Human Glioma Stem Cells Research Articles

Identification of RNA-Binding Protein LARP4B as a Tumor Suppressor in Glioma.

Transposon-based insertional mutagenesis is a valuable method for conducting unbiased forward genetic screens to identify cancer genes in mice. We used this system to elucidate factors involved in the malignant transformation of neural stem cells into glioma-initiating cells. We identified an RNA-binding protein, La-related protein 4b (LARP4B), as a candidate tumor-suppressor gene in glioma. LARP4B expression was consistently decreased in human glioma stem cells and cell lines compared with normal neural stem cells. Moreover, heterozygous deletion of LARP4B was detected in nearly 80% of glioblastomas in The Cancer Genome Atlas database. LARP4B loss was also associated with low expression and poor patient survival. Overexpression of LARP4B in glioma cell lines strongly inhibited proliferation by inducing mitotic arrest and apoptosis in four of six lines as well as in two patient-derived glioma stem cell populations. The expression levels of CDKN1A and BAX were also upregulated upon LARP4B overexpression, and the growth-inhibitory effects were partially dependent on p53 (TP53) activity in cells expressing wild-type, but not mutant, p53. We further found that the La module, which is responsible for the RNA chaperone activity of LARP4B, was important for the growth-suppressive effect and was associated with BAX mRNA. Finally, LARP4B depletion in p53 and Nf1-deficient mouse primary astrocytes promoted cell proliferation and led to increased tumor size and invasiveness in xenograft and orthotopic models. These data provide strong evidence that LARP4B serves as a tumor-suppressor gene in glioma, encouraging further exploration of the RNA targets potentially involved in LARP4B-mediatd growth inhibition. Cancer Res; 76(8); 2254-64. ©2016 AACR.

Optimized dissociation protocol for isolating human glioma stem cells from tumorspheres via fluorescence-activated cell sorting

Fluorescence-activated cell sorting (FACS) based on the surface marker CD133 is the most common method for isolating glioma stem cells (GSCs) from heterogeneous glioma cell populations. Optimization of this method will have profound implications for the future of GSC research. Five commonly used digestion reagents, Liberase-TL, trypsin, TrypLE, Accutase, and non-enzymatic cell dissociation solution (NECDS), were used to dissociate glioma tumorspheres derived from two primary glioma specimens (091214 and 090116) and the cell lines U87 and T98G. The dissociation time, cell viability, retention of CD133, and stemness capacity were assessed. The results showed that single cells derived from the Liberase-TL (200 µg/ml) group exhibited high viability and less damage to the antigen CD133. However, the efficiency of NECDS for dissociating the tumorspheres into single cells was fairly low. Meanwhile, the use of this digestion reagent resulted in obvious cellular and antigenic impairments. Taken together, Liberase-TL (200 µg/ml) is an ideal reagent for isolating GSCs from tumorspheres. In contrast, the use of NECDS for such a protocol should be carefully considered.

Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood–brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach.

The homing and inhibiting effects of hNSCs-BMP4 on human glioma stem cells.

Malignant gliomas patients have a poor survival rate, partially due to the inability in delivering therapeutic agents to the tumors, especially to the metastasis of human glioma stem cells (hGSCs). To explore whether the human neural stem cells (hNSCs) with an over-expression of BMP4 (hNSCs-BMP4) can trace and inhibit hGSCs, in this study, we examined the migration of hNSCs to hGSCs using transwell assay in vitro and performed the fluorescent tracer experiment in vivo. We examined the proliferation, differentiation, apoptosis and migration of hGSCs after co-culturing with hNSCs-BMP4 in vitro and tested the tropism and antitumor effects of hNSCs-BMP4 in the established brain xenograft models of hGSCs. We found that hNSCs-BMP4 could secrete BMP4 and trace hGSCs both in vitro and in vivo. When compared to the normal human astrocytes (NHAs) and hNSCs, hNSCs-BMP4 could significantly inhibit the invasive growth of hGSCs, promote their differentiation and apoptosis by activating Smad1/5/8 signaling, and prolong the survival time of the tumor-bearing nude mice. Collectively, this study suggested that hNSCs-BMP4 may help in developing therapeutic approaches for the treatment of human malignant gliomas.

Magnetofection Based on Superparamagnetic Iron Oxide Nanoparticles Weakens Glioma Stem Cell Proliferation and Invasion by Mediating High Expression of MicroRNA-374a.

Glioma stem cells belong to a special subpopulation of glioma cells that are characterized by strong proliferation, invasion and drug resistance capabilities. Magnetic nanoparticles are nanoscale biological materials with magnetic properties. In this study, CD133+ primary glioma stem cells were isolated from patients and cultured. Then, magnetic nanoparticles were used to mediate the transfection and expression of a microRNA-374a overexpression plasmid in the glioma stem cells. Transmission electron microscopy detected the presence of significant magnetic nanoparticle substances within the CD133+ glioma stem cells after transfection. The qRT-PCR and Northern blot results showed that the magnetic nanoparticles could be used to achieve the transfection of the microRNA-374a overexpression plasmid into glioma stem cells and the efficient expression of mature microRNA-374a. The MTT and flow cytometry results showed that the proliferation inhibition rate was significantly higher in cells from the microRNA-374a transfection group than in cells from the microRNA-mut transfection group; additionally, the former cells presented significant cell cycle arrest. The Transwell experiments confirmed that the overexpression of microRNA-374a could significantly reduce the invasiveness of CD133+ glioma stem cells. Moreover, the high expression of microRNA-374a mediated by the magnetic nanoparticles effectively reduced the tumourigenicity of CD133+ glioma stem cells in nude mice. The luciferase assays revealed that mature microRNA-374a fragments could bind to the 3'UTR of Neuritin (NRN1), thereby interfering with Neuritin mRNA expression. The qRT-PCR and Western blotting results showed that the overexpression of microRNA-374a significantly reduced the expression of genes such as NRN1, CCND1, CDK4 and Ki67 in glioma stem cells. Thus, magnetic nanoparticles can efficiently mediate the transfection and expression of microRNA expression plasmids in mammalian cells. The overexpression of microRNA-374a can effectively silence NRN1 expression, thereby inhibiting the proliferation, invasion and in vivo tumourigenicity of human glioma stem cells.

MiR-218-5p inhibits the stem cell properties and invasive ability of the A2B5⁺CD133⁻ subgroup of human glioma stem cells.

MicroRNAs (miRs) act as oncogenes or tumor-suppressor genes, and regulate the proliferation, apoptosis, invasion, differentiation, angiogenesis and behavior of glioma stem cells, which are important in glioma development and recurrence. The present study was performed to investigate the impact of miR-218-5p on stem cell properties and invasive ability of the A2B5+CD133- human glioma stem cell subgroup. qRT-PCR was used to detect miR-218-5p expression in non-cancerous brain and human glioma tissues, human glioma cell lines and human glioma stem cell lines. Lentivirus vectors encoding miR-218-5p and anti-miR-218-5p were constructed and stably transfected into A2B5+CD133- SHG-139s cells. Neurosphere formation Cell Counting Kit-8 (CCK-8) and Transwell assays, immunofluorescence and qRT-PCR analyses were used to explore the role of miR-218-5p in SHG-139s cells. qRT-PCR analysis showed that miR-218-5p expression was lower in human glioma tissues and cells than in non-cancerous brain tissues and normal human astrocyte cells, and lower in A2B5+CD133- (SHG-139s) cells than in CD133+ (SU2 and U87s) cells. The CCK-8 assay demonstrated that the growth curve was significantly inhibited in the miR-218-5p-SHG-139s cells compared to the miR-control, blank and anti-miR‑218-5p groups. The neurosphere formation assay indicated that upregulation of miR‑218-5p expression inhibited SHG-139s neurosphere formation. Immunofluorescence staining and qRT-PCR showed that miR-218-5p reduced stem cell marker (A2B5, nestin, PLAGL2, ALDH1 and Sox2) expression compared with the controls; however, immunofluorescence staining analysis showed that upregulation of miR-218-5p expression led to no difference in CD133 expression. miR‑218-5p reduced SHG-139s cell invasiveness in the Transwell assay and reduced MMP9 expression as detected in qRT-PCR and immunofluorescence analyses. All differences were statistically significant. miR-218-5p expression was lower in human glioma tissues, cells and the A2B5+CD133- human glioma stem cell subgroup. miR-218-5p may be a tumor-suppressor gene in glioma that functions by upregulating miR-218-5p expression, which inhibits the stem cell properties and invasive properties of SHG-139s cells.

MPTH-18STREAMS, VORTICES, AND NEUROSPHERES: IN VIVO NON-RANDOM LARGE SCALE SELF-ORGANIZATION OF GLIOBLASTOMA MULTIFORME AND THE ROLE OF NOVEL HISTOPATHOLOGICAL STRUCTURES

GBM (WHO IV) histological structures used in glioma diagnosis are pseudopalisades, microvascular proliferation, and necrosis. To these we can add Scherer's structures: the perivascular, perineuronal, white matter, and subpial growth of glioma cells. Though CXCL12 is important in the organization of Scherer's structures, the molecular and cellular basis of the other histopathological structures is not fully understood. To determine the large scale organization of gliomas we studied various models, including transplantable gliomas (mouse, human glioma stem cells), and genetically induced gliomas using the Sleeping Beauty Transposase system (NRas, LgT, sh-p53, PDGF, sh-ATRX, IDH1mut). Examination of all glioma models indicates the existence of streams, a hallmark of self-organization. Streams are 10-20 cells across, of various lengths, and distributed throughout the tumors. Within streams cells are fusiform and at the tumor border participate in glioma invasion. Streams can be understood mathematically using models from statistical mechanics. Experimentally, streams facilitated the migration of slower migrating glioma stem cells. We propose that streams accelerate glioma growth; inhibiting stream formation may be beneficial therapeutically. We also detected the presence of vortices in which cells appear as if rotating around a central axis. Vortices were found in murine transplantable models and in genetically induced gliomas -independently of individual genetic mutations-. Vortices can be studied mathematically using the theories of fluid dynamics. We propose that vortices are cores of glioma stem cells that nucleate brain tumor growth around them. Disrupting vortex formation could also lead to novel therapies. Our work demonstrates that gliomas display large scale, non-random, self-organization. We show the existence of novel histopathological structures in glioblastoma, and their potential role in glioma growth and invasion. We are currently working to understand their cellular, molecular, and mathematical basis as this will make them into novel so far unknown therapeutic targets.

CBIO-17EXPRESSION AND FUNCTIONAL ANALYSIS OF AN ONCOANTIGEN, DEPDC1, IN MALIGNANT GLIOMA

DEP domain containing 1 (DEPDC1) is a novel oncoantigen that is specically expressed protein in cancer cells and also have oncogenic activity and high immunogenicity. We demonstrated an overexpression of DEPDC1 in glioma tissues and are performing a clinical trial of vaccine therapy for recurrent malignant glioma using peptides of VEGF receptors and four oncoantigens of which DEPDC1 is a target molecule. In this study, we analyzed the expression and function of DEPDC1 in malignant glioma. mRNA and protein expression of DEPDC1 was shown to be increased in glioma cells and tumor tissues. Furthermore, we found an overexpression of DEPDC1 in human and mouse glioma stem cells. DEPDC1 was located in nucleus in glioma cells. Suppression of DEPDC1 by siRNA inhibited glioma cell proliferation and also interfered translocation of phosphorylated NFkB to nucleus. These results suggest that DEPDC1 may inhibit the apoptosis of glioma cells through NFkB signaling pathway and a possibility of molecular target therapy of DEPDC1 for the treatment of glioma.

STEM-15LONG NONCODING RNA REGULATION OF THE CANCER STEM CELL PHENOTYPE IN GLIOBLASTOMA MULTIFORME

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, with a two-year survival rate of less than 25%. The recurrence of GBM has been attributed to the presence of glioma stem cells (GSC), which are thought to play a central role in tumor development and progression. Long noncoding RNAs (lncRNAs) have been suggested to play a role in maintaining pluripotency, self-renewal, and differentiation in embryonic stem cells (ESCs). We hypothesized that lncRNAs functionally contribute to GBM development and tumor propagation by maintaining the cancer stem cell phenotype in glioblastoma. Initially, an in silico “nearest-neighbor” approach was employed to identify 112 lncRNA candidates that were close to the transcription factors that have been implicated in regulating self-renewal and pluripotency of ESCs or iPSCs, as well as factors that have been used to reprogram GSCs. Based on further in silico analyses and in vitro studies, we have identified three novel long noncoding RNAs, lincSox2, lincPOU5F1, and lincCTNNB1 that show differential expression in stem vs. differentiated normal human fetal neural stem cells (NSCs) and GSCs. The expression of lincSox2 and lincPOU5F1 significantly decreased in differentiated human NSCs compared to controls, and were significantly increased in differentiated GSCs compared to control GSCs. In comparison, the expression of lincCTNNB1 was increased in all differentiated cells, compared to their corresponding stem cell controls. Further knockdown experiments followed by in vivo studies will provide insight into functional relevance of these candidates in maintaining “stemness” in GSCs. Based on in silico and in vitro studies, we have identified two novel long noncoding RNAs that show differential expression in stem vs. differentiated NSCs and GSCs, and may functionally contribute to glioma biology by regulating the cancer stem cell phenotype. However, further characterization is needed to fully understand the role of lncRNAs in glioblastoma multiforme.

ATPS-77THE KETONE BODY ß-HYDROXYBUTYRATE RADIOSENSITIZES GLIOBLASTOMA MULTIFORME STEM CELLS

Glioblastoma multiforme (GBM) is a malignant brain tumor that typically recurs following standard treatment with surgery, radiation and chemotherapy. A rare population of radio-chemoresistant glioma stem cells (GSCs) is implicated in tumor repopulation and progression following treatment. Improvement in the prognosis of these patients requires the development of new therapeutic approaches. One emerging strategy is to target aberrant cell metabolism, a trait shared by virtually all tumor cells. The ketogenic diet (KD), a high fat, low carbohydrate and protein metabolic therapy has demonstrated anti-tumor effects alone, and in combination with radiation. The KD prolongs survival in animal glioma models, and when used in conjunction with radiation 9 of 11 mice were cured of their implanted tumors. We are now extending this work to determine if the ketone body, β-hydroxybutyrate (βHB) radiosensitizes human GSCs in vitro. The effects of βHB on growth were examined at various βHB concentrations in two GSC lines, L0 and L1. Both 5mM and 10mM βHB significantly reduced GSC growth. When coupled with 4Gy of radiation, 5mM βHB treatment significantly reduced L1 neurosphere formation and frequency, more than either βHB treatment or radiation alone. In L0, combination treatment significantly reduced neurosphere formation and frequency, more than radiation alone. To explore underlying mechanisms we are investigating the effect of βHB on DNA damage and repair in GSCs. Additionally, we are determining the effect of βHB on hypoxia within GSC neurospheres by analyzing expression of hypoxia inducible factor 1 alpha and the hypoxic marker carbonic anhydrase 9. We previously showed that the KD reduced the expression of these genes in a mouse glioma model. Taken altogether, these data suggest that βHB may sensitize GBMs to radiation by inhibiting growth of both glioma cells and GSCs, which may have implications for its use to treat tumor recurrence.

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro.

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

Abstract 2321: A hematopoietic stem cell factor drives brain tumor initiating cell genesis through Notch signaling

Abstract Glioblastomas (GBMs) maintain their cellular heterogeneity with glioma stem cells (GSCs) producing a variety of tumor cell types. Here, we interrogated the oncogenic roles of Lim domain only 2 (LMO2) in GBM and GSCs in mice and human. High expression of LMO2 was found in human patient-derived GSCs compared to the differentiated progeny cells. LMO2 is required for GSC growth both in vitro and in vivo, as shRNA-mediated LMO2 silencing attenuated tumor growth derived from human GSCs. Further, LMO2 is sufficient to induce stem cell characteristics (stemness) in mouse pre-malignant astrocytes, as forced LMO2 expression facilitated in vitro and in vivo growth of astrocytes derived from Ink4a/Arf null mice and acquisition of GSC phenotypes. A subset of mouse and human GSCs converted into vascular endothelial-like tumor cells both in vitro and in vivo, which phenotype was attenuated by LMO2 silencing and promoted by LMO2 overexpression. Mechanistically, the action of LMO2 for induction of glioma stemness is mediated by transcriptional regulation of Jagged1 resulting in activation of the Notch pathway, whereas LMO2 directly occupies the promoter regions of the VE-cadherin gene for a gain of endothelial cellular phenotype. Subsequently, selective ablation of human GSC-derived VE-cadherin-expressing cells attenuated vascular formation in mouse intracranial tumors, thereby significantly prolonging mouse survival. Clinically, LMO2 expression was elevated in GBM tissues and inversely correlated with prognosis of GBM patients. Taken together, our findings describe novel dual roles of LMO2 to induce tumorigenesis and angiogenesis, and provide potential therapeutic targets in MGs. Citation Format: Sunghak Kim, Eun-Jung Kim, Park Cheol Gyu, Se-Yeong Oh, Minata Mutsuko, Hyunggee Kim, Ichiro Nakano. A hematopoietic stem cell factor drives brain tumor initiating cell genesis through Notch signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2321. doi:10.1158/1538-7445.AM2015-2321

Abstract 510: A novel role for Olig2 in the regulation of glioma invasion

Abstract Glioblastoma multiforme (GBM) are highly infiltrative, aggressive and lethal primary brain tumors in adults that are resistant to conventional treatments. A major clinical obstacle in the treatment of GBMs is the ability of glioma cells to invade the surrounding brain parenchyma, preventing their complete surgical resection. Several studies have shown that invasive glioma stem cells (GSCs) extend beyond the surgically resected regions. These invasive cells are the cause for tumor recurrence and poor prognosis in GBM patients. The molecular and cellular mechanism involved in the regulation of the switch from proliferation to invasion/migration in GBM is poorly understood. We investigated the role of Olig2, a Central Nervous System (CNS) specific transcription factor, in promoting the switch from proliferating to invading glioma cell. Genetically relevant murine glioma model as well as patient-derived glioma stem cells (GSCs) were utilized to identify the effect of Olig2 on glioma invasion. Functionally, Olig2-mediated invasion was assessed by 3D-transwell invasion assays, organotypic slice cultures as well as PDX tumors in immunocompromised mice. In addition, murine and patient-derived GSCs were utilized to identify direct genetic targets of Olig2 involved in glioma invasion by ChIP-seq and RNA-seq. Our data demonstrate that Olig2-expressing murine glioma stem/progenitor-like cells are highly invasive as compared to Olig2 knockout cells. We further corroborated the requirement of Olig2 for glioma invasion in patient-derived GSCs by utilizing shRNA-mediated knockdown of Olig2. Olig2 depletion resulted in reduced glioma cell invasion in at least four independent human GSCs. Recent studies have shown that activation of transcription factors such as Zeb1 and Twist1 occurs in invasive high grade gliomas. We show that knockdown of Olig2 in murine as well as human GSCs leads to a significant decrease in the expression of invasion genes such as Zeb1, Vimentin, Twist1, and Stat3 as compared to control cells. In conclusion, our studies highlight a novel role for the Olig2 signalling network in promoting the switch from proliferation to invasion in GBM, and provide a plausible target for inhibiting glioma invasion. Citation Format: Shiv K. Singh, Nicole Giannonatti, Robert Kupp, Costanza Lo Cascio, Shwetal Mehta. A novel role for Olig2 in the regulation of glioma invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2015-510

Abstract 1174: Exploiting glioma stem cell metabolism to abrogate radioresistance in glioblastomas

Abstract Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and, even with aggressive treatment that includes surgical resection, radiation, and temozolomide administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem cells (GSCs) exist, which are thought to be radiation resistant and may be capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg effect. Experiments have shown that the cellular metabolism of GSCs differs from that of differentiated cells, making them an attractive target for novel therapeutic approaches. Much work has been done to analyze the metabolic profiles of GSCs with the goal of identifying potential therapeutic targets, but little data exists linking metabolic changes to radiation resistance. The purpose of this study is to determine the impact of radiation on the cellular metabolism of GSCs and to identify changes that can be exploited for treatment in conjunction with radiation therapy. To this end, we analyzed three human GSC lines harboring unique sets of genetic mutations that were hypothesized to differentially impact cellular metabolism. Experimental approaches include in vitro examination of metabolic rates and mitochondrial stress of the GSC lines via Seahorse extracellular flux analysis with and without radiation administration as well as analysis of tumor xenografts in mice via 13C MR spectroscopic imaging using hyperpolarized [1-13C] pyruvate as a tracer. Additionally, we are employing genomic and proteomic profiling of the GSC lines both in culture and in vivo to categorize metabolic changes following radiation treatment. We show that the basal metabolic rates of GSC lines with distinctive genetic mutations can be differentiated using Seahorse analysis. This is confirmed in vivo by 13C MR spectroscopic imaging, thus revealing a potential non-invasive diagnostic modality. The underlying mechanism behind these metabolic differences and how this relates to each GSC's response to radiation is currently being examined. We expect that this data will result in the identification of molecular targets within the metabolic pathways of cells that can be exploited with drugs in conjunction with radiation therapy to improve the prognosis and survival of patients presenting with GBMs in the clinic. Citation Format: Elizabeth I. Spehalski, Keita Saito, Tamalee Kramp, Anita Tandle, Barbara Rath, Murali Cherukuri, Kevin Camphausen. Exploiting glioma stem cell metabolism to abrogate radioresistance in glioblastomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1174. doi:10.1158/1538-7445.AM2015-1174

CRNDE affects the malignant biological characteristics of human glioma stem cells by negatively regulating miR-186.

The long non-coding RNA Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that activated early in colorectal neoplasia, but it is also up-regulated in many other solid tumors. Herein, the function and underlying mechanism of CRNDE in regulating glioma stem cells (GSCs) were investigated. We found that CRNDE expression was up-regulated while miR-186 expression was down-regulated in GSCs. Overexpression of CRNDE could promote the cellular proliferation, migration, invasion and inhibit the apoptosis in GSCs. Overexpression of miR-186 exerted functions of inhibiting the proliferation, migration and invasion of GSCs and promoting apoptosis. And CRNDE decreased the expression levels of XIAP and PAK7 by binding to miR-186 and negatively regulating it. In addition, miR-186 binded to XIAP and PAK7 3'UTR region, and decrease the expression of them, thus regulating the expression levels of downstream target proteins such as caspase 3, BAD, cyclin D1 and MARK2. The in vivo effect of CRNDE and miR-186 showed that the tumor formation rate was minimum in tumor-bearing nude mice with the knockdown of CRNDE and the overexpression of miR-186. In conclusion, CRNDE played an oncogenic role of GSCs through the negative regulation of miR-186. Both CRNDE and miR-186 could be regarded as potential targets in the glioma therapy.

MicroRNA-153/Nrf-2/GPx1 pathway regulates radiosensitivity and stemness of glioma stem cells via reactive oxygen species.

Glioma stem cells (GSCs) exhibit stem cell properties and high resistance to radiotherapy. The main aim of our study was to determine the roles of ROS in radioresistance and stemness of GSCs. We found that microRNA (miR)-153 was down-regulated and its target gene nuclear factor-erythroid 2-related factor-2 (Nrf-2) was up-regulated in GSCs compared with that of non-GSCs glioma cells. The enhanced Nrf-2 expression increased glutathione peroxidase 1 (GPx1) transcription and decreased ROS level leading to radioresistance of GSCs. MiR-153 overexpression resulted in increased ROS production and radiosensitization of GSCs. Moreover, miR-153 overexpression led to decreased neurosphere formation capacity and stem cell marker expression, and induced differentiation through ROS-mediated activation of p38 MAPK in GSCs. Nrf-2 overexpression rescued the decreased stemness and radioresistance resulting from miR-153 overexpression in GSCs. In addition, miR-153 overexpression reduced tumorigenic capacity of GSCs and increased survival in mice bearing human GSCs. These findings demonstrated that miR-153 overexpression decreased radioresistance and stemness of GSCs through targeting Nrf-2/GPx1/ROS pathway.

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation

To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry. The fraction of CD133(+) cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion-induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.

Sevoflurane promotes the expansion of glioma stem cells through activation of hypoxia-inducible factors in vitro

Growing evidences indicate that inhalational anaesthetics can enhance the growth and malignant potential of tumour cells and may affect tumour recurrence after surgery. Tumour stem cells play a vital role in tumour recurrence. This study investigates the effect of sevoflurane on glioma stem cells (GSCs) in vitro and the underlying molecular mechanisms in this process. Cultured GSCs were exposed to clinically relevant concentrations and durations of sevoflurane exposure. Cell proliferation and self-renewal capacity were determined. Expression of the stem cell marker CD133, vascular endothelial growth factor (VEGF), hypoxia-inducible factors (HIFs), and phosphorylated Akt, which is a protein kinase invoved in multiple cellular processes, were measured using western blotting. Small interfering RNAs and an Akt inhibitor were used to investigate specific pathways. Compared with controls, cells exposed to 2% sevoflurane for 6 h induced a larger number of proliferated cells (31.2±7.6% vs 19.0±5.8%; P<0.01). Levels of CD133, VEGF, HIF-1α, HIF-2α, and p-Akt were up-regulated by sevoflurane in a time- and concentration-dependent manner. Small interfering RNA against HIFs decreased the percentage of proliferating GSCs after sevoflurane exposure and pre-treatment of cells with an Akt inhibitor abrogated the expression of HIFs induced by sevoflurane. Sevoflurane can promote the expansion of human GSCs through HIFs in vitro. Inhaled anaesthetics may enhance tumour growth through tumour stem cells.

Loss of CX3CR1 increases accumulation of inflammatory monocytes and promotes gliomagenesis

The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b+CD45hiCX3CR1lowLy-6ChiLy-6G-F4/80-/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.

The LIM-only transcription factor LMO2 determines tumorigenic and angiogenic traits in glioma stem cells.

Glioblastomas (GBMs) maintain their cellular heterogeneity with glioma stem cells (GSCs) producing a variety of tumor cell types. Here we interrogated the oncogenic roles of Lim domain only 2 (LMO2) in GBM and GSCs in mice and human. High expression of LMO2 was found in human patient-derived GSCs compared with the differentiated progeny cells. LMO2 is required for GSC proliferation both in vitro and in vivo, as shRNA-mediated LMO2 silencing attenuated tumor growth derived from human GSCs. Further, LMO2 is sufficient to induce stem cell characteristics (stemness) in mouse premalignant astrocytes, as forced LMO2 expression facilitated in vitro and in vivo growth of astrocytes derived from Ink4a/Arf null mice and acquisition of GSC phenotypes. A subset of mouse and human GSCs converted into vascular endothelial-like tumor cells both in vitro and in vivo, which phenotype was attenuated by LMO2 silencing and promoted by LMO2 overexpression. Mechanistically, the action of LMO2 for induction of glioma stemness is mediated by transcriptional regulation of Jagged1 resulting in activation of the Notch pathway, whereas LMO2 directly occupies the promoter regions of the VE-cadherin gene for a gain of endothelial cellular phenotype. Subsequently, selective ablation of human GSC-derived VE-cadherin-expressing cells attenuated vascular formation in mouse intracranial tumors, thereby significantly prolonging mouse survival. Clinically, LMO2 expression was elevated in GBM tissues and inversely correlated with prognosis of GBM patients. Taken together, our findings describe novel dual roles of LMO2 to induce tumorigenesis and angiogenesis, and provide potential therapeutic targets in GBMs.