Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings, however, requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study, we show that once specified to neuro-epithelial lineage, hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However, our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes, namely, migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes, a new potential source for therapeutic use for brain tumors and other neurological diseases.

Evidence has pointed to brain tumor stem cells (BTSC) as culprits behind human high-grade glioma (hHGG) resistance to standard therapy. Pre-clinical rodent models are the mainstay for testing of new therapeutic strategies. The typical model involves the intracranial injection of human glioma cells into immunocompromised hosts, hindering the evaluation of tumor-host responses and resulting in non-infiltrative tumors. The CT-2A model is an immunocompetent mouse model with potential to overcome these disadvantages. In this study, we confirmed the highly infiltrative nature of intracranial CT-2A tumors and optimized reproducible injection parameters. We then generated neurospheres and established, for the first time, the stemness of this model. CT-2A expression of the BTSC marker, CD133, increased from 2% in monolayer cells to 31% in fully-formed neurospheres. Investigation of three stem cell markers (Oct4, Nanog and Nestin) revealed a distinct stemness signature with monolayer cells expressing Oct4 and Nestin (no Nanog), and neurospheres expressing all three. Additionally, CT-2A cells were more proliferative and invasive than U87 cells, while CT-2A neurospheres were significantly more proliferative and invasive than either monolayer cells in vitro. Taken together, our results show that this model is a valuable tool for pre-clinical testing of novel therapeutics against hHGG and also affords the opportunity for investigation of BTSC in an immunocompetent setting.

Glioblastoma is the most common, and at the same time, most aggressive type of high-grade glioma (HGG). The prognosis of glioblastoma patients treated with standard therapy including surgery, temozolomide and radiation therapy remains poor. Peroxisome proliferator-activated receptor-α (PPARα) agonists are in widespread clinical use for the treatment of hyperlipidemia. Recent evidence has suggested a potential role in various cancers including glioblastoma. In this study, we characterized the effects of PPARα agonist, fenofibrate, directly on HGG cells and glioma stem cells (GSC). Fenofibrate exhibited dose-dependent p53-independent anti-proliferative effects on HGG starting at 25 μM and pro-apoptotic effects starting at 50 μM, suggesting that the anti-proliferative actions are present only at 25 μM. PPARα was expressed in all HGG cell lines. Inhibition of PPARα with specific inhibitor GW6471 did not affect either proliferation or apoptosis suggesting that these are PPARα-independent effects. Fenofibrate treatment of HGG cells robustly diminished the expression of key signaling pathways, including NF-κB and cyclin D1. Phosphorylation of Akt was also diminished, with no change in total Akt. Effects on apoptotic signaling molecules, Bax and Bcl-xL, had a trend towards pro-apoptotic effects. With respect to GSC, fenofibrate treatment at 25 μM significantly decreased invasion in association with a decrease in CD133 and Oct4 expression. Overall, results support consideration of fenofibrate as an anti-glioma agent and establish its potential as an adjunct treatment strategy for HGG. Translation to the clinical setting could be rapid given its current use as a clinical agent and its low toxicity profile.

The prognosis of patients with human high-grade gliomas (HGGs) remains dismal despite major advances in their management, due mainly to the high resistance of these infiltrative tumor cells to programmed cell death (PCD). Most therapeutic strategies for HGGs are aimed to maximize PCD type I, apoptosis or type II, autophagy. These are predominantly distinctive processes, but many studies suggest a cross-talk between the two. A better understanding of the link between PCD types I and II might allow development of more effective therapies for HGGs. In this study, we examined whether there is a common upstream signaling event responsible for both apoptotic and autophagic PCD using 3 chemotherapeutic agents in human HGG cells. Our study shows that each agent caused a significant decrease in cell viability in each of the HGG cell lines tested. The increase rate of apoptosis and autophagy varied among cell lines and chemotherapeutic agents used. Increased expression of cytidine-cytidine-adenosine-adenosine-thymidine (C)/ enhancer binding protein (EBP) homologous transcription factor C/EBP homologous protein (CHOP)/ growth arrest and DNA damage-inducible gene 153 (GADD153) was documented after use of either proautophagic or pro-apoptotic agents. The involvement of CHOP/GADD153 in both type I and type II PCD was confirmed by overexpression and gene-silencing studies. Gene silencing by small-interfering RNAmediated CHOP/GADD153 resulted in increased cell viability, decreased upregulation of microtubule-associated protein light-chain 3 ′ type II (LC3II) and cleaved caspase-3, and inhibition of apoptosis and autophagy. Exogenous expression of CHOP/GADD153 triggered apoptosis and autophagy in the absence of other stimuli. The clinical significance of these findings was supported by the evidence that celecoxib, a nonsteroidal anti-inflammatory drug known to induce GADD153-mediated apoptosis, strongly increases both type I and type II PCD in HGG cells when combined with another inducer of GADD153. These data suggest that CHOP/ GADD153 should be investigated as a novel targetable signaling step to improve therapies for HGGs.Keywords: apoptosis, autophagy, CHOP/GADD153, cell viability, gliomas. High-grade gliomas (HGGs), the most frequent types of gliomas, are associated with poor prognosis. Among these, glioblastoma multiforme (GBM), accounting for 50% of gliomas, is associated with a 5-year survival rate of less than 5% and a median survival rate of approximately 14 months, even after use of aggressive surgical tumor resection and postoperative radiation therapy combined with temozolomide (TMZ) chemotherapy is used.2 This is due mainly to high resistance of these infiltrative tumor cells to programmed cell death (PCD).HGG cells show only partial sensitivity to therapies that induce apoptosis, i.e., PCD type I.3 -6 Recent studies indicate that HGG cells are more sensitive to treatments that induce autophagy, i.e., PCD type II. pro-apoptotic and pro-autophagic actions. In particular, TMZ, the oral alkylating agent currently con...

Objectives The gene encoding glucose transporter 3 (GLUT3, SLC2A3 ) is present in the human population at variable copy number. An overt disease phenotype of SLC2A3 copy number variants has not been reported; however, deletion of SLC2A3 has been previously reported to protect carriers from rheumatoid arthritis, implicating GLUT3 as a therapeutic target in rheumatoid arthritis. Here we aim to perform functional analysis of GLUT3 copy number variants in immune cells, and test the reported protective association of the GLUT3 copy number variants for rheumatoid arthritis in a genetic replication study. Methods Cells from genotyped healthy controls were analyzed for SLC2A3 /GLUT3 expression and glycolysis capacity. We genotyped the SLC2A3 copy number variant in four independent cohorts of rheumatoid arthritis and controls and one cohort of multiple sclerosis and controls. Results Heterozygous deletion of SLC2A3 correlates directly with expression levels of GLUT3 and influences glycolysis rates in the human immune system. The frequency of the SLC2A3 copy number variant is not different between rheumatoid arthritis, multiple sclerosis and control groups. Conclusions Despite a robust SLC2A3 gene copy number dependent phenotype, our study of large groups of rheumatoid arthritis cases and controls provides no evidence for rheumatoid arthritis disease protection in deletion carriers. These data emphasize the importance of well powered replication studies to confirm or refute genetic associations, particularly for relatively rare variants.