia/macrophage can be regulated by ABT898 and this regulation is in part CD36-dependent. In addition, treatment with ABT898 significantly reduced vessel density in xenograft tumors. In vitro ABT898 induced apoptosis of brain endothelial cells (ECs) and inhibited tubulomorphogenesis in collagen gels, both in a dose-dependent manner, and both were reversed by treatment with CD36 neutralizing antibody, Collectively, these data suggest that ABT-898 offers the novel advantage over Bevacizumab of decreasing stromal cell recruitment to the tumor and thus should be considered as an alternative anti-angiogenic therapy for GBM.

Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible involvement in radioresistance.

Glioblastomas (GBM) are some bad prognosis brain tumors despite a conventional treatment associating surgical resection and subsequent radio-chemotherapy. Among these heterogeneous tumors, a subpopulation of chemo- and radioresistant GBM stem-like cells appears to be involved in the systematic GBM recurrence. Moreover, recent studies showed that differentiated tumor cells may have the ability to dedifferentiate and acquire a stem-like phenotype, a phenomenon also called plasticity, in response to microenvironment stresses such as hypoxia. We hypothesized that GBM cells could be subjected to a similar dedifferentiation process after ionizing radiations (IRs), then supporting the GBM rapid recurrence after radiotherapy. In the present study we demonstrated that subtoxic IR exposure of differentiated GBM cells isolated from patient resections potentiated the long-term reacquisition of stem-associated properties such as the ability to generate primary and secondary neurospheres, the expression of stemness markers and an increased tumorigenicity. We also identified during this process an upregulation of the anti-apoptotic protein survivin and we showed that its specific downregulation led to the blockade of the IR-induced plasticity. Altogether, these results demonstrated that irradiation could regulate GBM cell dedifferentiation via a survivin-dependent pathway. Targeting the mechanisms associated with IR-induced plasticity will likely contribute to the development of some innovating pharmacological strategies for an improved radiosensitization of these aggressive brain cancers.

Abstract 1213: Comparing protein pathway activation mapping portraits between gliobastoma patient-matched primary tumor, xenografts and neurospheres: implications for precision medicine

Abstract Background: Most current targeted therapeutics are directed against the activated protein kinase-driven signaling pathway architecture. While guiding therapy selection based on protein phosphorylation is biochemically rational given these proteins represent the direct drug targets it is not certain which protein networks/signaling protein targets are the causal driver of any patient's tumor. Thus, the availability of a system that recapitulates the human in vivo setting and allows for credentialing and identification of which activated signaling networks are causally necessary for tumor viability would be extremely important. Methods: To test two such systems and determine how they resemble the cell signaling architecture of the human primary tumor we compared eight snap-frozen laser capture microdissected (LCM) primary glioblastoma multiforme (GBM) samples with tumor-matching LCM mouse xenografts as well as matching neurospheres (NP). Reverse phase protein microarrays were utilized wherein the total level or phosphorylated level of 70 key signal transduction proteins known to be the direct targets for a number of important targeted therapies and implicated in GBM tumor biology were measured. Results: Overall, the signaling architecture of the xenografts more closely resembled the matched primary LCM tumors in this study set. However, unsupervised hierarchical clustering of the data revealed instances where pathway information such as total c-MET, phospho RAF (S259), phospho GSK3αβ (S21/9) and total EGFR was retained across matched LCM primary tumor, xenografts and NP. In addition there were a number of instances where the signaling architecture of the primary human tumor did not resemble the matched NP and xenograft signature. Conclusion: These pilot data indicate that 3D cell culture conditions may effectively recapitulate in vivo signaling network activation in certain instances, although these preliminary results also suggest that xenografts more closely resemble the cell architecture of matching primary GBM tumors. These data highlight the importance and impact of the microenvironment and culture conditions on tumor cell signal transduction network activation. However, two caveats to this approach remain: (a) drug studies in GBM xenografts and NP are limited to those targeting signaling pathways that are faithfully reproduced from the human in vivo setting and (b) the time required to develop a patient matched xenograft model for individualized drug testing may be too long for a fast-progressing disease such as GBM. Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Laila Poisson, Valerie Calvert, Andrew Borgman, David M. Cherba, Mary E. Winn, Emanuel F. Petricoin. Comparing protein pathway activation mapping portraits between gliobastoma patient-matched primary tumor, xenografts and neurospheres: implications for precision medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1213. doi:10.1158/1538-7445.AM2014-1213

Abstract 447: The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection

Abstract Introduction: In GBM IDH mutations are associated with relatively prolonged survival compared to other subtypes. The mutations in both IDH1 result in the abnormal production of 2-hydroxyglutarate (2-HG). Although the subject of intense study, the function of 2-HG in GBM is not entirely understood. The investigation of IDH mutant tumors has been hampered by the lack of appropriate models. We have now utilized the neurosphere culture method to generate cells from 7 different tumors bearing IDH1 mutations. This study tests the validity of these lines. We utilize both these primary cultures and mouse neurospheres to test the hypotheses that mutant IDH1 is pro-proliferative and influences cell fate. Methods: We successfully propagated and obtained microarray expression data on 7 neurosphere lines bearing IDH1 mutations and 61 IDH1 wildtype cell lines for comparison. We additionally performed methylation sequencing on 3 IDH1 mutant and 3 IDH1 wildtype lines to identify sets of genes that are consistently methylated and down-regulated in these IDH1mutant cell lines. We treated 6 IDH1 mutant lines with a small molecule IDH1 mutant inhibitor that prevents 2-HG formation to to determine effects on proliferation and whether down-regulated genes were rescued by the inhibition of 2-HG formation. Finally we treated fetal murine neural progenitors with exogenous 2-HG to determine the effects on differentiation status as well as on gene expression. Results: Gene expression studies of the 7 IDH1 mutant neurosphere lines revealed 103 genes that were 4-fold down-regulated in IDH1mutant tumors. Of those 103 genes, 32 were also found to be methylated and down-regulated in IDH1mutant brain tumor samples found in public databases. Methylation sequencing revealed that 31 of these 32 genes were heavily methylated in our three IDH1mutant cell lines, while only 12 of these genes were methylated in the IDH1WT cell lines. Treatment of IDH1mutant neurosphere cultures with a small molecule inhibitor resulted in diminished growth capacity. Microarray analysis demonstrated that only one of the downregulated genes was was consistently rescued, Inhibitor of DNA binding 2 (ID2). Application of exogenous 2-HG to murine neurosphere cultures promototed the appearance of oligodendrocytes and neurons, and also induced downregulation of ID2 and upregulation of Olig 2. Discussion: Our data suggest that there is likely a conserved set of “key gateway genes” that are frequently down-regulated in these IDH1mutant tumors and that patient-derived glioma cell lines maintain these epigenetic changes. Inhibition of 2-HG formation may be an effective therapy evidenced by the fact that tumor cells grow slower without 2-HG production. However inhibition of 2-HG production did not appear to reverse the majority of epigenetic changes in gene expression. The Id2 gene is consistently rescued by 2-HG inhibition and may be an important target for future tumor therapy. Citation Format: Matthew C. Garrett, Jack Mottahedeh, Jantzen Sperry, Ascia Eskin, Giovanni Coppola, Ya-shin Shih, Albert Lai, Arthur Chou, Linda Liau, Rob Prins, Timothy Cloughesy, Hong Wu, Stanley Nelson, Harley Kornblum. The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 447. doi:10.1158/1538-7445.AM2014-447

Directing mouse embryonic neurosphere differentiation toward an enriched neuronal population

Neural stem cells (NSC) are self-renewing multipotent cells that have emerged as a powerful tool to repair the injured brain. These cells can be cultured as neurospheres, which are floating aggregates of neural stem/progenitor cells (NSPCs). Despite their high clonal expansion capacity, it has been suggested that in neurospheres, only a small percentage of cells are capable of proliferation and that this system is not efficient in terms of neurogenic competence. Thus, our aim was to develop a neurosphere culture method with a highly proliferative stem/progenitor cell population and particularly with a prominent neurogenic potential, surpassing some of the claimed weaknesses of the neurosphere assay. In our model, mouse neurospheres were harvested from neural tissue at E15 and after only 4 days in vitro (DIV), we have achieved highly proliferative primary neurospheres (81% Sox2 and 76% Ki67 positive cells) and a rather low number of cells expressing glial and neuronal markers (∼10%). After inducing differentiation, we have attained an enriched neuronal population (45% β-III-tubulin positive cells at 15 DIV). Using a simple methodology, we have developed a NSPC model that can provide a valuable source of neuronal precursors, thus offering a potential starting point for cell replacement therapies following CNS injury.

EPIGENETIC REGULATION OF GBM CELL STEMNESS AND TUMOR PROPAGATING CAPACITY BY OCT4 AND SOX2

BACKGROUND: (blind field). METHODS: Experiments utilized human glioblastoma neurosphere lines and low passage (<5) primary neurospheres derived from clinical GBM specimens enriched in GBM sphere-forming stem cells. The Adherent GBM cell lines that lack stem-like tumor-propagating cells were used to test phenotype reprogramming by Oct4 and Sox2. Lentiviral and retroviral systems were used for Oct4/Sox2 and micro-RNA gene transfer, respectively. Brain cancer miRNA PCR Arrays (SABiosciences) and quantitative RT-PCR were used to evaluate miRNA expression. OCT4 and Sox2 transcriptional targets were identified and validated by ChIP-PCR and luciferase reporter assays. Micro-RNA promoter methylation was evaluated using bisulfite sequencing. GBM cell stemness was evaluated by sphere-forming assays and analyses of stem cell marker expression by qRT-PCR, immunoblotting and flow cytometry. Tumor xenograft propagating capacity was assayed in immune-deficient mice. RESULTS: In this study, we identify a novel molecular circuit linking reprogramming transcription factors, DNA methylation and microRNA that regulates glioblastoma cell stemness and tumor-propagating capacity. We show that the Oct4 and Sox2 transcription factors induce human glioma cells to transition to a stem-like tumor-propagating state through the direct induction of DNA methyl transferases leading to the methylation-dependent transcriptional regulation of microRNAs. Specifically, miR-148a expression is shown to correlate inversely with GBM cell stemness, to be repressed by Oct4/Sox2-driven DNA methylation, and to inhibit GBM cell stemness and tumor propagation by GBM sphere-forming cells. CONCLUSIONS: These results identify a new methylation-dependent transcriptional axis by which reprogramming transcription factors regulate tumor cell stemness and tumor propagating capacity. Our results also identify miR-148a as a novel tumor-suppressing inhibitor of glioma cell stemness and suggest new approaches for treating GBM by targeting stem-like tumor propagating cell populations. SECONDARY CATEGORY: n/a.

Age-dependent and differential effects of Smad7ΔEx1 on neural progenitor cell proliferation and on neurogenesis

We recently reported that young (3 to 4months old) mice lacking Exon 1 of the Smad7 gene (S7ΔEx1 mice) show enhanced proliferation of neural stem and progenitor cells (NPCs) in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ) of the lateral ventricles. It remained unclear, however, whether this phenotype would persist along aging, the latter typically being associated with a profound decrease in neurogenesis. Analysis of NPCs' proliferation based on the cell cycle marker PCNA in 12month-old S7ΔEx1 mice revealed a reversal of the phenotype. Hence, in contrast to their younger counterparts, 12month-old S7ΔEx1 mice had a reduced number of proliferating cells, compared to wildtype (WT) mice. At the same time, the survival of newly generated cells was enhanced in the aged transgenic animals. 12month-old S7ΔEx1 mice further displayed a reduced level of neurogenesis based on the numbers of cells expressing doublecortin (DCX), a marker for newborn neurons. The reduced neurogenesis in aged S7ΔEx1 mice was not due to a stem cell depletion, which might have occurred as a consequence of hyperproliferation in the young mice, since the number of Nestin and Sox2 positive cells was similar in WT and S7ΔEx1 mice. Instead, Nestin positive cells in the DG as well as primary neurosphere cultures derived from 12month-old S7ΔEx1 mice had a reduced capability to proliferate. However, after passaging, when released from their age- and niche-associated proliferative block, neurospheres from aged S7ΔEx1 mice regained the hyperproliferative property. Further, pSmad2 antibody staining intensity was elevated in the DG and SVZ of 12-month old transgenic compared to WT mice, indicating increased intracellular TGF-beta signaling in the aged S7ΔEx1 mice. In summary, this points toward differential effects of S7ΔEx1 on neurogenesis: (i) a hyperproliferation in young animals caused by a cell autonomous mechanism, and (ii) a TGF-beta dependent modulation of neurogenesis in aged S7ΔEx1 animals that abrogates the cell-intrinsic hyperproliferative properties and results in reduced proliferation, increased stem cell quiescence, and enhanced survival of newly generated cells.

Imaging of molecular oxygen at subcellular resolution in mammalian cell culture: from 2D to 3D models (764.1)

Oxygen monitoring is necessary for studying cell metabolism, stem cell development and ROS production, however most of the current methods suffer from low sensitivity and signal drift (electrodes), lack of quantitative context and end‐point nature (hypoxia stains) or inability to probe O2 inside the cell (extracellular probes). Phosphorescence lifetime imaging microscopy (PLIM) allows for quantitative, sensitive and specific detection of O2, by means of phosphorescent indicator dyes. We developed phosphorescent O2 probe (Pt‐Glc) which demonstrates efficient cytosolic staining of different cell lines (2D cultures), and 3D tissue models such as spheroids (primary neurospheres) and live brain slices from embryonic tissue. The PLIM analysis of neurospheres from rat brain cortex with Pt‐Glc confirmed presence of hypoxic niches. We correlated oxygenation map of spheroids with their size, distribution of live cell (cholera toxin, TMRM), proliferating (BrdU) and stem cell markers (nestin), thus performing multiplexed metabolic imaging of live neurosphere. A comparison with pimonidazole, HIF‐a immunostaining and nanoparticle probes was also made. Thus, Pt‐Glc represents valuable tool for studying stem cell niche within ex vivo and in vitro 3D models.Grant Funding Source: Supported by the Science Foundation Ireland.

Protective effects of ziprasidone on the damage of hippocampal-derived neural stem cell induced by lipopolysaccharide

Objective To investigate the effects of ziprasidone on the hippocampal-derived neural stem cells (NSCs) viability and apoptosis injured.by LPS. Methods The NSCs were derived from the hippocampus of fetal rats, after the primary neurospheres passaged, the cells were treated with LPS (200 μg/L) and different concentrations of ziprasidone for 48 h or 72 h. The cell viability and the level of LDH were measured by the kit of WST-8 and LDH detected kit, respectively. Furthermore, the apoptosis rate of each dosage group was measured by using Annexin-V-FLUOS Staining Kit. Results (1) Cell viability test showed that the cell viability of LPS group (OD value, 0.265±0.047) was significantly reduced than that of sham (OD value, 0.380±0.029), LPS+ 5 μM (OD value, 0.316±0.008) and LPS+ 10 μM (OD value, 0.321±0.006) group after ziprasidone treatment for 48 h. There were also significant difference of cell viability between sham, LPS+ 5 μM, LPS+ 10 μM and LPS group after ziprasidone treatment for 72 h. (2) LDH test showed that the LDH level of LPS group was significantly decreased than that of sham, LPS+ 5 μM and LPS+ 10 μM after 48 hour's ziprasidone treatment (P<0.01). The LDH level of LPS group was also significantly reduced than that of sham, LPS+ 5 μM and LPS+ 10 μM after 72 hour's ziprasidone treatment (P<0.01). (3) The apoptosis of LPS+ 10 μM((17.87±2.29)%), LPS+ 5 μM ((18.45±3.84)%) treated group or sham group((9.15±1.54)%) was significantly lower than LPS group((22.67±2.15)%) by using flow cytometry after 48 h treatment.Furthermore, the apoptosis of LPS+ 10 μM ((10.52±1.42)%) treated group or sham group((8.45±1.04)%) was significantly lower than LPS group((15.70±2.97)%) after 72 h treatment. Conclusion The cellular damage of NSCs injured by LPS can be restrained by ziprasidone, and this effect might be one of the cellular mechanism of neural protective effect of ziprasidone. Key words: Ziprasidone; Neural stem cells; Protective effect; Lipopolysaccharide

A fast and simple differentiation protocol to study the pro-neurogenic activity of soluble factors in neurospheres

Sphere-forming assays are widely used for the propagation, characterization and manipulation of adult brain-derived stem- and progenitor cells. However despite the broad application of this cell culture system in neural stem cell- and brain tumor research, no standardized protocols exist. Variations in experimental procedures not only concern the use of media components but also cell density, the number of passages the cells are propagated before analysis and, in cases where the neurogenic or gliogenic potential of the cells is investigated, the duration that the cells are allowed to differentiate. The latter deserves consideration because the proportion of differentiated cells obtained at the endpoint of the experiment depends not only on the absolute number of cells that differentiate at a given time, but also on the number of cell divisions prior to differentiation and the rate of cell death in the cultures. In the present study we describe a fast and simple differentiation protocol to investigate the pro-neurogenic potential of soluble factors added to subventricular zone (SVZ)-derived neurospheres. The assay relies on the use of primary neurospheres and very short differentiation times, thereby largely excluding the contribution of cell proliferation and cell death to the results. We use this modified assay to test the consequence of pharmacological inhibition of the EGF receptor-, Erk1/2-, Protein Kinase B/AKT-, and Sonic Hedgehog-pathways on neuronal differentiation of SVZ-neurosphere cultures.

MRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Abstract A254: Systemic injection of human neural stem cells expressing anti-cancer agent targets invasive gliomas and induces tumor regression in combination with a cardiac glycoside.

Abstract Glioblastoma multiforme (GBM) is the most common and most aggressive malignant form of brain tumors in human. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as an anticancer agent, however, a considerable number of tumor types, including GBM, are resistant to TRAIL. Also, TRAIL therapeutic use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney and a poor penetration across the blood-brain barrier. The tumor-tropic properties of neural stem cells (NSCs) could serve as a novel strategy to deliver therapeutic genes to tumors in the brain. Furthermore, we have previously reported that the cardiac glycoside lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis. In this study, we engineered human neural stem cell to synthesize and secrete an active form of TRAIL (NSC-sTRAIL) as well as the secreted Gaussia luciferase (Gluc) as a blood marker for cell proliferation. We showed that these cells selectively migrate toward GBM stem-like cells (GSCs) in a transwell assay. However, GSCs’ growth was only decreased by 11.6%. When this co-culture was treated with lanatoside C, GSCs’ growth was inhibited by 81.5% in a caspase-mediated manner. We then tested this combined therapy in an invasive orthotopic model using primary GSC neurospheres expressing firefly luciferase. Upon systemic injection, Gluc blood assay showed that at the next day at least 30% of NSC-sTRAIL, and after 5 weeks up to 0.5% of these cells survived. Histological analysis showed that the NSC-sTRAIL cells crossed the blood-brain barrier and migrated toward invasive GSC tumors. Bioluminescence imaging showed that tumors in mice treated with NSC-sTRAIL cells in combination with lanatoside C resulted in significant tumor regression and mouse survival elongation as compared to single therapy. Taken together, this study demonstrated that systemic injection of neural stem cells targets invasive brain tumors and could be used to deliver an anticancer agent such as sTRAIL which yields significant tumor regression when used in combination with lanatoside C. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A254. Citation Format: Jian Teng, Seyedali Hejazi, Christian E. Badr, Bakhos A. Tannous. Systemic injection of human neural stem cells expressing anti-cancer agent targets invasive gliomas and induces tumor regression in combination with a cardiac glycoside. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A254.

Sox6 Up-Regulation by Macrophage Migration Inhibitory Factor Promotes Survival and Maintenance of Mouse Neural Stem/Progenitor Cells

Macrophage migration inhibitory factor (MIF) has important roles in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs), but downstream effectors remain unknown. We show here that MIF robustly increases the expression of Sox6 in NSPCs in vitro. During neural development, Sox6 is expressed in the ventricular zone of the ganglionic eminence (GE) of mouse brains at embryonic day 14.5 (E14.5), cultured NSPCs from E14.5 GE, and NSPCs in the subventricular zone (SVZ) around the lateral ventricle (LV) of the adult mouse forebrain. Retroviral overexpression of Sox6 in NSPCs increases the number of primary and secondary neurospheres and inhibits cell differentiation. This effect is accompanied with increased expression of Hes1 and Bcl-2 and Akt phosphorylation, thus suggesting a role for Sox6 in promoting cell survival and/or self-renewal ability. Constitutive activation of the transcription factor Stat3 results in up-regulation of Sox6 expression and chromatin immunoprecipitation analysis showed that MIF increases Stat3 binding to the Sox6 promoter in NSPCs, indicating that Stat3 stimulates Sox6 expression downstream of MIF. Finally, the ability of MIF to increase the number of primary and secondary neurospheres is inhibited by Sox6 gene silencing. Collectively, our data identify Sox6 as an important downstream effector of MIF signaling in stemness maintenance of NSPCs.

Abstract 2169: SapC-DOPS induces lethal mitophagy in glioblastoma.

Abstract The goal of this study is to evaluate SapC-DOPS, a novel cancer nanotherapeutic, for glioblastoma multiforme (GBM). SapC-DOPS delivered intravenously (i.v) was found to specifically target intracranial tumors in mice bearing spontaneous brain tumor, as well in nude mice intracranially implanted with human GBM cells. Treatment of tumor bearing mice with SapC-DOPS (i.v.) significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P&amp;lt;.0001). Western blot analysis of primary GBM neurospheres treated with SapC-DOPS did not show activation of apoptosis as measured by cleaved caspase 9 and cleaved PARP, and lacked activation of DNA damage markers phosphor-ATM and γ-H2AX. Consistent with this, treatment with a pan-caspase inhibitor Z-VAD-FMK did not rescue SapC-DOPS-induced killing (P&amp;gt;0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was also confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed punctuated GFP expression following treatment, indicative of autophagosome formation. Quantification of GFP punctated cells showed a significant increase in SapC-DOPS treated cells compared to control (P&amp;lt;.001). Analysis of red/green fluorescence following acridine orange staining showed an induction of acidic vesicular organelles indicative of autophagolysosomes. In addition, inhibition of autophagosome formation using 3-methyladeneine or inhibition of auotphagic vacuole maturation with bafilomycin A1 resulted in a significant rescue of SapC-DOPS-induced killing (P&amp;lt;.001). Knockdown of ATG5 using siRNA also resulted in a rescue of SapC-DOPS-induced cell death and autophagy induction (P&amp;lt;.001). Interestingly, we did not observe a decrease in the activation of mTOR as determined by the phosphorylation of 4EBP1 and p70S6K which are typically induced during autophagy. This led us to test the use of rapamycin, a known inhibitor of mTOR and inducer of autophagy, in combination with SapC-DOPS. By using the Chou Talalay analysis, we observed strong synergy for multiple drug combinations in primary GBM neurospheres (combination index &amp;lt; .4). To investigate whether SapC-DOPS induced autophagy could be preferentially targeting mitochondria (mitophagy), we utilized MitoTracker Green (mitochondria) and LysoTracker Red (autophagolysosomes). By using confocal microscopy analysis, we were able to observe a decrease in mitochondrial mass in cells treated with SapC-DOPS as well as co-localization of mitochondria with autophagolysosomes. In addition to this, we observed a significant decrease in ATP levels following SapC-DOPS treatment (P&amp;lt;.01). These findings suggest therapeutic implications for treating GBM by using SapC-DOPS alone and in combination with an AKT/mTOR inhibitor. Citation Format: Jeffrey Wojton, Naduparambil K. Jacob, Nicholas Denton, Nina Dmitrieva, Hiroshi Nakashima, Chang-Hyuk Kwon, Lionel Chow, Ennio A. Chiocca, Arnab Chakravarti, Balveen Kaur, Xiaoyang Qi. SapC-DOPS induces lethal mitophagy in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2013-2169

Abstract 2067: BMN 673 potentiates efficacy of temozolomide in sensitive and resistant glioblastoma models.

Abstract Poly (ADP-ribose) polymerase (PARP) plays an important role in several DNA repair processes, and previous studies with various PARP inhibitors have demonstrated robust enhancement of sensitivity to temozolomide (TMZ) chemotherapy in pre-clinical models of glioblastoma multiforme (GBM). The focus of this study was to evaluate the antitumor effects of a novel PARP inhibitor, BMN 673, as a single agent and in combination with TMZ in the established U251 glioma cell line, neurosphere cultures from the primary Mayo GBM xenograft line GBM12, and TMZ-resistant cells derived from either line (U251TMZ, GBM12TMZ(MGMT high) and GBM12TMZ(MGMT low)). In a biochemical assay of PARP inhibition, BMN 673 potently suppressed poly-ADP-ribosylation of cellular proteins at concentrations of 3 nM following hydrogen peroxide treatment of U251 cells. Single agent treatment had modest anti-tumor activity with 30 nM BMN 673 reducing absorbance in a CyQUANT cell proliferation assay relative to control treatment in both U251 and U251TMZ cells (Table 1). BMN 673 co-treatment also enhanced the efficacy of TMZ in both U251-derivative lines at a clinically relevant concentration of TMZ. Similarly, treatment with BMN673 alone or in combination with TMZ inhibited primary neurosphere formation in all three GBM12 models. Notably, the addition BMN 673 clearly re-sensitized both resistant GBM12 sub-lines to relatively low concentrations of TMZ. Consistent with a mechanism involving disruption of DNA repair, combined treatment with BMN 673 and TMZ resulted in robust activation of ATM-mediated KAP1 phosphorylation (Ser824) and ATR-mediated Chk1 phosphorylation (Ser345) as compared to TMZ or BMN 673 alone 24 hours after treatment. In conclusion, BMN673 is a promising PARP inhibitor with high potency and cytotoxicity in vitro against both TMZ sensitive and TMZ resistant GBM cells. Table 1 - In vitro antitumor effects of TMZ combined with BMN673 TMZ, microM 0 nM BMN673 30 nM BMN673 CyQUANT U251 Relative absorbance Relative absorbance 0 1.00 0.58 30 0.80 0.30 U251TMZ 0 1.00 0.69 30 1.02 0.21 Primary Neurosphere Assay GBM12 Relative Count Relative Count 0 1.00 0.66 5 0.32 0 10 0.01 0 GBM12TMZ (MGMT high) 0 1.00 0.32 5 1.11 0.02 10 1.04 0.01 GBM12TMZ (MGMT low) 0 1.00 0.25 5 0.86 0.03 10 0.75 0 Citation Format: Sani H. Kizilbash, Ryo Kawashima, Shiv Gupta, Ann C. Mladek, Yuqiao Shen, Jann N. Sarkaria. BMN 673 potentiates efficacy of temozolomide in sensitive and resistant glioblastoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2067. doi:10.1158/1538-7445.AM2013-2067

Abstract 3431: βIII-tubulin in glioblastoma: An emerging multifactorial survival factor role in chemotherapy response and tumor formation.

Abstract Glioblastoma (GBM) is an aggressive primary brain tumour with a very poor prognosis. Current treatment is usually surgical resection, radiotherapy and chemotherapy, however, GBM's highly resistant nature renders treatment ineffective. ßIII-tubulin is a neuronal specific microtubule protein that is aberrantly expressed in GBM and it has been implicated in a range of epithelial cancers with poor prognosis and high aggressiveness1. In gliomas, its high expression correlates with higher tumour grade2, however the functional relevance of this expression is unknown. Our aim was to investigate the functional and mechanistic role of ßIII-tubulin in GBM drug sensitivity and tumorigenesis. Methods: To study the functional role of βIII-tubulin in GBM, siRNA gene knockdown was used in two GBM cell lines, U87 and U87vIII and in a primary GBM neurosphere culture. Non-targeting siRNA was used as a control. Knockdown levels were assessed by western blotting. Apoptosis was measured by AnnexinV staining. Drug sensitivity of cell lines was tested using drug-treated clonogenic assays. Senescence was assessed by using cytoplasmic staining for sa-β-galactosidase. Neurosphere formation was assessed by soft-agar growth assay. Results: Specific βIII-tubulin knockdown achieved a 94% and 52% reduction in ßIII-tubulin levels in GBM cell lines and neurospheres respectively, compared to control siRNA. βIII-tubulin knockdown in U87vIII cells resulted in a significant increase in sensitivity to both the DNA-damaging agent temozolomide (TMZ) and tubulin binding agents epothilone B and paclitaxel compared to control (p&amp;lt;0.01). Increased sensitivity to TMZ following βIII-tubulin knockdown in U87 and U87vIII cells was associated with a significant increase in drug-induced apoptosis (p&amp;lt;0.01). TMZ is known to induce senescence3,4 and βIII-tubulin knockdown significantly increased TMZ-induced senescence in U87 (βIII-tub siRNA: 84.1±2.6%, p&amp;lt;0.05, compared to ctrl siRNA: 58.1±7.1%) and U87vIII (βIII-tub siRNA: 83.9±4.5%, p&amp;lt;0.01, compared to ctrl siRNA: 56.3±4.3%) cells. Further validation using a 3D-growth model of primary human GBM neurospheres, showed that neurosphere formation was significantly reduced (30.7%, p&amp;lt;0.01) upon βIII-tubulin knockdown compared to ctrl siRNA. Conclusions: Our data strongly suggests that aberrant expression of βIII-tubulin in glioblastoma may play an important role in intrinsic drug sensitivity to broad classes of chemotherapy drugs. Targeting βIII-tubulin increases temozolomide sensitivity by enhancing drug-induced apoptosis and senescence. Furthermore, we have identified a potential role for βIII-tubulin in GBM tumorigenesis. 1 Kavallaris. Nature Rev Cancer, 10:194-204, 2010 2 Katsetos et al. Arch Pathol Lab Med, 125:613-624, 2001 3 Mhaidat. Br J Cancer, 97:1225-1233, 2007 4 Gunther. Br J Cancer, 88:463-469, 2003 Citation Format: Gorjana Mitic, Maria Tsoli, David S. Ziegler, Maria Kavallaris. βIII-tubulin in glioblastoma: An emerging multifactorial survival factor role in chemotherapy response and tumor formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3431. doi:10.1158/1538-7445.AM2013-3431

Abstract 5033: Modeling Ewing's sarcoma and tolerance of EWS-FLI1 with neural crest stem cells.

Abstract Ewing's sarcoma (ES) is a highly malignant bone and soft tissue tumor that primarily affects children and young adults. These tumors are characterized by chromosomal translocations, most commonly t(11;22)(q24;q12), which generates the EWS-FLI1 fusion oncogene. Despite its oncogenic potential, the fusion protein induces cell cycle arrest or apoptosis in most non-transformed cells unless mutations in tumor suppressors such as p16 are also present. However, only a minority of patients (∼25%) have such mutations at diagnosis, suggesting other mechanisms for cellular tolerance must be present in the cell of origin. Currently, the cell of origin of these tumors is controversial, with data supporting both a mesenchymal and/or neural crest origin. To assess the potential to model ES using mouse neural crest stem cells (NCSCs) and elucidate the mechanism(s) of EWS-FLI1 tolerance, primary fetal and adult mouse NCSCs were transduced with a V5-tagged EWS-FLI1 2a eGFP construct. Both wild-type fetal and postnatal NCSCs tolerated EWS-FLI1 expression, with 40-60% of the stem cell-derived neurospheres positive for GFP. There was no difference in neurosphere forming ability between cells infected with EWS-FLI1 or an empty vector, suggesting EWS-FLI1 expression was not toxic to these cells. EWS-FLI1 expression was maintained as the stem cells self-renewed, with GFP-positive primary neurospheres giving rise to GFP-positive secondary and tertiary neurospheres but there was no difference in the frequency of GFP-positive and -negative secondary neurospheres. EWS-FLI1 expression was confirmed by quantitative PCR and the V5 tag was localized to the nucleus by immunocytochemistry, consistent with transcription factor function. EWS-FLI1-mediated gene expression analysis is currently ongoing. Interestingly, the tolerance of EWS-FLI1 was dramatically reduced in Bmi-1 deficient NCSCs. Bmi-1 is known to function cooperatively with multiple other oncogenes to mediate transformation but its role in tolerance has not been studied. In the absence of Bmi-1 only 8% of the fetal neurospheres were positive for GFP, a large decrease compared to 40% of the wild-type neurospheres. Taken together, these data are consistent with a neural crest origin for ES and demonstrate a critical role for Bmi-1-mediated tolerance to EWS-FLI1. Work is ongoing to determine whether EWS-FLI1 can transform these cells and to assess the full utility of this novel system to model ES and genetically assess important regulators of EWS-FLI1 function. Citation Format: Jack T. Mosher, Victor S. Chen, Elizabeth R. Lawlor. Modeling Ewing's sarcoma and tolerance of EWS-FLI1 with neural crest stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2013-5033

Abstract 3240: Pre-clinical investigation of the Wee1 inhibitor, MK1775, in glioblastoma multiforme.

Abstract Wee1 is a key regulator of both G2/M transition and DNA replication fork stability. In this study, the efficacy of the Wee1 inhibitor MK1775 was evaluated in the Mayo primary glioblastoma multiforme (GBM) xenograft model. Based on western blot expression of Wee1, GBM22, GBM12 and GBM6 were selected for study based on low, intermediate and high-level expression, respectively. Wee1-mediated phosphorylation of Y15-Cdc2 was robustly suppressed in vitro in both GBM22 and GBM6 at 300 nM MK1775. At this concentration, MK1775 had marked anti-proliferative effects on GBM22 with 80±2.5% suppression of absorbance in a CyQUANT cell proliferation assay, while similar treatment of GBM6 had minimal effect on growth (26±21% suppression of absorbance). In a similar primary neurosphere formation assay, 300 nM completely inhibited neurosphere formation in GBM22 and GBM12, while having no effect on GBM6 neurosphere formation. Consistent with the cytotoxicity data, single agent treatment of GBM22 with MK1775 resulted in a marked S-phase arrest 24 hours after treatment (60% vs. 40% cells in S with MK1775 vs. control, respectively) and subsequent accumulation of cells in G2/M 48 hours after treatment (95% vs. 9.5%, respectively). In contrast, relatively minimal cell cycle perturbations were observed with MK1775 in GBM6 (24 hour S-phase accumulation 27% vs. 21%, respectively). Combination studies with MK1775 and temozolomide (TMZ) in a neurosphere formation assay demonstrated enhancement of treatment benefit only in GBM22 and GBM12 but not GBM6 (relative neurosphere numbers for TMZ, MK1775, or combined treatment compared to untreated were 34%, 16% and 7%, respectively for GBM22, 41%, 95% and 15%, respectively, for GBM12; while the same combination had no effect on GBM6 neurosphere formation). In anticipation of evaluating treatment efficacy in animal models, pharmacokinetic modeling of MK1775 brain delivery was performed in nude mice. Following a single MK1775 dose of 50 mg/kg, time to maximum blood concentration was 1.0 hours, AUC was 29.8 microM·h, and Cmax was 7.8 microM in whole blood and 0.24 microM in brain parenchyma. To evaluate whether limitations in brain delivery affected efficacy, parallel in vivo GBM12 heterotopic and orthotopic xenograft efficacy studies were performed with a 5 day dosing regimen of TMZ and MK1775 repeated every 28 days for 3 cycles. In the orthotopic model, MK1775 provided no survival benefit with median survival being 16, 15, 88 and 92 days for placebo, MK1775 alone, TMZ alone, or MK1775 + TMZ treatment, respectively. In contrast, there is some suggestion of MK1775 efficacy in the flank tumor model with median time to reach 1000 mm3 being 45, 56, 101 and 112 days, respectively. Collectively, these data suggest that despite promising in vitro activity for MK1775, limited drug delivery into the brain may reduce the efficacy of MK1775 therapy in an orthotopic GBM xenograft model. Citation Format: Jenny Pokorny, Vincenzo Pucci, Stuart Shumway, Bennett Ma, Katrina Bakken, Mark Schroeder, Brett Carlson, Jann N. Sarkaria. Pre-clinical investigation of the Wee1 inhibitor, MK1775, in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2013-3240