Human Glioblastoma Multiforme Cells Research Articles

Abstract B13: Reprogramming glioblastoma multiforme cells to neurons by protein kinase inhibitors

Abstract Reprogramming healthy somatic cells into pluripotent stem (iPS) cells with defined factors has been intensively investigated. However, reprogramming cancer cells has fallen much behind. Direct reversion of cancers into normal tissues is an ideal strategy for cancer treatment. Recent reports have showed that defined factors can induce reprogramming of cancer cells into iPS cells, supporting this notion. Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans. Despite multimodal therapy, the outcome for patients with GBM is still poor, with a median survival of just 14.2 months. Therefore, developing novel therapeutic strategy is an emergent task. The tumor suppressor p53 is considered today the most important tumor suppressor protein in humans and it suppresses both reprogramming and oncogenic transformation. p53 inactivation or mutations are the most common in GBM, and this p53 deficient GBM subgroup is currently the most difficult to treat, typically associated with rapid local recurrence after treatment. p53 mutations result in loss-of-function, gain-of-function, and dominant-negative mutational effects for p53. Thus, different p53 mutants may have various effects on reprogramming, promoting or inhibiting GBM reprogramming. Furthermore, p53 activity is often inactivated in GBM with wild-type p53 that may also affect reprogramming. We have demonstrated that the depletion of p53 reprograms human somatic cells into neurons by regulation of a set of transcription factors (Nature Communications, 2014; Cell Reports, 2014, International Journal of Stem Cell Research & Therapy, 2016). Integration-free reprogramming cancer cells would be safer and preferable for clinical use. Along those lines, we screened a protein kinase inhibitor library and found that the kinase inhibitors can substitute for all transcription factors and convert human p53-deficient GBM cells into “normal” neurons (functional neurons). In vitro and in vivo tumorigenesis assays showed that induced neurons lose tumorigenicity. Moreover, the inhibitor reprogramming prevents GBM local recurrence in mice. Currently these inhibitors are used as anti-tumor drugs in patients, so this reprogramming strategy has significant potential to move rapidly toward clinical trials. Note: This abstract was not presented at the conference. Citation Format: Jie Yuan, Fan Zhang, Meng You, Qin Yang. Reprogramming glioblastoma multiforme cells to neurons by protein kinase inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B13.

Gold nanotriangles: scale up and X-ray radiosensitization effects in mice.

In the past decade, there has been considerable interest in radiosensitization using gold nanoparticles that accumulate specifically in cancerous tissue while sparing normal tissues. Despite this interest, it remains unclear which nanoparticle morphologies, cellular uptake, or cytoplasmic distribution elicit optimal radiosensitization. We introduce gold nanotriangles (AuNTs) as a possible X-ray radiotherapy sensitizer. In this study, we first explored a large-scale synthetic method for the production of high quality monodisperse AuNTs. Second, we conducted in vitro and in vivo experiments to evaluate the effect of PEGylated AuNTs (pAuNTs) on cellular uptake, cytotoxicity, bio-distribution, and radiosensitization on radiation-resistant human Glioblastoma Multiforme (GBM) cells. Our results suggest that the new scale up synthesis methodology consistently produced high quality AuNTs and pAuNTs which had nonspecific cellular uptake without any obvious cytotoxicity and exhibited excellent radiosensitization.

PP80. CAVEOLIN-1 IMPLICATED AS A PRO-INVASIVE GENE IN HIGH-GRADE GLIOMA CELL MODELS: IMPLEMENTATION OF A 3D SPHEROID MATRIX INVASION ASSAY

INTRODUCTION: The poor prognosis associated with Glioblastoma multiforme (GBM) is multifactorial but includes the capacity of residual tumour cells not removed by surgery and resistant to radio-/chemo-therapy undergoing diffuse invasion into the surrounding brain tissue. Caveolin-1 (Cav-1) is the major structural and functional component of caveolae. In a number of tumour types Cav-1 is recognised to participate in cytoskeletal rearrangement, integrin-mediated adhesion and/or matrix remodelling. We proposed Cav-1 serves to promote invasion of GBM cells. To investigate this we have employed in an in-vitro 3D cellular invasion assay. METHOD: The human GBM cell lines, UP007 and UP029 established from primary cultures of biopsy-derived brain tumours (University of Portsmouth), U-87 MG (ECACC) and U-373 MG (ECACC) were genetically modified to stably knock-out Cav-1 using a Lentiviral Cav-1 shRNA approach; corresponding stably transfected non-target (NT) shRNA cell lines were generated as controls. Neuropheres were formed and embedded within an extracellular matrix (Matrigel™). Over a two-/four-day period (depending on cell line) the migration of cells away from the neurophere core (CORE) was quantified by image capture and processing (Image J) using a custom-developed MatLab script for pixel density analysis indicative of the density of migrating cellular material. RESULTS: Cav-1 knockout resulted in significant (P 0.05) towards reduced invasion. Depending upon the cell line the Cav-1 knockdown also resulted in reduced size and cellular density of the neurosphere core (UP007 and UP029) indicative of reduced proliferation and/or cell survival capacity. CONCLUSION: Using an in-vitro 3D cellular invasion assay we have found Cav-1 expression in a series of three GBM cell lines to promote cellular invasion capacity. Ongoing studies are addressing signalling mechanisms and the influence of the microenvironment.

Influence of vascular endothelial growth factor and radiation on gap junctional intercellular communication in glioblastoma multiforme cell lines.

Glioblastoma multiforme (GBM) is a highly aggressive glial brain tumor with an unfavorable prognosis despite all current therapies including surgery, radiation and chemotherapy. One characteristic of this tumor is a strong synthesis of vascular endothelial growth factor (VEGF), an angiogenesis factor, followed by pronounced vascularization. VEGF became a target in the treatment of GBM, for example with bevacizumab or the tyrosine kinase inhibitor axitinib, which blocks VEGF receptors. To improve patients’ prognosis, new targets in the treatment of GBM are under investigations. The role of gap junctions in GBM remains unknown, but some experimental therapies affect these intercellular channels to treat the tumor. Gap junctions are composed of connexins to allow the transport of small molecules between adjacent cells through gap junctional intercellular communication (GJIC). Based on data derived from astrocytes in former studies, which show that VEGF is able to enhance GJIC, the current study analyzed the effects of VEGF, radiation therapy and VEGF receptor blockade by axitinib on GJIC in human GBM cell lines U-87 and U-251. While VEGF is able to induce GJIC in U-251 cells but not in U-87 cells, radiation enhances GJIC in both cell lines. VEGF receptor blockade by axitinib diminishes radiation induced effects in U-251 partially, while increases GJIC in U-87 cells. Our data indicate that VEGF and radiation are both modifying components of GJIC in pathologic brain tumor tissue.

Cytotoxic activity of the aqueous extract of Micromeria fruticosa (L.) Druce subsp. serpyllifolia on human U-87 MG cell lines

Micromeria fruticosa (L.) Druce subsp. serpyllifolia, which is widely used in folk medicine as a medicinal herbal tea, is grown in different areas of Turkey and the Mediterranean region. The present study was conducted to evaluate the aqueous extract of Micromeria fruticosa subsp. serpyllifolia for its antioxidant and antiproliferative activity on a human glioblastoma multiforme cell line (U-87 MG), which has not been reported before. Here, the extract was added to cultures at 8 different concentrations (0-200 ?g/mL). Cell viability and cell membrane damage was determined using the MTT and LDH assays for 48 h, respectively. To examine the oxidative effects, total antioxidant capacity (TAC) and total oxidant status (TOS) levels were measured. The extract displayed considerable antiproliferative activities at the high concentrations of 175 and 200 ?g/mL. Furthermore, the extract caused a significant increase in the release of the lactate dehydrogenase (LDH) enzyme in a concentration-dependent manner; 200 ?g/mL of extract enhanced the release of LDH. Treatments with extract at higher doses increased TOS levels and decreased TAC levels in human U-87 MG cells. Our study suggests that the aqueous extract of Micromeria fruticosa ssp. serpyllifolia was capable of inducing growth inhibition of cancer cells. These results encourage further research to assess the value of the extract in modern phytotherapy.

PROX1 promotes human glioblastoma cell proliferation and invasion via activation of the nuclear factor-κB signaling pathway

Prospero homeobox protein 1 (PROX1) is highly expressed in high-grade malignant astrocytic gliomas. However, the role of PROX1 in the pathogenesis of glioblastoma multiforme (GBM) remains unclear. The present study overexpressed PROX1 in human GBM cell lines and examined its effects on cell growth, tumorigenesis, and invasiveness. In addition, the involvement of the nuclear factor‑κB (NF‑κB) signaling pathway in the action of PROX1 was examined. It was identified that overexpression of PROX1 significantly increased the proliferation and colony formation of glioblastoma cells, compared with empty vector‑transfected controls. Furthermore, ectopic expression of PROX1 promoted the growth of GBM xenograft tumors. Western blot analysis revealed that PROX1 overexpression induced nuclear accumulation of NF‑κB p65 and upregulated the expression levels of the NF‑κB responsive genes cyclin D1 and matrix metallopeptidase9. An NF‑κB reporter assay demonstrated that PROX1‑overexpressing glioblastoma cells had significantly greater NF‑κB‑dependent reporter activities compared with empty vector‑transfected controls. Transfection of a dominant inhibitor of κBα mutant into PROX1‑overexpressing cells significantly impaired their proliferation and invasion capacities, which was accompanied by reduced levels of nuclear NF‑κB p65. Collectively, these data indicated that PROX1 serves an oncogenic role in GBM and promotes cell proliferation and invasiveness potentially via activation of the NF‑κB signaling pathway. Therefore, PROX1 may represent a potential target for the treatment of GBM.

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs.

Selective Toxicity of a Highly Potent Camptothecin Analogue: A Pilot Study with Glioblastoma Multiforme Cells.

We developed a novel camptothecin analogue, CPT417, that yields reduced toxicity compared to other analogues used in chemotherapeutic regimens. In this pilot study, we assessed the activity of CPT417 against glioblastoma multiforme (GBM) cells and glioma stem cells. The human U251 GBM cell line and normal human astrocytes were cultured in parallel for clonogenic survival analysis following exposure to increasing concentrations of CPT417. Cell viability of a glioma stem cell line was assessed 5 days after exposure to a range of CPT417 concentrations. CPT417 completely inhibited clonogenic survival of GBM cells at 10 nM, whereas this concentration only inhibited astrocytes by approximately 50%. Cell viability analysis of glioma stem cell cultures yielded a half-maximal response at 15 nM. CPT417 acts selectively against GBM cells at concentrations that are at least an order of magnitude below reported values for related alkylating agents in clinical use.

IMST-54. GLIOBLASTOMA EXTRACELLULAR VESICLES INDUCE IMMUNOSUPPRESSION VIA PD-L1

Glioblastoma multiforme (GBM) induces potent immunosuppression both within the tumor and systemically. The mechanisms underlying this have not been fully elucidated. GBM shed large numbers of extracellular vesicles (EV’s). Here, we test the hypothesis that GBM-EV’s contribute to immunosuppression. EV’s were isolated from cultured human GBM cell lines, GBM patient serum, and healthy donor serum by serial centrifugation. Nanosight nanoparticle tracking was used to determine EV frequency and size distribution. Western blot and ELISA were used to determine tumor-associated antigen and PD-L1 expression. GBM EV’s from wild type and PD-L1-overexpressing cells were cultured with healthy donor CD14+ monocytes or CD3+ T cells. Binding was determined by confocal microscopy. Induction of CD14+/HLA-DR- monocytic myeloid-derived suppressor cells (MDSC’s) and CD4+ and CD8+ regulatory T cells (Treg) were determined by flow cytometry. T cell proliferation was assessed by flow cytometry and CFSE staining. GBM’s shed large amounts of EV’s including both exosomes (<100nm) and microvesicles (100-500nm) in vitro. EV’s with similar size distribution are found in GBM patients’ serum at increased frequency compared with healthy donors. Tumor-associated antigens (ErbB2, MAGEA3) can be detected in GBM patients’ serum EV’s. Some GBM-derived EV’s express the immunosuppressive protein PD-L1 and bind both T cells and monocytes through PD-1. GBM EV’s induce MDSC differentiation in normal monocytes in vitro(27.5%+/-5.62%) compared to media (5.24%+/-0.77; P=0.0078) and this was further increased by PD-L1 overexpressing EV’s (54.28%+/-5.48%; P=0.0143). GBM EV-induced MDSC’s inhibited T cell proliferation in response to anti-CD3 (65.98%+/-5.64% vs. 40.40%+/-7.99%; P=0.0399). GBM EV’s also showed trends towards inducing both CD4 and CD8 Treg’s in a PD-L1-dependent fashion. GBM EV’s contain both immunosuppressive molecules and tumor-associated antigens. Their presence in GBM patients’ serum suggests potential to mediate systemic immunosuppression. In keeping with this, GBM EV’s induce immunosuppressive changes in leukocytes in a PD-L1-dependent fashion.

Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells.

Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl–Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl–Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling.

Topiramate induces acute intracellular acidification in glioblastoma.

Reversal of the intracellular/extracellular pH gradient is a hallmark of malignant tumors and is an important consideration in evaluating tumor growth potential and the effectiveness of anticancer therapies. Glioblastoma multiforme (GBM) brain tumors have increased expression of the carbonic anhydrase (CA) isozymes CAII, CAIX and CAXII that contribute to the altered regulation of intracellular pH (pHi). The anti-epileptic drug topiramate (TPM) inhibits CA action and may acidify the tumor intracellular compartment. In-vivo detection of acute tumor acidification could aid in cancer diagnosis and monitoring treatment response. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) has been used to measure tissue pH. Using a recently developed CEST-MRI method called amine/amide concentration independent detection (AACID), we have previously shown intracellular acidification caused by single dose of lonidamine. The current study aims to evaluate the intracellular acidification induced by a single dose of the clinically approved drug TPM. Brain tumors were induced in NU/NU mice by injecting 105 U87 human glioblastoma multiforme cells into the right frontal lobe. Using a 9.4T MRI scanner AACID measurements were acquired, before and after administration of TPM (dose: 120mg/kg, intraperitoneal), 15 ± 2days after tumor cell implantation. TPM administration induced acute intracellular acidification (average ± SD: baseline AACID = 1.14 ± 0.05; post AACID = 1.19 ± 0.05, paired ttest p = 0.02) in implanted brain tumors. In contrast, contralateral tissue showed no change in AACID value. These results suggest that topiramate can rapidly induce a tumor specific physiological change detectable by AACID CEST. This pH challenge paradigm could be exploited to aid in tumor detection and monitoring treatment response.

Biological effect of an alternating electric field on cell proliferation and synergistic antimitotic effect in combination with ionizing radiation.

Alternating electric fields at an intermediate frequency (100~300 kHz), referred to as tumour-treating fields (TTF), are believed to interrupt the process of mitosis via apoptosis and to act as an inhibitor of cell proliferation. Although the existence of an antimitotic effect of TTF is widely known, the proposed apoptotic mechanism of TTF on cell function and the efficacy of TTF are controversial issues among medical experts. To resolve these controversial issues, a better understanding of the underlying molecular mechanisms of TTF on cell function and the differences between the effects of TTF alone and in combination with other treatment techniques is essential. Here, we report experimental evidence of TTF-induced apoptosis and the synergistic antimitotic effect of TTF in combination with ionizing radiation (IR). For these experiments, two human Glioblastoma multiforme (GBM) cells (U373 and U87) were treated either with TTF alone or with TTF followed by ionizing radiation (IR). Cell apoptosis, DNA damage, and mitotic abnormalities were quantified after the application of TTF, and their percentages were markedly increased when TTF was combined with IR. Our experimental results also suggested that TTF combined with IR synergistically suppressed both cell migration and invasion, based on the inhibition of MMP-9 and vimentin.

Thymoquinone synergistically potentiates temozolomide cytotoxicity through the inhibition of autophagy in U87MG cell line.

Glioblastoma multiforme (GBM) is one of the most lethal forms of human cancer and temozolomide (TMZ) is currently part of the standard treatment for this disease. Combination therapy using natural substances can enhance the anti-cancer activity of TMZ. The purpose of this study was to evaluate the effect of TMZ in combination with thymoquinone (TQ) on human GBM cell line (U87MG). The cell line was treated with TMZ and/or TQ. Cell viability was assessed using trypan blue and MTT assay. The effect of TMZ and/or TQ on colony-forming ability of the cells was investigated. Apoptosis and autophagy were quantified by fluorescent dye staining. The expression level of two autophagy related genes (ATG) were assessed using RT-PCR. Furthermore, nitric oxide (NO) production was detected by Griess reaction. After treatment with TMZ and/or TQ, the cell viability was reduced in a time- and dose-dependent manner, and the cell survival fraction (SF) was significantly decreased (P=0.000). Apoptosis index of U87MG cells was also significantly increased (P=0.000). Autophagy was significantly increased by TMZ (P=0.000) and decreased by TQ (P=0.018). Also TMZ and/or TQ significantly decreased NO production by U87MG cell (P=0.000). TQ enhanced the anti-cancer activity of TMZ by inhibition of autophagy at the transcriptional level and decreased the colony-forming ability and NO production of U87MG cell line.

Targeting MT1-MMP as an ImmunoPET-Based Strategy for Imaging Gliomas

BackgroundA critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models.MethodsAn anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments.Results89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models.ConclusionA new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM.

In Vivo Antitumor Effect of Supercritical CO2 Extract of Mango Ginger ( Curcuma amada Roxb) in U-87MG Human Glioblastoma Nude Mice Xenografts.

Glioblastoma multiforme (GBM) is one the most aggressive and lethal human neoplasms with poor prognosis and very limited positive treatment options. The antitumor effect of supercritical CO2 extract of mango ginger ( Curcuma amada Roxb) (CA) with and without irinotecan (IR) was analyzed in U-87MG human glioblastoma multiforme (GBM) cells in vitro and in nude mice xenografts. CA is highly cytotoxic to GBM cells and is synergistic with IR as indicated by the combination index values of <1 in the CompuSyn analysis. CA inhibits tumor growth rate in GBM xenografts, the inhibition rate being higher than in IR treated group. GBM xenograft mice treated with IR + CA combination showed almost complete inhibition of tumor growth rate. Gene expression analysis of xenograft tumors indicated that IR + CA treatment significantly downregulated anti-apoptotic (Bcl-2 and mutant p53), inflammation-associated (COX-2) and cell division-associated (CCNB2) genes and upregulated pro-apoptotic genes (p21 and caspase-3). These results confirmed the therapeutic efficiency of IR + CA combination against GBM and the need for further clinical investigations.

Abstract 466: MR prediction of tumor burden in Patient-Derived Mouse Xenografts model of glioblastoma using an adaptive model

Abstract Introduction: In human glioblastoma multiforme (GBM), infiltrating cells are found in remote locations, even in the hemisphere contralateral to the primary lesion. Magnetic Resonance Imaging (MRI) allows approximation of the extent of tumor cell infiltration. However, the actual extent of infiltration may be greater or less than the edema, and there is no standard MRI practice that allow exploring the infiltrating tumor burden. This pilot study investigates the feasibility of using a set of MR modalities for the development of an MRI estimate of infiltrating tumor burden in Patient-Derived Mouse Xenografts model of GBM using an adaptive model. Material and Methods: 8 mice implanted with GBM CSC HF2927 were studied. MRI studies were performed in a Direct Drive Varian 7 Tesla. The following image sets were acquired: high-res. T1-weighted; T2-weighted (TE/TR = (20, 40, 60, 80)/3000 ms); MT-weighted fast spin-echo. Magnevist (0.25 mmol/kg i.p) was injected about 5 minutes before the post-contrast T1-weighted image set was acquired. The animal was sacrificed immediately after MRI and stained for the presence of human GBM cells. We used the following MRI sequences to establish a basis set for training the AM: pre- and post-contrast (Magnevist, I.P.) T1-weighted, 4-echo T2, MT, 3-direction, 3 b-value diffusion-weighted. Maps of T2 and proton density (T2-PD) were produced by fitting the T2 data. The histology image was warped and co-registered to the T2-PD image using mouse brain anatomical landmarks. MR image sets were normalized to the white matter area of the brain, and each voxel profile extracted from the 9 image set was normalized to the summation of two normalized T2 images (echo 2 and 3), following which the normalized profile along with the co-registered histology was used for training and testing of an artificial neural network (ANN) with Multi-Layer perceptron architecture to predict the presence of local tumor burden in form of tumorous cell density. Results and Conclusions: The ANN was successfully trained and validated using K-Fold Cross Validation technique (KFCV) and the 9 MR modalities as its input set. Results imply that the chosen MRI feature set contains adequate information content for training the ANN. The predictive power of the ANN was ∼ 0.81. The correlation coefficient for the association between the predicted map and histology was ∼ 0.85. Given the success of training an ANN to predict infiltrating tumor burden, it may be possible to identify similar or different basis set of MRI images that can robustly predict both infiltrating and solid tumor burden in human GBM. Citation Format: Hassan Bagher-Ebadian, Ana deCarvalho, Azimeh NV Dehkordi, Tavarekere Nagaraja, Susan Irtenkauf, Swayamparva Panda, Meser M. Ali, Arbab S. Ali, Hamid Soltanian-Zadeh, Robert Knight, James R. Ewing. MR prediction of tumor burden in Patient-Derived Mouse Xenografts model of glioblastoma using an adaptive model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 466.

Voltage-Gated Proton Channel in Human Glioblastoma Multiforme Cells.

Solid tumors tend to have a more glycolytic metabolism leading to an accumulation of acidic metabolites in their cytosol, and consequently, their intracellular pH (pHi) turns critically lower if the cells do not handle the acid excess. Recently, it was proposed that the voltage gated proton channels (HV1) can regulate the pHi in several cancers. Here we report the functional expression of voltage gated proton channels in a human glioblastoma multiforme (GBM) cell line, the most common and lethal brain tumor. T98G cells presented an outward, slow activating voltage-dependent proton current, which was also ΔpH-dependent and inhibited by ZnCl2, characterizing it as being conducted by HV1 channels. Furthermore, blocking HV1 channels with ZnCl2 significantly reduced the pHi, cell survival, and migration, indicating an important role for HV1 for tumor proliferation and progression in GBM. Overall, our results suggest that HV1 channels can be a new therapeutic target for GBM.

The mechanisms of malic enzyme 2 in the tumorigenesis of human gliomas

The high level of resistance of glioblastoma multiforme (GBM) to currently used chemotherapies and other conventional therapies, its invasive characteristics and the presence of stem-like cells are the major factors that make the treatment of GBM difficult. Recent studies have demonstrated that the homeostasis of energy metabolism, glycolysis and mitochondrial oxidation of glucose are important for GBM cell growth and chemo-resistance. However, it is not clear which specific gene(s) are involved in the homeostasis of energy metabolism and invasiveness of GBM cells. We performed a preliminary analysis of data obtained from Gene Expression Omnibus profiles and determined that malic enzyme 2 (ME2) expression was positively associated with WHO grade in human primary gliomas. Hence, we evaluated the detailed working mechanisms of ME2 in human GBM cell processes, including proliferation, cell cycle, invasion, migration, ROS, and ATP production. Our data demonstrated that ME2 was involved in GBM growth, invasion and migration. ME2 has two cofactors, NAD+ or NADP+, which are used to produce NADH and NADPH for ATP production and ROS clearance, respectively. If the catalytic activity of ME2 is determined to be critical for its roles in GBM growth, invasion and migration, small molecule inhibitors of ME2 may be valuable drugs for GBM therapy. We hope that our current data provides a candidate treatment strategy for GBM.

Lead Optimization of 2-Phenylindolylglyoxylyldipeptide Murine Double Minute (MDM)2/Translocator Protein (TSPO) Dual Inhibitors for the Treatment of Gliomas.

In glioblastoma multiforme (GBM), translocator protein (TSPO) and murine double minute (MDM)2/p53 complex represent two druggable targets. We recently reported the first dual binder 3 possessing a higher anticancer effect in GBM cells than the standards PK11195 1 or Nutlin-3 2 singularly applied. Herein, through a structure-activity relationship study, we developed derivatives 4-10 with improved potencies toward both TSPO and MDM2. As a result, compound 9: (i) reactivated the p53 functionality; (ii) inhibited the viability of two human GBM cells; (iii) impaired the proliferation of glioma cancer stem cells (CSCs), more resistant to chemotherapeutics and responsible of GBM recurrence; (iv) sensitized GBM cells and CSCs to the activity of temozolomide; (v) directed its effects preferentially toward tumor cells with respect to healthy ones. Thus, 9 may represent a promising cytotoxic agent, which is worthy of being further developed for a therapeutic approach against GBM, where the downstream p53 signaling is intact and TSPO is overexpressed.

587. Genetic Delivery of a miRNA Cluster with Polycistronic siRNAs Reduces Expression of Epidermal Growth Receptor in Human Glioblastoma Cells

Glioblastoma multiforme (GBM), the most common malignancy of the brain, is a grade IV astrocytoma. It is one of the most rapidly growing and invasive types of glial tumors. The standard therapy includes surgical removal, radiation and chemotherapy with a median survival of about 14 months. In addition, systemic therapies are limited by the blood-brain barrier. To bypass the barrier, we are developing a gene delivery strategy to inhibit the expression of tyrosine kinase receptors (TKR), which are commonly upregulated in GBM. One TKR, epidermal growth factor receptor (EGFR), is overexpressed in GBM leading to uncontrolled growth and proliferation. Our approach is to recruit the RNA interference pathway. Although small interfering RNAs (siRNAs) are often utilized to silence gene expression, exogenously expressed siRNAs are not an effective strategy to treat human disease due to both extracellular and intracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we use a natural miRNA cluster genetic background to effectively deliver the DNA encoding multiple anti-EGFR siRNAs by inserting them into the tertiary structure of the miRNA cluster, miR-17-92. The anti-EGFR polycistronic miRNA cluster (pAAV-miR-IP1) expresses six siRNAs directed against EGFR, specifically targeting the extracellular ligand binding domain, transmembrane domain, intracellular tyrosine kinase domain and 3’ untranslated region of the EGFR transcript. The vector, pAAV-miR-IP1, was transfected into the human GBM cell lines, A172 and U87MG. Results demonstrate that pAAV-miR-IP1 was expressed at high levels with a subsequent reduction in EGFR mRNA expression. Additional strategies include using the polycistronic delivery mechanism to target multiple TKRs in addition to EGFR.