Glioma-initiating Cells Research Articles

Interferon-β exposure induces a fragile glioblastoma stem cell phenotype with a transcriptional profile of reduced migratory and MAPK pathway activity.

BackgroundType I interferons (IFN-α/β) are cytokines that are typically expressed in response to double-stranded RNA associated with viral infections. Glioblastomas are the most common malignant primary brain tumors, characterized by an infiltrative growth pattern and prominent angiogenic activity, and thought to be maintained by a subpopulation of glioma-initiating (stem-like) cells (GICs). The growth of human GIC lines is highly sensitive to IFN-β.MethodsRepetitive pulse stimulation with IFN-β1a (IS) was used to generate IS sublines that had acquired resistance to IFN-β-induced suppression of sphere formation. These cell lines were characterized by analyses of type 1 IFN signaling, growth patterns, and transcriptomic profiles.ResultsHere we report that repetitive IFN-β1a stimulation (IS) induces a stable phenotype (referred to as IS) at the level of maintaining sphere formation, although classical IFN signaling defined by the expression of both IFN receptors, myxovirus resistance protein A (MxA) accumulation, and STAT1 induction is unaffected. Furthermore, this stably altered IS phenotype is characterized by constitutively decreased sphere formation capacity and morphological features of senescence and autophagy. Transcriptional profiling reveals increased type I IFN signaling in these IS cells, but decreased expression of genes involved in receptor signaling and cell migration.ConclusionsAltogether, these data suggest a role for promoting IFN-β signaling in glioblastoma and might provide clues to design future therapeutic approaches.

CBMS-03 PHOSPHORYLATION STATE OF OLIG2 REGULATES PROLIFERATION OF GLIOMA STEM CELLS

The Cancer Genome Atlas project described a robust gene expression-based molecular classification of glioblastoma with the functional and biological significance of the subclasses yet to be determined. Here, we show that a comprehensive analysis of a panel of glioma initiating cell (GIC) lines can identify a group of stem cells with high OLIGg2 expression as in Proneural-like GBM subtype. In vitro differentiation studies showed that proneural GIC lines possess the potential to differentiate into astrocytic, neuronal, and oligodendrocytic lineages, whereas mesenchymal GICs exhibited limited potential for neural lineage differentiation following retinoic acid induction. A considerable decline of OLIG2 in proneural GIC lines was observed following retinoic acid treatment. We also showed that OLIG2 is a functional marker associated with cell proliferation in Olig2-high GIC lines. In addition, OLIG2 inhibition disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and OLIG2. Inhibition of CDK2/CDK4 activity disrupted OLIG2-CDK2/CDK4 interactions and attenuated OLIG2 protein stability. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high OLIG2 expression.

BOT-02 2-METHYLTHIO MODIFICATION OF N6-ISOPENTENYLADENOSINE IN MITOCHONDRIAL TRNAS BY CDK5RAP1 PROMOTES THE MAINTENANCE OF GLIOMA-INITIATING CELLS

2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) is an evolutionally conserved modification that is found in mitochondrial (mt)-tRNAs. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) specifically converts N6-isopentenyladenosine (i6A) to ms2i6A at position A37 of four mt-DNA-encoded tRNAs, and the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Here, we report that the ms2 conversion mediated by CDK5RAP1 in mt-tRNAs is required to sustain glioma-initiating cell (GIC)-related traits. CDK5RAP1 maintained the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs. This regulation was not related to the translational control of mt-proteins. CDK5RAP1 abrogated the antitumor effect of i6A by converting i6A to ms2i6A and protected GICs from excessive autophagy triggered by i6A. The elevated activity of CDK5RAP1 contributed to the amelioration of the cytotoxic effect of i6A and promoted GIC maintenance. The hypoxic microenvironment in the tumor core activated CDK5RAP1, whose activity was inversely correlated with the oxygen concentration because of two [4Fe-4S] clusters in the enzyme. This work demonstrates that CDK5RAP1 is crucial for the detoxification of endogenous i6A and that GICs readily utilize this mechanism for survival.

Secretome analysis of patient-derived GBM tumor spheres identifies midkine as a potent therapeutic target

Glioblastoma (GBM) is the most lethal primary brain tumor with few treatment options. The survival of glioma-initiating cells (GICs) is one of the major factors contributing to treatment failure. GICs frequently produce and respond to their own growth factors that support cell proliferation and survival. In this study, we aimed to identify critical autocrine factors mediating GIC survival and to evaluate the anti-GBM effect of antagonizing these factors. Proteomic analysis was performed using conditioned media from two different patient-derived GBM tumor spheres under a growth factor-depleted status. Then, the antitumor effects of inhibiting an identified autocrine factor were evaluated by bioinformatic analysis and molecular validation. Proteins secreted by sphere-forming GICs promote cell proliferation/survival and detoxify reactive oxygen species (ROS). Among these proteins, we focused on midkine (MDK) as a clinically significant and pathologically relevant autocrine factor. Antagonizing MDK reduced the survival of GBM tumor spheres through the promotion of cell cycle arrest and the consequent apoptotic cell death caused by oxidative stress-induced DNA damage. We also identified PCBP4, a novel molecular predictor of resistance to anti-MDK treatment. Collectively, our results indicate that MDK inhibition is an important therapeutic option by suppressing GIC survival through the induction of ROS-mediated cell cycle arrest and apoptosis.

EXTH-63. LIPOSOMAL DELIVERY OF FERRITIN HEAVY CHAIN (FTH1) siRNA RESULTS IN INCREASED RADIATION SENSITIVITY IN PATIENT DERIVED GLIOMA INITIATING CELLS

Abstract Glioblastoma (GBM) is the most lethal and incurable brain cancer. Despite maximum treatment, high recurrence rates result in median survival of less than 2 years. A subset of treatment resistant cells within GBM known as glioma initiating cells (GICs) are implicated in recurrence and exhibit ferritin overexpression. High expression of ferritin is known to be associated with poor overall survival in GBM patients. Our previous finding showed that downregulation of the ferritin heavy chain subunit (FTH1) enhanced treatment sensitivity in astrocytoma cells. Recently, functional importance of FTH1 in GICs was demonstrated by shRNA silencing leading to ablation of tumorigenic ability. We therefore posited that disrupting iron metabolism through FTH1 knockdown would radiosensitize GICs. Thus we developed multivalent cationic liposomes that enabled delivery of FTH1 siRNA to patient derived GICs isolated from pro-neural (PN) and mesenchymal (Mes) GBM. In both subtypes, FTH1 loss led to increased apoptosis and reduced cell viability. When exposed to radiation, PN GICs were unable to repair and recover from DNA damage, leading to a further decrease in cell viability and a complete loss of reproductive clonogenic potential unlike their more radioresistant Mes counterparts. Moreover, expression of stem cell marker Nestin was markedly reduced in PN but not Mes GICs. Thus, FTH1 loss is detrimental to both GIC subtypes, it seems to be critical for maintaining stemness and radiation resistance of PN GICs. These findings further support the viability of FTH1 as a therapeutic target in GBM.

TMIC-12. TUMOR EDGE-DESTINED CELLS IN GBM CELLS IN A CELL-INTRINSIC MECHANISM

Abstract The highly infiltrative nature of glioblastoma (GBM) underscores limited response to current therapies and subsequent unfavorable clinical outcome. Despite the gross total resection of tumors located in the enhancing lesions, GBMs inevitably recur from the areas adjacent to the resection cavity that retains tumor cells with tumor-initiating capacity with therapy resistant nature (glioma-initiating cells: GICs). Here, we identified, in clinical GBM tumors, two mutually-exclusive glioma-initiating cell subpopulations in two different regions of GBM tumors, core- and edge-located glioma-initiating cells that co-exist in single tumors (Minata et al. Cell Reports. 2019). Following this observation, we further established patient-derived GBM clones from both tumor core and edge tissues, termed core-GICs and edge-GICs, and uncovered their distinct molecular signatures. Unexpectedly, we found that these two distinct GIC subpopulations retain the spatial identity, meaning that the core GICs locate themselves in the injected site, whereas the edge GICs initiated to form edge-like lesions, when xenografted into mouse brains. Through OMICs analyses, we identified CD38 as a key molecule to determine the edge phenotype both in vitro and in vivo. Collectively, our findings indicate, for the first time, that GBM cells are heterogeneous to be composed of tumor cells destined to be located in distinct regions of the tumors in a molecularly-defined manner.

DDIS-01. NEW DRUG DISCOVERY WITH DRUG RE-POSITIONING SYSTEM TOWARD GBM TREATMENT

Abstract INTRODUCTION Invasion of Glioblastoma (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 cells. Therefore, we hypothesized that the inhibition of actin polymerization with existing medication could lead to clinical GBM treatment directly in short time. The Drug Repositioning system is also known as drug repurposing or drug reprofiling and understood as the process of redeveloping a compound for use in a different disease. In this time, we would show the new direction of drug development with drug repositioning. MATERIALS AND METHODS 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. RESULTS Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing FAK signaling. Fluvoxamine showed no drug toxicity. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same doze of anti-depressant effect. CONCLUSIONS The SSRI group has big potential for clinical GBM treatment with low cost, short time and low side effect.

CSIG-06. DEPATUXIZUMAB MAFODOTIN (ABT-414)-INDUCED GLIOBLASTOMA CELL DEATH REQUIRES EGFR OVEREXPRESSION, BUT NOT PHOSPHORYLATION

Abstract Glioblastomas commonly (40%) exhibit epidermal growth factor receptor (EGFR) amplification, half of these tumors carry the EGFRvIII deletion variant characterized by an in-frame deletion of exons 2–7, resulting in constitutive EGFR activation. Although EGFR tyrosine kinase inhibitors had only modest effects in glioblastoma, novel therapeutic agents targeting amplified EGFR or EGFRvIII continue to be developed. Depatuxizumab mafodotin (ABT-414) is an antibody drug conjugate consisting of the monoclonal antibody 806 and, as its toxic payload, monomethyl auristatin F, designed to target EGFR-overexpressing tumor cells. Since long-term glioma cell lines and patient-derived glioma-initiating cell lines appeared to express too little EGFR in vitro to be sensitive to ABT-414, we generated glioma sublines overexpressing EGFR or EGFRvIII to explore the determinants of ABT-414-induced glioma cell death. Overexpression of EGFRvIII induces sensitization to ABT-414 more readily than overexpression of EGFR. There is no bystander killing of cells devoid of EGFR or EGFRvIII expression. Surprisingly, either exposure to EGF or to EGFR tyrosin kinase inhibitors reduce EGFR protein levels and are thus no strategies to promote ABT-414-induced cell killing. Furthermore, glioma cells overexpressing kinase-dead EGFR or EGFRvIII retain binding of mAb 806 and sensitivity to ABT-414, allowing to dissociate EGFR phosphorylation from the emergence of the “active” EGFR conformation required for ABT-414 binding. The combination of EGFR-targeting antibody drug conjugates with EGFR tyrosine kinase inhibitors carries a high risk of failure. Promoting mere EGFR expression rather than phosphorylation should result in glioblastoma cell sensitization to ABT-414 or related agents.

DDIS-04. INTERFERON-β SENSITIZES HUMAN GLIOBLASTOMA CELLS TO THE CYCLIN-DEPENDENT KINASE INHIBITOR, TG02

Abstract Novel treatments for glioblastoma, the most common malignant primary brain tumor, are urgently needed. Type I interferons (IFN) are natural cytokines primarily involved in defense against viral infections, but may also play a role in the control of cancer, notably in the suppression of the cancer stem cell phenotype. TG02 is a novel orally available cyclin-dependent kinase 9 inhibitor which induces glioma cell apoptosis without profound caspase activation and is currently being explored in early clinical trials in newly diagnosed and recurrent glioblastoma. Here we used human glioma-initiating cell line models to explore whether IFN-β modulate the anti-glioma activity of TG02. Pre-exposure to IFN-β sensitized human glioma models to a variable extent to subsequent exposure to TG02. Combination treatment was associated with increased DEVD-amc cleaving activity that was blocked by BCL2. However, BCL2 did not protect from the synergistic effects of IFN and TG02 on glioma cell growth, furthermore, although IFN strongly induced pro-apoptotic XIAP-associated factor (XAF) expression, disrupting XAF expression did not abrogate the synergy with TG02 either. Consistent with that, caspase 3 gene silencing did not abrogate effects of TG02 or IFN-β alone or in combination. Finally, we observed that IFN-β may indeed modulate the effects of TG02 up-stream in the signaling cascade since inhibition of RNA polymerase II phosphorylation, a direct readout of TG02 pharmacodynamic activity, was facilitated in glioma cells pre-exposed to IFN-β. In summary, these data suggest that type I IFN may be combined with TG02 to limit glioblastoma growth but the well characterized effects of IFN and TG02 on apoptotic signaling are dispensable for synergistic control of tumor growth. Instead, exploring how IFN signaling primes glioma cells for TG02-mediated direct target inhibition may help to design novel, more effective pharmacological approaches to glioblastoma.

Genetic and Lineage Classification of Glioma-Initiating Cells Identifies a Clinically Relevant Glioblastoma Model.

The Cancer Genome Atlas (TCGA) project described a robust gene expression-based molecular classification of glioblastoma (GBM), but the functional and biological significance of the subclasses has not been determined. The present comprehensive analysis of 25 glioma-initiating cell (GIC) lines classifies GIC lines into four subtypes (classical, mesenchymal, proneural, and neural) that are closely related to the TCGA GBM subclasses and display distinct lineage characteristics and differentiation behavior that recapitulate neural development. More importantly, the GIC subtypes exhibit distinct biological phenotypes in relation to self-renewal capacity, proliferation, invasiveness, and angiogenic potential in vitro and in vivo. In addition, the GIC subtypes exhibit divergent patterns of signaling pathway activation and deactivation of the Wnt, Notch, and TGF-β pathways. These results will improve drug discovery targeting certain genetic mutation in glioblastoma and improve the development of precision medicine.

2-Methylthio Conversion of N6-Isopentenyladenosine in Mitochondrial tRNAs by CDK5RAP1 Promotes the Maintenance of Glioma-Initiating Cells.

Summary2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) is an evolutionally conserved modification found in mitochondrial (mt)-tRNAs. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) specifically converts N6-isopentenyladenosine (i6A) to ms2i6A at position A37 of four mt-DNA-encoded tRNAs, and the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Here, we report that the ms2 conversion mediated by CDK5RAP1 in mt-tRNAs is required to sustain glioma-initiating cell (GIC)-related traits. CDK5RAP1 maintained the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs. This regulation was not related to the translational control of mt-proteins. CDK5RAP1 abrogated the antitumor effect of i6A by converting i6A to ms2i6A and protected GICs from excessive autophagy triggered by i6A. The elevated activity of CDK5RAP1 contributed to the amelioration of the tumor-suppressive effect of i6A and promoted GIC maintenance. This work demonstrates that CDK5RAP1 is crucial for the detoxification of endogenous i6A and that GICs readily utilize this mechanism for survival.

Identification of KLRC2 as a candidate marker for brain tumor-initiating cells

ABSTRACTObjective: Brain tumor-initiating cells are characterized by their features of self-renewal, multi-lineage differentiation, and tumorigenicity. We analyzed the gene expression of brain tumor-initiating cells to identify their novel cellular markers.Methods: We performed cDNA microarray, in silico expressed sequence tags (ESTs), RT-PCR, and q-PCR analyses.Results: We identified 10 genes that were more highly expressed in brain tumor-initiating cells than in neural stem cells. In addition, we identified 10 other genes that were more highly expressed in brain tumor-initiating cells than in glioma cell line cells from the cDNA microarray analysis. Using the EST database, we looked to see if the 20 genes were expressed more highly in gliomas, compared with normal adult brains. Among the 20 genes, five (KLRC2, HOXB2, KCNJ2, KLRC1, and COL20A1) were expressed more than twice in glioma samples, compared with normal adult brains, and, therefore, were referred for further evaluation. RT-PCR was conducted using cDNA samples obtained from neural stem cells, normal brain tissue, fetal brain tissue, glioma cell lines, and glioma tumor-initiating cell lines. KLRC2, a transmembrane activating receptor in natural killer cells, was expressed more highly in glioma-initiating cells than in neural stem cell lines or normal adult brain tissue. The q-PCR analysis revealed that expression of KLRC2 was significantly higher in brain tumor-initiating cells compared to normal brain controls.Conclusion: KLRC2 could be a novel cellular marker for brain tumor-initiating cells.

Tyrosine kinase Eph receptor A6 sensitizes glioma-initiating cells towards bone morphogenetic protein-induced apoptosis.

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation‐ and apoptosis‐inducing effects on glioma‐initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin‐producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP‐2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP‐2‐induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK‐2, which was associated with BMP‐induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain‐of‐function of EPHA6 together with BMP‐2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP‐2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.

Liposomal delivery of ferritin heavy chain 1 (FTH1) siRNA in patient xenograft derived glioblastoma initiating cells suggests different sensitivities to radiation and distinct survival mechanisms.

Elevated expression of the iron regulatory protein, ferritin heavy chain 1 (FTH1), is increasingly being associated with high tumor grade and poor survival outcomes in glioblastoma. Glioma initiating cells (GICs), a small population of stem-like cells implicated in therapeutic resistance and glioblastoma recurrence, have recently been shown to exhibit increased FTH1 expression. We previously demonstrated that FTH1 knockdown enhanced therapeutic sensitivity in an astrocytoma cell line. Therefore, in this study we developed a liposomal formulation to enable the in vitro delivery of FTH1 siRNA in patient xenograft derived GICs from glioblastomas with pro-neural and mesenchymal transcriptional signatures to interrogate the effect of FTH1 downregulation on their radiation sensitivity. Transfection with siRNA decreased FTH1 expression significantly in both GICs. However, there were inherent differences in transfectability between pro-neural and mesenchymal tumor derived GICs, leading us to modify siRNA: liposome ratios for comparable transfection. Moreover, loss of FTH1 expression resulted in increased extracellular lactate dehydrogenase activity, executioner caspase 3/7 induction, substantial mitochondrial damage, diminished mitochondrial mass and reduced cell viability. However, only GICs from pro-neural glioblastoma showed marked increase in radiosensitivity upon FTH1 downregulation demonstrated by decreased cell viability, impaired DNA repair and reduced colony formation subsequent to radiation. In addition, the stemness marker Nestin was downregulated upon FTH1 silencing only in GICs of pro-neural but not mesenchymal origin. Using liposomes as a siRNA delivery system, we established FTH1 as a critical factor for survival in both GIC subtypes as well as a regulator of radioresistance and stemness in pro-neural tumor derived GICs. Our study provides further evidence to support the role of FTH1 as a promising target in glioblastoma.

P11.45 Synergistic growth inhibition mediated by dual PI3K/mTOR pathway targeting and genetic or direct pharmacological AKT inhibition in human glioblastoma models

Abstract BACKGROUND Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have had limited success to date. MATERIAL AND METHODS Here we analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition, and effects on proliferation, and clonogenicity or spherogenicity as readouts, in an extended panel of human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC). RESULTS Glioma cells, including GIC in particular, were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist in vitro. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. In vivo studies confirmed the anti-glioma activity of PQR309 in the orthotopic LN-229 glioma model. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3ß) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition. CONCLUSION These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.

Mesenchymal transition and increased therapy resistance of glioblastoma cells is related to astrocyte reactivity

Grade IV astrocytoma/glioblastoma multiforme (GBM) is essentially incurable, partly due to its heterogenous nature, demonstrated even within the glioma-initiating cell (GIC) population. Increased therapy resistance of GICs is coupled to transition into a mesenchymal (MES) cell state. The GBM MES molecular signature displays a pronounced inflammatory character and its expression vary within and between tumors. Herein, we investigate how MES transition of GBM cells relates to inflammatory responses of normal astroglia. In response to CNS insults astrocytes enter a reactive cell state and participate in directing neuroinflammation and subsequent healing processes. We found that the MES signature show strong resemblance to gene programs induced in reactive astrocytes. Likewise, astrocyte reactivity gene signatures were enriched in therapy-resistant MES-like GIC clones. Variable expression of astrocyte reactivity related genes also largely defined intratumoral GBM cell heterogeneity at the single-cell level and strongly correlated with our previously defined therapy-resistance signature (based on linked molecular and functional characterization of GIC clones). In line with this, therapy-resistant MES-like GIC secreted immunoregulatory and tissue repair related proteins characteristic of astrocyte reactivity. Moreover, sensitive GIC clones could be made reactive through long-term exposure to the proinflammatory cytokine interleukin 1 beta (IL1β). IL1β induced a slow MES transition, increased therapy resistance, and a shift in DNA methylation profile towards that of resistant clones, which confirmed a slow reprogramming process. In summary, GICs enter through MES transition a reactive-astrocyte-like cell state, connected to therapy resistance. Thus, from a biological point of view, MES GICs would preferably be called 'reactive GICs'. The ability of GBM cells to mimic astroglial reactivity contextualizes the immunomodulatory and microenvironment reshaping abilities of GBM cells that generate a tumor-promoting milieu. This insight will be important to guide the development of future sensitizing therapies targeting treatment-resistant relapse-driving cell populations as well as enhancing the efficiency of immunotherapies in GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 2621: OLIG2 regulates stem cell maintenance and cell cycle in glioma stem cells

Abstract The Cancer Genome Atlas project described a robust gene expression-based molecular classification of glioblastoma with the functional and biological significance of the subclasses yet to be determined. Here, we show that a comprehensive analysis of a panel of glioma initiating cell (GIC) lines can identify a group of stem cells with high OLIGg2 expression as in Proneural-like GBM subtype. In vitro differentiation studies showed that proneural GIC lines possess the potential to differentiate into astrocytic, neuronal, and oligodendrocytic lineages, whereas mesenchymal GICs exhibited limited potential for neural lineage differentiation following retinoic acid induction. A considerable decline of OLIG2 in proneural GIC lines was observed following retinoic acid treatment. We also showed that OLIG2 is a functional marker associated with cell proliferation in Olig2-high GIC lines. In addition, OLIG2 inhibiton disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and OLIG2. Inhibition of CDK2/CDK4 activity disrupted OLIG2-CDK2/CDK4 interactions and attenuated OLIG2 protein stability. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high OLIG2 expression. Citation Format: Norihiko Saito, Nozomi Hirai, Kazuya Aoki, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. OLIG2 regulates stem cell maintenance and cell cycle in glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2621.

Co-cultures of Glioma Stem Cells and Primary Neurons, Astrocytes, Microglia, and Endothelial Cells for Investigation of Intercellular Communication in the Brain.

Intercellular communication within complex biological and pathological systems via extracellular vesicles (EVs) and secreted factors is a highly attractive area of research. However, cell models enabling investigation of such communication in vitro are limited. Commonly utilized is the supplementation of hyper-concentrated EVs or other extracellular factors to the recipient cell cultures. This approach requires purification of the secreted complexes and is confounded by the contamination of media components. Two-chamber co-cultures of donor and recipient cells separated by a pore membrane may represent a more physiological and better-controlled system for the investigation of intercellular communication. Yet, distinct culture conditions for different neural cell types often make them incompatible for co-culturing. Here we optimized short-term co-cultures of patient-derived low-passage glioma-initiating stem cells with normal cells of the brain microenvironment, such as primary neurons, astrocytes, microglia, and brain endothelial cells. We demonstrate the culture compatibility of these cell types and internalization of glioma-derived extracellular RNA by the normal recipient cells. The presented protocols are valuable for the investigation of intercellular communication between glioma brain tumor and cells of its microenvironment, including but not limited to the EVs-mediated communication.RESEARCH IN CONTEXTCell-to-cell communication is essential in normal physiology and implicated in disease; however, experimental systems for its modeling in vitro are limited. Particularly, the investigation of communication between brain tumors and normal cells of the brain microenvironment has been challenged by the lack of adequate culture models. Here we developed co-cultures of glioma stem cells with various types of normal brain cells, including primary neurons, astrocytes, microglia, and brain endothelial cells, and demonstrated their utility for the study of intercellular communication. Detection of proposed markers in the recipient cells confirmed RNA transfer in these co-cultures.

Radiosensitization of orthotopic GIC-driven glioblastoma by doxycycline causes skin damage

Doxycycline (DXC) is a tetracycline antibiotic which has been proposed as a breast cancer radiosensitizer by specifically reducing the expression of the DNA repair enzyme DNA PK in breast cancer initiating cells. Since DXC presents favorable pharmacokinetics properties including the capacity to cross the blood-brain barrier, it has been hypothesized that it could radiosensitize brain tumors as well. We have investigated the radiosensitizing capacity of DXC towards human glioma initiating cells (GIC)-driven orthotopic glioblastomas (GB) in NOD/SCID mice that faithfully mimic the growth properties of the clinical tumors of origin. DXC at 10 mg/Kg body weight was subcutaneously delivered daily, 5 days/week for 4 weeks. At the same time, radiotherapeutic fractions of 0.25 Gy to the head were delivered every 3–4 days (twice/week) for 15 weeks. No survival advantage was observed in DXC-treated mice as compared to vehicle-treated mice by this radiosensitizing protocol. On the contrary, skin damage with hair loss and deep ulcers were observed after 4 weeks in DXC-treated mice leading to discontinuation of drug treatment. These results do not support the use of DXC as a radiosensitizer for brain tumors and indicate skin damage as an important side effect of DXC.

Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

Emerging evidence reveals enrichment of glioma-initiating cells (GICs) following therapeutic intervention. One factor known to contribute to this enrichment is cellular plasticity—the ability of glioma cells to attain multiple phenotypes. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open-chromatin marks in known astrocytic enhancers for interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p = 0.001) and positively correlated with that of genes associated with the GIC phenotypes, such as KLF4, c-Myc, and HIF2α (p < 0.001). Immunohistochemical analysis of patient samples demonstrated elevated IL-8 expression in about 60% of recurrent GBM tumors relative to matched primary tumors and this expression also positively correlates with time to recurrence. Exposure to IL-8 significantly enhanced the self-renewing capacity of PDX GBM (average threefold, p < 0.0005), as well as increasing the expression of GIC markers in the CXCR2 population. Furthermore, IL-8 knockdown significantly delayed PDX GBM tumor growth in vivo (p < 0.0005). Finally, guided by in silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that autocrine IL-8 alters cellular plasticity and mediates alterations in histone status. These findings suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.