Glioblastoma Multiforme Proliferation Research Articles

CXCR4 antagonism sensitizes cancer cells to novel indole-based MDM2/4 inhibitors in glioblastoma multiforme

Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization. Notably, CXCR4 and MDM2-4 cooperate in promoting tumour invasion and progression. Although CXCR4 actively promotes MDM2 activation leading to p53 inactivation, MDM2-4 knockdown induces the downregulation of CXCR4 gene transcription.Our study aimed to assess if the CXCR4 signal blockade could enhance glioma cells’ sensitivity to the inhibition of the p53-MDMs axis. Rationally designed inhibitors of MDM2/4 were combined with the CXCR4 antagonist, AMD3100, in human GBM cells and GBM stem-like cells (neurospheres), which are crucial for tumour recurrence and chemotherapy resistance. The dual MDM2/4 inhibitor RS3594 and the CXCR4 antagonist AMD3100 reduced GBM cell invasiveness and migration in single-agent treatment and mainly in combination. AMD3100 sensitized GBM cells to the antiproliferative activity of RS3594. It is noteworthy that these two compounds present synergic effects on cancer stem components: RS3594 inhibited the growth and formation of neurospheres, AMD3100 induced differentiation of neurospheres while enhancing RS3594 effectiveness preventing their proliferation/clonogenicity. These results confirm that blocking CXCR4/MDM2/4 represents a valuable strategy to reduce GBM proliferation and invasiveness, acting on the stem cell component too.

MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways.

Glioblastoma multiforme (GBM) is a devastating disease yet no effective drug treatment has been established to date. Glioblastoma stem-like cells (GSCs) are insensitive to treatment and may be one of the reasons for the relapse of GBM. Maternal embryonic leucine zipper kinase gene (MELK) plays an important role in the malignant proliferation and the maintenance of GSC stemness properties of GBM. However, the therapeutic effect of targeted inhibition of MELK on GBM remains unclear. This study analyzed the effect of a MELK oral inhibitor, OTSSP167, on GBM proliferation and the maintenance of GSC stemness. OTSSP167 significantly inhibited cell proliferation, colony formation, invasion, and migration of GBM. OTSSP167 treatment reduced the expression of cell cycle G2/M phase-related proteins, Cyclin B1 and Cdc2, while up-regulation the expression of p21 and subsequently induced cell cycle arrest at the G2/M phase. OTSSP167 effectively prolonged the survival of tumor-bearing mice and inhibited tumor cell growth in in vivo mouse models. It also reduced protein kinase B (AKT) phosphorylation levels by OTSSP167 treatment, thereby disrupting the proliferation and invasion of GBM cells. Furthermore, OTSSP167 inhibited the proliferation, neurosphere formation and self-renewal capacity of GSCs by reducing forkhead box M1 (FOXM1) phosphorylation and transcriptional activity. Interestingly, the inhibitory effect of OTSSP167 on the proliferation of GSCs was 4-fold more effective than GBM cells. In conclusion, MELK inhibition suppresses the growth of GBM and GSCs by double-blocking AKT and FOXM1 signals. Targeted inhibition of MELK may thus be potentially used as a novel treatment for GBM.

Gradient hydrogels for screening stiffness effects on patient-derived glioblastoma xenograft cellfates in 3D.

Brain cancer is a devastating disease given its extreme invasiveness and intricate location. Glioblastoma multiforme (GBM) is one of the most common forms of brain cancer, and cancer progression is often correlated with significantly altered tissue stiffness. To elucidate the effect of matrix stiffness on GBM cell fates, previous research is largely limited to 2D studies using immortalized cell lines, which has limited physiological relevance. The objective of the study is to develop gradient hydrogels with brain-mimicking stiffness range as a 3Din vitro GBM model for screening of the effects of matrix stiffness on GBM. To increase the physiological relevance, patient-derived tumor xenograft (PDTX) GBM cells were used. Our gradient platform allows formation of cell-containing hydrogels with stiffness ranging from 40 Pa to 1,300 Pa within a few minutes. By focusing on a brain-mimicking stiffness range, this gradient hydrogel platform is designed for investigating brain cancer. Increasing stiffness led to decreased GBM proliferation and less spreading, which is accompanied by downregulation of matrix-metalloproteinases (MMPs). Using temozolomide (TMZ) as a model drug, we demonstrate that increasing stiffness led to higher drug resistance by PDTX GBM cells in 3D, suggesting matrix stiffness can directly modulate how GBM cells respond to drug treatment. While the current study focuses on stiffness gradient, the setup may also be adapted for screening other cancer niche cues such as how biochemical ligand gradient modulates brain cancer progression and drug responses using reduced materials and time.

The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme.

The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

Largazole is a Brain-Penetrant Class I HDAC Inhibitor with Extended Applicability to Glioblastoma and CNS Diseases.

Largazole is a potent class I selective histone deacetylase inhibitor prodrug with anticancer activity against solid tumors in preclinical models. Largazole possesses in vitro activity against glioblastoma multiforme (GBM) cells and sufficiently crosses the blood-brain barrier based on measurement of the active species, largazole thiol, to achieve therapeutically relevant concentrations in the mouse brain. The effective dose resulted in pronounced functional responses on the transcript level based on RNA sequencing and quantitative polymerase chain reaction after reverse transcription (RT-qPCR), revealing desirable expression changes of genes related to neuroprotection, including Bdnf and Pax6 upregulation, extending the applicability of largazole to the treatment of brain cancer and neurodegenerative disorders. The largazole-induced modulation of Pax6 unifies both activities, since Pax6 expression suppresses GBM proliferation and invasion and inversely correlates with GBM tumor grade, while it is also implicated in neurogenesis, neuronal plasticity, and cognitive ability. Our results suggest that largazole could be repurposed for diseases of the brain.

MicroRNA-128-3p Enhances the Chemosensitivity of Temozolomide in Glioblastoma by Targeting c-Met and EMT

Temozolomide is a first line anti-tumor drug used for the treatment of patients with Glioblastoma multiforme (GBM). However, the drug resistance to temozolomide limits its clinical application. Therefore, novel strategies to overcome chemoresistance are desperately needed for improved treatment of human GBM. Recent studies have demonstrated that miRNAs are closely related to resistance to cancer chemotherapy. This study aimed to further validate the biological role of miR-128-3p and to investigate whether miR-128-3p can enhance the chemosensitivity of glioblastoma to temozolomide (TMZ) and the underlying mechanisms. The effects of miR-128-3p and TMZ on the proliferation of glioblastoma cells were investigated by cell counting kit-8 (cck8). Transwell and intracerebral invasion assays were applied to determine the effects of the combination of miR-128-3p and TMZ on the invasion and migration of glioblastoma in vitro and in vivo. Flow cytometry was used to detect apoptosis in each group, and immunofluorescence was used to determine the expression levels of EMT-related proteins. RT-PCR and Western-blot were applied to detect EMT-transformed proteins (c-Met, PDGFRα, Notch1, and Slug) and EMT phenotype-associated proteins (Vim, CD44, and E-cadherin) at both mRNA and protein levels. Based on the microRNA.org database, we predicted the target genes of miR-128-3p. The target-relationship between miR-128-3p and c-Met and PDGFRα was verified by dual luciferase reporter gene. The tumor volume, weight and the expression levels of the proteins described above were measured in subcutaneously transplanted tumor model in nude mice. We found that the expression of miR-128-3p was down-regulated in glioblastoma tissue samples and cell lines. miR-128-3p suppressed the proliferation, migration, and invasion of GBM both in vitro and in vivo; miR-128-3p enhanced the therapeutic effect of TMZ via inhibition of proliferation, invasion and migration of glioblastoma cells and induction of apoptosis. Overexpression of miR-128-3p down-regulated the expression levels of EMT-transformed proteins (c-Met, PDGFRα, Notch1 and Slug) to enhance the effect of TMZ. In addition, we found that miR-128-3p targeted and bound c-Met. More importantly, the upregulation of c‐Met significantly prompted U87 and U251 cell proliferation. This effect could be abolished when c‐Met was silenced. The investigation in tumor bearing nude mice showed that miR-128-3p in combination with TMZ reduced tumor volume and the invasion extent, and increased the sensitivity of glioblastoma to TMZ. miR-128-3p is capable of enhancing the sensitivity of glioblastoma to TMZ through regulating c-Met/EMT.

Inhibition of glioblastoma and macrophage phagocytosis using sialic acid-grafted tamoxifen-carmustine-polyethyleneimine-poly(lactic-co-glycolic acid) nanoparticles

Management of glioblastoma multiforme (GBM), a cerebral glioma with highest-grade malignancy, is a crucial challenge to current anticancer treatment because the lack of GBM-targeting capability of antitumor drug, such as carmustine (BCNU) with a short half-life, along with low blood-brain barrier (BBB) permeability to achieve the clinical efficacy. Hence, polyethyleneimine-poly(lactic-co-glycolic acid) nanoparticles comprising stable core-shell structure with tamoxifen were crosslinked with sialic acid (SA-TAM-PEI-PLGA NPs) as nanocarriers to improve bioavailability of BCNU for pharmacotherapy. The brain-targeted experiments revealed that an increasing SA level improved BBB permeability for BCNU, and was associated with a reduction in transendothelial electrical resistance and an increase in propidium iodide penetration of endothelial monolayer. Immunocytochemical staining images evidenced that surface SA was critical in triggering transcytosis through N-acetylglucosamine on human cerebral microvessel endothelia, and surface crosslinking restrained macrophage phagocytosis. A high TAM level on the carrier surface reduced the survival rate of human U87, and western blots showed that conjugated TAM induced apoptosis of the cancer cells. The use of SA-TAM-PEI-PLGA NPs carrying BCNU as a pharmaceutical preparation is an effective strategy to overcome the BBB restriction and activate apoptosis of tumor cells to prevent the proliferation of GBM.

Targeted delivery of miRNA based therapeuticals in the clinical management of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive (WHO grade IV) form of diffuse glioma endowed with tremendous invasive capacity. The availability of narrow therapeutic choices for GBM management adds to the irony, even the post-treatment median survival time is roughly around 14-16 months. Gene mutations seem to be cardinal to GBM formation, owing to involvement of amplified and mutated receptor tyrosine kinase (RTK)-encoding genes, leading to dysregulation of growth factor signaling pathways. Of-late, the role of different microRNAs (miRNAs) in progression and proliferation of GBM was realized, which lead to their burgeon potential applications for diagnostic and therapeutic purposes. miRNA signatures are intricately linked with onset and progression of GBM. Although, progression of GBM causes significant changes in the BBB to form BBTB, but still efficient passage of cancer therapeutics, including antibodies and miRNAs are prevented, leading to low bioavailability. Recent developments in the nanomedicine field provide novel approaches to manage GBM via efficient and brain targeted delivery of miRNAs either alone or as part of cytotoxic pharmaceutical composition, thereby modulating cell signaling in well predicted manner to promise positive therapeutic outcomes.

The CXCL12/CXCR4 axis confers temozolomide resistance to human glioblastoma cells via up-regulation of FOXM1

Glioblastoma multiforme (GBM) is the most common primary malignancy in the adult central nervous, and is characterized by high aggressiveness and a high mortality rate. The high mortality rate is largely due to the development of drug resistance. Temozolomide (TMZ) resistance is considered to be one of the major reasons responsible for GBM therapy failure. CXCL12/CXCR4 has been demonstrated to be involved in cell proliferation, migration, invasion, angiogenesis, and radioresistance in GBM. However, its role in TMZ resistance in GBM is unknown. In this study, we aimed to evaluate the role of CXCL12/CXCR4 in mediating the TMZ resistance to GBM cells and explore the underlying mechanisms. We found that the CXCL12/CXCR4 axis enhanced TMZ resistance in GBM cells. Further study showed that CXCL12/CXCR4 conferred TMZ resistance and promoted the migration and invasion of GBM cells by up-regulating FOXM1. This resistance was partially reversed by suppressing CXCL12/CXCR4 and FOXM1 silencing. Our study revealed the vital role of CXCL12/CXCR4 in mediating the resistance of GBM cells to TMZ, and suggested that targeting CXCL12/CXCR4 axis may attenuate the resistance to TMZ in GBM.

Gene Delivery of RNA Therapeutics for the Treatment of Brain Tumors

The proliferation of glioblastoma multiforme (GBM) is often coupled to the dysregulation, amplification and overexpression of tyrosine kinase receptors (TKRs) including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) among others. TKRs are cell‐surface receptors that bind to their cognate ligand, peptide hormones known as growth factors. Binding of the growth factor ligand stimulates TKRs, activating a signal‐transduction cascade that leads to cell growth, migration and the formation of tumor vasculature. A look at TKRs reveals a general architecture of the receptor. They are comprised of three distinct domains: an extracellular, transmembrane and an intracellular domain. As these receptors are often upregulated in GBM, an RNA Therapy to modulate the expression, inhibit the translation of critical TKR domains or reduce its activation would reduce the functionality of these oncogenic transcripts and proteins in GBM. Our research focuses on the development of AAV gene therapy vectors that encode novel RNA therapeutics to reduce the expression of oncogenic transcripts as well as inhibit their activation in GBM, the most common malignant primary brain tumor in adults. Individuals diagnosed with GBM have a short life expectancy of 12–15 months. The standard care and treatment as well as the developmental pipeline of new therapeutic strategies are often limited by the blood‐brain barrier (BBB), restricting systemically administered therapies from reaching the brain (Hicks et al., 2015). Thus, a novel strategy that circumvents this barrier is necessary to effectively reach the CNS tumor microenvironment. Direct AAV delivery of genes that encode RNA therapeutics at the time of tumor resection would bypass the BBB to deliver a persistent and constant dosage from the inside‐out. With the advancement of nanopore sequencing technology, we are able to read longer transcripts (>200kb). With this technology, we are able to examine the architecture of nascent pre‐mRNA transcripts, detect lower abundant alternatively spliced and polyadenylated isoforms, as well take advantage of RNA structurome analysis to reveal RNA elements within TKRs which are more susceptible to RNA anti‐sense directed therapies. To this end, we have developed a strategy to identify novel targets within oncogenic transcripts and incorporate these therapies directed against these targets into our AAV directed RNA gene therapy platforms. In addition, we use RNA mimics to take advantage of the RNA interference pathway to modulate and reduce expression of oncogenic transcripts. This research has the potential to identify novel RNA elements and alternatively spliced and polyadenylated isoforms of TKRs in health and disease. The ultimate goal of our research project is to build on current strategies to identify even better RNA elements of the EGFR and VEGFR2 transcripts which are structurally available for both microRNA degradation and anti‐sense directed alternative splicing therapy.

RNA Therapy Recruitment of hnRNP H to Alter the pre‐mRNA Splicing of the Epidermal Growth Factor Receptor Transcript in Human Glioblastoma

Glioblastoma Multiforme (GBM) is a prominent form of brain cancer that encompasses the formation of gliomata in astrocytes and oligodendrocytes of the central nervous system. GBM has a mortality rate of 15 months due to resistance to current standard care. Tyrosine Kinase Receptors (TKRs) are key players in the proliferation and development of GBM. Examples of TKRs include, vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR). VEGFR is responsible for the formation of new blood vessels while EGFR signals for the increase in tumor size. DNA transcription occurs within the nucleus to produce pre‐messenger RNA (pre‐mRNA) that is modified by capping, splicing and polyadenylation to form mature mRNA, which is translated into protein by the ribosomal complex. Our strategy aims to deliver a gene therapy that will be targeted to the nucleus of intra‐tumoral cells where the therapy will be transcribed to take on the RNA function to alter pre‐mRNA splicing of TKR genes. The focus of this project involves alternative splicing of EGFR exons 15 to 16, disrupting the expression of the transmembrane domain of exon 17. Without its transmembrane domain, EGFR will bind to its ligand without activating the EGFR pathway. This dominant negative receptor is termed soluble decoy due to its ability to sequester ligands without causing physiological changes within the cell. To ensure that our therapy interacts with hetero‐ribonucleoprotein H to further block splice site recognition, EMSA experiments are performed to confirm RNA‐protein binding affinity and the formation of RNA‐protein complexes. Mammalian HEK 293T cell cultures are grown and transfected with a DNA plasmid to produce hnRNP H. Cell lysis is followed by protein isolation and dialysis. The RNA therapy was generated by T7 transcription of cDNA, followed by RNA extraction and purification via RNA 4% polyacrylamide gels. Varying concentrations of therapeutic RNA were incubated with protein H for 30 minutes at 37 degrees Celsius to evaluate optimal binding concentration. Binding reactions were run through a non‐denaturing gel to separate molecules and verify the formation of RNA‐protein complexes. Results of gel‐shift assays show that the RNA therapy binds the hnRNP H protein forming a complex. This demonstrates RNA therapy recruitment of the splicing silencer to the pre‐mRNA target.

Gibbs point field model quantifies disorder in microvasculature of U87-glioblastoma

Microvascular proliferation in glioblastoma multiforme is a biological key mechanism to facilitate tumor growth and infiltration and a main target for treatment interventions. The vascular architecture can be obtained by Single Plane Illumination Microscopy (SPIM) to evaluate vascular heterogeneity in tumorous tissue. We make use of the Gibbs point field model to quantify the order of regularity in capillary distributions found in the U87 glioblastoma model in a murine model and to compare tumorous and healthy brain tissue. A single model parameter Γ was assigned that is linked to tissue-specific vascular topology through Monte-Carlo simulations. Distributions of the model parameter Γ differ significantly between glioblastoma tissue with mean 〈ΓG〉=2.1±0.4, as compared to healthy brain tissue with mean 〈ΓH〉=4.9±0.4, suggesting that the average Γ-value allows for tissue differentiation. These results may be used for diagnostic magnetic resonance imaging, where it has been shown recently that Γ is linked to tissue-inherent relaxation parameters.

CBMS-04 SIGNIFICANT ROLE OF HYPOXIA IN THE EXPRESSION AND FUNCTION OF OSTEOPONTIN IN CD44-HIGHLY EXPRESSED GLIOMA STEM-LIKE CELLS IN TUMOR PROGRESSION OF GLIOBLASTOMA

The poor prognosis of glioblastoma multiforme (GBM) may be due to the surviving glioma stem-like cells (GSCs) in the tumor periphery after tumor resection. We demonstrated that CD44-expressed GSCs existed much more in the tumor periphery of high invasive (HI) type GBM than low invasive (LI) type GBM. The HI type was significantly associated with worse outcome, but how GSCs with high CD44 expression relate to tumor progression remains unknown. In this study, we investigated effects of hypoxia on CD44-directed signal pathways, leading to tumor invasion and proliferation in GBM. We focused on the CD44 ligand osteopontin (OPN) because it is known hypoxia affects the interaction of CD44 and OPN which promotes stemness and proliferation of cancer stem cells. We examined mRNA expressions of hypoxia inducible factor (HIF)-1a, HIF-2a, CD44 and OPN in tumor tissues of GBM and investigated effects of hypoxia (1%O2:severe or 5%O2:moderate) on the expression of these molecules using cultured GSCs that were established from tumor tissues showing high CD44 expression in the periphery of GBMs. In addition, we analyzed the effects of OPN on invasive, migratory and proliferative activities of GSCs under the hypoxic conditions. OPN was much higher expressed in the tumor periphery of LI type GBM than HI type GBM. Severe hypoxia significantly increased the expressions of HIF-1a and CD44 but did not OPN. On the other hands, moderate hypoxia promoted the expressions of HIF-2a and OPN. Knockdown of HIF-2a significantly inhibited OPN expression. In addition, the more OPN was expressed in the cultured GSCs under moderate hypoxia, the more the GSCs proliferated and decreased their invasive and migratory activities. In conclusion, GSCs existing in the tumor periphery of GBM can migrate or proliferate by changing CD44-directed signal pathways. Moderate hypoxia promoted HIF-2a/OPN/CD44 pathway, resulting in phenotypic transition to high proliferative tumors.

Lipid accumulation and oxidation in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor in adults. Despite the multimodal standard treatments for GBM, the median survival is still about one year. Analysis of brain tissues from GBM patients shows that lipid droplets are highly enriched in tumor tissues while undetectable in normal brain tissues, yet the identity and functions of lipid species in GBM are not well understood. The aims of the present work are to determine how GBM utilizes fatty acids, and assess their roles in GBM proliferation. Treatment of U138 GBM cells with a monounsaturated fatty acid, oleic acid, induces accumulation of perilipin 2-coated lipid droplets containing triglycerides enriched in C18:1 fatty acid, and increases fatty acid oxidation. Interestingly, oleic acid also increases glucose utilization and proliferation of GBM cells. In contrast, pharmacologic inhibition of monoacylglycerol lipase attenuates GBM proliferation. Our findings demonstrate that monounsaturated fatty acids promote GBM proliferation via triglyceride metabolism, suggesting a novel lipid droplet-mediated pathway which may be targeted for GBM treatment.

High doses of sodium ascorbate interfere with the expansion of glioblastoma multiforme cells in vitro and in vivo

AimsConstant development of chemotherapeutic strategies has considerably improved the efficiency of tumor treatment. However, adverse effects of chemotherapeutics enforce premature treatment cessation, which leads to the tumor recurrence and accelerated death of oncologic patients. Recently, sodium ascorbate (ASC) has been suggested as a promising drug for the adjunctive chemotherapy of glioblastoma multiforme (GBM) and prostate cancer (PC). To estimate whether ASC can interfere with tumor recurrence between the first and second-line chemotherapy, we analyzed the effect of high ASC doses on the expansion of cells in vitro and in vivo. Main methodsBrightfield microscopy-assisted approaches were used to estimate the effect of ASC (1–14 mM) on the morphology and invasiveness of human GBM, rat PC and normal mouse 3T3 cells, whereas cytostatic/pro-apoptotic activity of ASC was estimated with flow cytometry. These assays were complemented by the in vitro CellROX-assisted analyses of intracellular oxidative stress and in vivo estimation of GBM tumor invasion. Key findingsASC considerably decreased the proliferation and motility of GBM and PC cells. This effect was accompanied by intracellular ROS over-production and necrotic death of tumor cells, apparently resulting from their “autoschizis”. In vivo studies demonstrated the retardation of GBM tumor growth and invasion in the rats undergone intravenous ASC administration, in the absence of detectable systemic adverse effects of ASC. SignificanceOur data support previous notions on anti-tumor activity of high ASC doses. However, autoschizis-related cell responses to ASC indicate that its application in human adjunctive tumor therapy should be considered with caution.

Abstract 250: Tumor treating fields increases membrane permeability in glioblastoma cells

Abstract INTRODUCTION: Glioblastoma multiforme (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are often ineffective. Novel approaches need consideration. We investigated two such novel approaches: Withaferin A and Tumor Treating Fields (TTFields). Withaferin A is a steroidal lactone originating from extracts of the winter cherry plant (Withania somnifera). Extracts such as AshwaMAX can contain up to 4.3% (w/w) Withaferin A. Recently, the U.S. Food and Drug Administration approved another approach to treating glioblastoma: tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but mechanisms of action are not fully understood. METHODS: To investigate these two therapies, we used human glioblastoma cells (GBM2, GBM39, and U87-MG) isolated from primary tumors. Cells were modified to express firefly luciferase. Proliferation was monitored by: bioluminescent imaging, cell counting via hemocytometer, or cell titer blue viability assay. TTFields were imposed on cell cultures by the inovitroTM device from Novocure Ltd. RESULTS: Both AshwaMAX and Withaferin A inhibited growth of GBM cell lines (IC50 ~0.25 µM). In exploring Withaferin A and TTFields interventions in GBM cell culture, we discovered that the steroid worked synergistically with TTFields (p<0.01, n=3 experiments) to inhibit GBM proliferation. Current theories about TTFields’ action involve it disrupting mitosis through interference with mitotic spindle assembly. In the present studies, we show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. In GBM, increased permeability was indicated through increased bioluminescence with TTFields exposure (p<0.0001, TTFields vs no TTFields), as well as with increased binding and ingress of membrane-associating reagents such as Dextran-FITC (p<0.0001, TTFields vs no TTFields). Increased permeability was observed and with scanning electron microscopy showing augmented number (54+19 vs. 24+11 per ROI, TTFields vs no TTFields, p=0.0002) and sizes of holes (241+92 vs. 130+32 nm2, TTFields vs no TTFields, p=0.0005) in the GBM cellular membrane. Such phenomena were reversible after cessation of TTFields. CONCLUSIONS: In summary, increased membrane permeability by TTFields helps to explain previous reports of additive vs. synergistic effects between TTFields and chemotherapies, including Withaferin A. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies. Citation Format: Edwin Chang, Chirag Patel, Christoph Pohling, Caroline Young, Jonathan Song, Thomas A. Flores, Yitian Zeng, Lydia Marie Joubert, Hamed Arami, Arutselvan Natarajan, Robert Sinclair, Sanjiv S. Gambhir. Tumor treating fields increases membrane permeability in glioblastoma 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 250.

Combined Applications of Repurposed Drugs and Their Detrimental Effects on Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with high rates of recurrence. This study aimed to investigate the effect of repurposed drug combinations on GBM. Viability of U87 MG and 11ST patient-derived GMB cell lines, after valproic acid, tranylcypromine or riluzole alone, in different combinations, as well as combined with standard temozolomide chemotherapy was examined using the MTT assay. Proliferation, mRNA level of tissue factor pathway inhibitor 2 (TFPI2), and cell invasion were evaluated using anti-Ki-67 antibody staining, reverse transcriptase-polymerase chain reaction and xCELLigence system. The strongest effect on cell viability was achieved by the combination of riluzole with valproic acid (U87MG: 27.2%, 11ST: 25.99%). Tranylcypromine significantly enhanced the effect of temozolomide when used in combination, as did valproic acid. The normally high proliferation of GBM significantly declined under treatment with valproic acid with tranylcypromine (p=0.01). Finally, we observed reduction of invasion comparing single tranylcypromine to its combination with valproic acid or riluzole. These results support the idea that combinations of drugs could increase the treatment efficiency of GBM.

151. Hyperthermia, generated by focused ultrasound, in combination with radiations for a more effective glioblastoma therapy

Purpose The existing transcranial focused ultrasound device (Insightec Exablate 4000 Neuro) can induce hyperthermia in a brain region ten times larger than was recently covered with ablation [1] . The purpose of this work was to theoretically derive how a relatively small volume of glioblastoma multiforme (GBM) can be effectively treated by combining focused ultrasound based hyperthermia with an external beam radiotherapy. Methods We applied a mathematical descriptions of GBM proliferation and diffusion, in the frame of the reaction diffusion theory. We formulated equations capturing of the impact of radiotherapy and heat on GBM in the reaction diffusion equation, including the regrowth induced by stem cells [2] . Results Our calculated results show that the expected patient survival could be significantly improved by this treatment [3] . For the future, due to the lower power required for a single heating point, a somewhat lower frequency could be hypothesized, without risks of cavitation. This modification would allow the heating of larger tumors in less central regions. Conclusions The proposed methodology, now limited to a small tumors in a quite central location, can be easily improved in several different respects. These results advocate thorough experiments to validate this very promising theoretical therapeutic benefit.

Investigating the role of CRIPTO-1 (TDGF-1) in glioblastoma multiforme U87 cell line.

Cripto-1 has been implicated in a number of human cancers. Although there is high potential for a role of Cripto-1 in glioblastoma multiforme (GBM) pathogenesis and progression, few studies have tried to define its role in GBM. These studies were limited in that Cripto-1 expression was not studied in detail in relation to markers of cancer initiation and progression. Therefore, these correlative studies allowed limited interpretation of Criptos-1's effect on the various aspects of GBM development using the U87 GBM cell line. In this study, we sought to delineate the role of Cripto-1 in facilitating pathogenesis, stemness, proliferation, invasion, migration and angiogenesis in GBM. Our findings show that upon overexpressing Cripto-1 in U87 GBM cells, the stemness markers Nanog, Oct4, Sox2, and CD44 increased expression. Similarly, an increase in Ki67 was observed demonstrating Cripto-1's potential to induce cellular proliferation. Likewise, we report a novel finding that increased expression of the markers of migration and invasion, Vimentin and Twist, correlated with upregulation of Cripto-1. Moreover, Cripto-1 exposure led to VEGFR-2 overexpression along with higher tube formation under conditions promoting endothelial growth. Taken together our results support a role for Cripto-1 in the initiation, development, progression, and maintenance of GBM pathogenesis. The data presented here are also consistent with a role for Cripto-1 in the re-growth and invasive growth in GBM. This highlights its potential use as a predictive and diagnostic marker in GBM as well as a therapeutic target.

CD163, a novel therapeutic target, regulates the proliferation and stemness of glioma cells via casein kinase 2.

Glioma is a devastating cancer with a dismal prognosis and there is an urgent need to discover novel glioma-specific antigens for glioma therapy. Previous studies have identified CD163-positive tumour cells in certain solid tumours, but CD163 expression in glioma remains unknown. In this study, via an analysis of public datasets, we demonstrated that CD163 overexpression in glioma specimens correlated with an unfavourable patient prognosis. CD163 expression was increased in glioma cells, especially primary glioma cells. The loss of CD163 expression inhibited both cell cycle progression and the proliferation of glioblastoma multiforme (GBM) cell lines and primary glioma cells. CD163 interacted directly with casein kinase 2 (CK2) and CD163 silencing reduced AKT/GSK3β/β-catenin/cyclin D1 pathway activity via CK2. Moreover, CD163 was upregulated in CD133-positive glioma stem cells (GSCs), and CD163 downregulation decreased the expression of GSC markers, including CD133, ALDH1A1, NANOG and OCT4. The knockdown of CD163 impaired GSC stemness by inhibiting the CK2/AKT/GSK3β/β-catenin pathway. Finally, a CD163 antibody successfully induced complement-dependent cytotoxicity against glioma cells. Our findings indicate that CD163 contributes to gliomagenesis via CK2 and provides preclinical evidence that CD163 and the CD163 pathway might serve as a therapeutic target for glioma.