Human Glioma Tumor Research Articles

HERV-W envelope protein is present in microglial cells of the human glioma tumor microenvironment and differentially modulates neoplastic cell behavior

Gliomas are the most common parenchymal tumors of the central nervous system (CNS). With regard to their still unclear etiology, several recent studies have provided evidence of a new category of pathogenic elements called human endogenous retroviruses (HERVs) which seem to contribute to the evolution and progression of many neurological diseases such as amyotrophic lateral sclerosis (ALS), schizophrenia, chronic inflammatory polyneuropathy (CIDP) and, particularly, multiple sclerosis (MS). In these diseases, HERVs exert effects on cellular processes such as inflammation, proliferation, and migration. In previous studies, we demonstrated that in MS, the human endogenous retrovirus type-W envelope protein (HERV-W ENV) interferes with lesion repair through the activation of microglia (MG), the innate myeloid immune cells of the CNS. Here, we now show that HERV-W ENV is also present in the microglial cells (MG) of the tumor microenvironment (TME) in gliomas. It modulates the behavior of glioblastoma (GBM) cell lines in GBM/MG cocultures by altering their gene expression, secreted cytokines, morphology, proliferation, and migration properties and could thereby contribute to key tumor properties.

Preliminary studies on changes in the amount of tryptophan metabolites in human glioma tissues

BackgroundWe have developed and validated methods for the determination of three major tryptophan metabolites metabolized by the kynurenine pathway, namely kynurenine (KYN), 3-hydroxykynurenine (3-HK), and 3-hydroxyanthranilic acid (3-HAA). KYN and 3-HK were determined using RP-HPLC-UV, and 3-HAA using RP-HPLC-FL. We then developed a comparative method based on CE-UV. The developed methods were validated and 36 samples of human brain glioma tissue homogenates were assayed in all 4 grades of malignancy, and the concentration levels of assayed metabolites were compared with available clinical data. ResultsEach of the methods is characterized by high precision, accuracy and repeatability, and the determined LOQ values indicate the possibility of performing quantitative analysis on the available samples of human glioma tumors (36 samples in grades G1–G4). The concentration values of selected metabolites obtained using HPLC methods were subjected to statistical analysis and preliminary clinical data processing. We found statistically significant differences in the concentrations of KYN, 3-HK and 3-HAA between the various grades of the disease, and characterized these differences more precisely by means of the Dunn–Bonferroni post hoc test. We did not find that the patient's environment or habits significantly affected the metabolites concentration of the study samples population. In addition, we showed a high positive correlation between KYN, 3-HK and 3-HAA, which appears to be a characteristic that describes metabolic changes of Trp in relation to KYN, 3-HK and 3-HAA, and indicates potential diagnostic value. SignificanceThe preliminary studies carried out contribute new knowledge on the molecular basis of human brain glioma. They also provide valuable information useful for the development of glioma diagnostics, differentiation of disease grades and assessment of the patient's condition. The obtained relationships between metabolite concentrations and the grade of malignancy of the disease and correlations between metabolite concentrations constitute the basis for further broader biochemical and clinical analysis.

Abstract LB193: Enhanced single cell DNA methylation analysis using combinatorial indexing

Abstract The epigenetic landscape of the human brain undergoes a plethora of changes during malignant transformation. In recent years, DNA methylation-based epigenetic modifications have been widely studied using traditional techniques like bisulfite sequencing and enzymatic methyl sequencing (EM-seq). However, these methods analyze bulk cell populations and lack the granularity of single-cell analysis. Although advances in single-cell analysis have revolutionized our understanding of cellular heterogeneity and functional diversity within complex biological systems, they are still costly, low throughput, and laborious. To address these challenges, ScaleBio has pioneered combinatorial indexing technology, enabling a significant increase in cell throughput. This method utilizes the cell itself as a compartment to perform 2-3 rounds of sequential barcoding in a plate-based workflow, eliminating the need for complex instrumentation. This technology has been successfully adapted to assess DNA methylation at the single-cell level offering a robust, affordable, high-throughput protocol that enhances yield, diversity, and coverage. In this study we used ScaleBio's single-cell methylation kit to investigate DNA methylation patterns during oncogenesis using cancer cells with widespread DNA methylation changes, such as human isocitrate dehydrogenase (IDH) mutant glioma cells at the single-cell level. The IDH gene family, comprising of IDH1, IDH2, and IDH3, encodes enzymes involved in the tricarboxylic acid (TCA) cycle. IDH1/2 mutations are present in over 80% of low-grade gliomas while IDH-mutant gliomas constitute about 1/5th of all adult diffuse gliomas, thus making them one of the most common brain tumor subtypes. As such, IDH mutations have emerged as attractive therapeutic targets for glioma treatment. By uncovering DNA methylation patterns at the single cell level, we provide here an epigenetic map to better understand this complex disease and better inform clinical discoveries. We achieved high cell recovery and robust cytosine coverage throughout our analysis of single cell methylomes isolated from human glioma tumor tissue. Using this data we generated a ranked list of the top hypo- and hypermethylated genomic regions and identified cell type specific clusters seen in different pathological states by looking at Differentially Methylated Regions (DMR) uncovering unique single-cell methylation profiles that may be obscured by bulk or pseudo-bulk analysis. These data show that the ScaleBio single-cell methylation workflow offers increased sensitivity, specificity, and accuracy in identifying DNA methylation sites when compared to other techniques while offering a comprehensive view of methylomes and providing insights into cellular heterogeneity and trajectories. Citation Format: Sanika Khare, Dominic Skinner, Maggie Nakamoto, Hosu Sin, Ashley Woodfin, Eric Pu, Lin Lin, Jason Koth, Beth Walczak. Enhanced single cell DNA methylation analysis using combinatorial indexing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB193.

Non-Detection of HCMV Total Genomic DNA in Human Glioma Cells Genome.

To demonstrate if the human cytomegalovirus (HCMV) genome, that is involved in the pathogenesis of gliomas, is part of the genomic DNA of glioma cells or not. The study included U87MG glioblastoma cell culture and tumor samples from glioma patients. The genomic DNA of tumor samples and U87MG cells were extracted and real-time quantitative PCR was used to assess the presence of the human cytomegalovirus genomic DNA. Consequently, HCMV positivity was not detected in the tumor and cell line genomic DNA under the aforementioned experimental conditions. We found that the genomic DNA of all the samples was negative for HCMV genomic DNA. Thus, HCMV could not be detected in human glioma tumors and we put forward that HCMV genomic DNA was not incorporated into the genomic DNA of glioma cells. Thus, total viral DNA is not involved in the pathogenesis of glioma; however, small viral particles or specific genes might be incorporated into the genomic DNA of glioma cells, leading to cancer development. This prompts further studies for verification.

MODL-41. LOSS OF DISTAL CHROMOSOME 6Q AND ASSOCIATED PDE10A INDUCES AN AGGRESSIVE GLIOMA PHENOTYPE BY FACILITATING PRONEURAL TO MESENCHYMAL TRANSITION

Abstract Somatic copy number alterations (SCNAs) are common in glioblastoma, with some SCNAs having prognostic implications. Identifying and characterizing SCNA-related genes acting as oncogenes or tumor suppressors may potentially provide new therapeutic avenues for gliomas. In this study, we sought to uncover novel SCNA-related genes by employing an unbiased log rank analysis screen of genomic regions associated with glioblastoma survival. We identified 6q27 loss as an independent poorly-prognostic SCNA across three separate glioblastoma cohorts. An in vitro CRISPR screen using human neural stem cells identified five genes in the 6q27 region that caused increased cellular proliferation. One of these genes, phosphodiesterase 10A (PDE10A), had a gene dosage effect on expression and decreased expression in human and mouse glioma tumor tissue compared to normal brain, which are both common tumor suppressor phenotypes. Knockdown of Pde10a in mouse neural stem cells in vitro validated the human cell CRISPR screen phenotype. Suppression of Pde10a in RCAS/Tva mouse glioma in vivo caused decreased survival with increased tumor grade, mitotic activity, necrosis, Vegfa expression, and AKT/pS6 signaling. Single cell RNA-sequencing of mouse gliomas revealed that Pde10a suppression results in a shift from proneural to mesenchymal-like state. In human glioma tissue, PDE10A loss is also associated with an increased mesenchymal signature. Top activated pathways in Pde10a suppressed gliomas involved cell-cell interaction, including upregulation of cell adhesion molecules and downregulation of focal adhesion complex disassembly genes. Functional analysis in vitro confirmed that Pde10a suppression results in increased extracellular matrix adhesion and decreased migration. Relevant to standard glioblastoma treatment, PDE10A loss was associated with decreased MGMT promoter methylation, decreased DNA methyltransferase gene expression, and it potentiated radiation resistance in vitro. In summary, we leveraged several multiomic and experimental platforms to reveal PDE10A as a tumor suppressor in glioblastoma, predominantly by inducing a proneural to mesenchymal transition phenotype.

NIMG-19. TUMOR PARAMETERS AFFECTED BY NANOCOMBRESTATIN THERAPY IN AN EXPERIMENTAL RAT MODEL OF GLIOMA

Abstract INTRODUCTION: of polymeric nanoparticles in cancer therapeutics is widely investigated since nanomedicine often enables the intratumoral delivery of drugs with increased efficacy with minimal side effects. In this study magnetic resonance imaging (MRI) monitoring was employed to study the therapeutic effect of nanocombretastatin (G3-CA4) in an orthotopic glioma model. Water insoluble combretastatin (CA4) was conjugated to a small-sized water soluble G3-succinimic acid PAMAM dendrimer. Tumor growth and vascular parameters were assessed non-invasively using MRI. In the brain, intravenously (iv) administered magnetic resonance (MR) contrast agents (CAs) can be used to measure the biomarkers of tumor perfusion - the blood-to-brain transvascular transfer constant (K-trans), tumor plasma volume (Vp), the extravascular extracellular space (Ve) and the blood volume (VD) in cerebral tumors. K-trans, Vp, Ve and VD showed the discrimination of tumorous vasculature responding to CA4 and G3-CA4 therapies from tumorous tissues. The average K-trans, Vp, Ve and VD decreased significantly 24 hours after G3-CA4 treatment. The results showed that G3-CA4 caused a change in tumor vasculature as measured with DCE-MRI. These effects were homogenous throughout the U-251 tumor. However, CA4 did not show any change in tumor vasculature. Imaging, histological and molecular signatures of the primary glioblastoma were compared. Large necrotic area was found together with destroyed blood vessels at the core of the tumor leaving viable tissue at the rim of the tumor. In vivo studies proved that the cytotoxicity of G3-CA4 conjugate was more effective against human U-251 tumor while CA4 alone was ineffective. Therefore, we have accomplished the effective transvascular delivery of G3-CA4 into human U251 glioma tumor with a compromised blood brain tumor barrier. In vivo studies proved that the cytotoxicity of G3-CA4 conjugate is more effective against human U-251 tumor than that of free CA4.

Small molecule antagonists of PTPmu identified by artificial intelligence-based computational screening block glioma cell migration and growth.

PTPmu (PTPμ) is a member of the receptor protein tyrosine phosphatase IIb family that participates in both homophilic cell-cell adhesion and signaling. PTPmu is proteolytically downregulated in glioblastoma generating extracellular and intracellular fragments that have oncogenic activity. The intracellular fragments, in particular, are known to accumulate in the cytoplasm and nucleus where they interact with inappropriate binding partners/substrates generating signals required for glioma cell migration and growth. Thus, interfering with these fragments is an attractive therapeutic strategy. To develop agents that target these fragments, we used the AI-based AtomNetⓇ model, a drug design and discovery tool, to virtually screen molecular libraries for compounds able to target a binding pocket bordered by the wedge domain, a known regulatory motif located within the juxtamembrane portion of the protein. Seventy-four high-scoring and chemically diverse virtual hits were then screened in multiple cell-based assays for effects on glioma cell motility (scratch assays) and growth in 3D culture (sphere assays), and PTPmu-dependent adhesion (Sf9 aggregation). We identified three inhibitors (247678835, 247682206, 247678791) that affected the motility of multiple glioma cell lines (LN229, U87MG, and Gli36delta5), the growth of LN229 and Gli36 spheres, and PTPmu-dependent Sf9 aggregation. Compound 247678791 was further shown to suppress PTPmu enzymatic activity in an in vitro phosphatase assay, and 247678835 was able to inhibit the growth of human glioma tumors in mice. We propose that these three compounds are PTPmu-targeting agents with therapeutic potential for treating glioblastoma.

Cystathionine gamma-lyase (CTH) inhibition attenuates glioblastoma formation

PurposeGlioblastoma (GBM) is the most common type of adult brain tumor with extremely poor survival. Cystathionine-gamma lyase (CTH) is one of the main Hydrogen Sulfide (H2S) producing enzymes and its expression contributes to tumorigenesis and angiogenesis but its role in glioblastoma development remains poorly understood. Methodsand Principal Results: An established allogenic immunocompetent in vivo GBM model was used in C57BL/6J WT and CTH KO mice where the tumor volume and tumor microvessel density were blindly measured by stereological analysis. Tumor macrophage and stemness markers were measured by blinded immunohistochemistry. Mouse and human GBM cell lines were used for cell-based analyses. In human gliomas, the CTH expression was analyzed by bioinformatic analysis on different databases.In vivo, the genetic ablation of CTH in the host led to a significant reduction of the tumor volume and the protumorigenic and stemness transcription factor sex determining region Y-box 2 (SOX2). The tumor microvessel density (indicative of angiogenesis) and the expression levels of peritumoral macrophages showed no significant changes between the two genotypes. Bioinformatic analysis in human glioma tumors revealed that higher CTH expression is positively correlated to SOX2 expression and associated with worse overall survival in all grades of gliomas. Patients not responding to temozolomide have also higher CTH expression. In mouse or human GBM cells, pharmacological inhibition (PAG) or CTH knockdown (siRNA) attenuates GBM cell proliferation, migration and stem cell formation frequency. Major ConclusionsInhibition of CTH could be a new promising target against glioblastoma formation.

Comparison of Near-Infrared Imaging Agents Targeting the PTPmu Tumor Biomarker.

Maximal, safe resection of solid tumors is considered a critical first step in successful cancer treatment. The advent of fluorescence image-guided surgery (FIGS) using non-specific agents has improved patient outcomes, particularly in the case of glioblastoma. Molecularly targeted agents that recognize specific tumor biomarkers have the potential to augment these gains. Identification of the optimal combination of targeting moiety and fluorophore is needed prior to initiating clinical trials. A 20-amino acid peptide (SBK2) recognizing the receptor protein-tyrosine phosphatase mu (PTPmu)-derived tumor-specific biomarker, with or without a linker, was conjugated to three different near-infrared fluorophores: indocyanine green (ICG), IRDye® 800CW, and Tide Fluor™ 8WS. The in vivo specificity, time course, and biodistribution were evaluated for each using mice with heterotopic human glioma tumors that express the PTPmu biomarker to identify component combinations with optimal properties for FIGS. SBK2 conjugated to ICG demonstrated excellent specificity for gliomas in heterotopic tumors. SBK2-ICG showed significantly higher in vivo tumor labeling compared to the Scram-ICG control from 10min to 24h, p < 0.01 at all timepoints, following injection, as well as a significantly higher ex vivo tumor signal at 24h, p < 0.001. Inserting a six-amino acid linker between the targeting peptide and ICG increased the clearance rate and resulted in significantly higher in vivo tumor signal relative to its linker-containing Scrambled control from 10min to 8h, p < 0.05 at all timepoints, after dosing. Agents made with the more hydrophilic IRDye® 800CW and Tide Fluor™ 8WS showed no specific tumor labeling relative to the controls. The IRDye 800CW-conjugated agents cleared within 1h, while the non-specific fluorescent tumor signal generated by the Tide Fluor 8WS-conjugated agents persists beyond 24h. The SBK2 PTPmu-targeting peptide conjugated to ICG specifically labels heterotopic human gliomas grown in mice between 10min and 24h following injection. Similar molecules constructed with more hydrophilic dyes demonstrated no specificity. These studies present a promising candidate for use in FIGS of PTPmu biomarker-expressing tumors.

CTULIP: application of a human-based RNA-seq primary tumor classification tool for cross-species primary tumor classification in canine.

The domestic dog, Canis familiaris, is quickly gaining traction as an advantageous model for use in the study of cancer, one of the leading causes of death worldwide. Naturally occurring canine cancers share clinical, histological, and molecular characteristics with the corresponding human diseases. In this study, we take a deep-learning approach to test how similar the gene expression profile of canine glioma and bladder cancer (BLCA) tumors are to the corresponding human tumors. We likewise develop a tool for identifying misclassified or outlier samples in large canine oncological datasets, analogous to that which was developed for human datasets. We test a number of machine learning algorithms and found that a convolutional neural network outperformed logistic regression and random forest approaches. We use a recently developed RNA-seq-based convolutional neural network, TULIP, to test the robustness of a human-data-trained primary tumor classification tool on cross-species primary tumor prediction. Our study ultimately highlights the molecular similarities between canine and human BLCA and glioma tumors, showing that protein-coding one-to-one homologs shared between humans and canines, are sufficient to distinguish between BLCA and gliomas. The results of this study indicate that using protein-coding one-to-one homologs as the features in the input layer of TULIP performs good primary tumor prediction in both humans and canines. Furthermore, our analysis shows that our selected features also contain the majority of features with known clinical relevance in BLCA and gliomas. Our success in using a human-data-trained model for cross-species primary tumor prediction also sheds light on the conservation of oncological pathways in humans and canines, further underscoring the importance of the canine model system in the study of human disease.

Silencing myelin protein zero-like 1 expression suppresses cell proliferation and invasiveness of human glioma cells by inhibiting multiple cancer-associated signal pathways

Glioma is the most common primary malignant tumor of the adult central nervous system. It has high morbidity and poor survival. Myelin protein zero-like protein 1 (MPZL1) is a cell surface glycoprotein that activates numerous adhesion-dependent signaling pathways. MPZL1 plays important roles in human cancers that include metastatic process; however, it is not clear if MPZL1 plays a role in human glioma. Therefore, this study aimed to determine if silencing MPZL1 impacted the cell proliferative features of human glioma cells. First, MPZL1 expression was investigated in human glioma samples and tumor cell lines. Then the effects of small interfering RNA (siRNA)-targeting MPZL1 were analyzed on proliferation, colony formation, cell cycle progression, and invasion of human glioma cells. The results from this study demonstrated that MPZL1 was highly expressed in human glioma tissues and glioma cell lines. In addition, knockdown of MPZL1 significantly inhibited cell proliferation, colony formation, and invasiveness of glioma cells, and effectively induced cell cycle arrest at the G1 phase. Western blotting analysis indicated that silencing MPZL1 expression downregulated the expression of matrix metalloproteinase-2 (MMP-2), WNT1, caspase-3, cyclin A1, epidermal growth factor receptor (EGFR), and signal transducer and activator of transcription 3 (STAT3), and upregulated p53. The results from this study suggest that MPZL1 might be a marker for tumors and could be a potential therapeutic target for human glioma.

Abstract 3175: FAT1 and the immunosuppressive milieu in glioblastoma tumors

Abstract INTRODUCTION: Glioblastoma tumors harbor an immunosuppressive microenvironment which is sustained by key players such as TGF-β1/2 and IL-10 cytokines; and mediators of the PD-1/PD-L1/2 pathway. Previously, our lab has shown the role of a new gene, FAT1, in promoting gliomagenesis by supporting the production of pro-inflammatory cytokines (IL-1β, IL-6, etc.) in glioma cells. Here, we show a potent role of FAT1 gene in regulation of anti-inflammatory genes in human glioma tumors, cell lines and primary cultures. Our findings indicate an additional way in which FAT1 might contribute to gliomagenesis. METHODS: q-PCR was used to analyse transcript levels of FAT1 and genes of the TGF-β1/2, IL-10 and PD-L1/2 pathways in fresh-frozen glioblastoma samples relative to normal brain RNA. Gene expression correlation was analysed using Spearman's test and semi-supervised clustering. TCGA expression data was extracted to analyse the correlation of FAT1 with anti-inflammatory genes. Primary tumor cultures and cell lines were transfected with FAT1-specific siRNA to check the effect on expression of anti-inflammatory cytokines, as well as effect on migratory properties of monocytes towards FAT1-knockdown tumor cells (supernatants). RESULTS: Significant positive correlation was found between upregulation of FAT1 and that of anti-inflammatory genes in fresh-frozen glioblastoma samples as well as TCGA datasets. Upon siRNA-mediated knockdown of FAT1 in in-vitro glioma cell cultures, we observed decreased anti-inflammatory genes' expression. We also found increased transmigration of monocytes towards the supernatants from siFAT1-transfected cells. CONCLUSION: FAT1 favors the immunosuppressive milieu in glioblastoma by supporting expression of anti-inflammatory genes. This is an emerging role of the new oncogene FAT1 and crucial to our understanding of the contributions of FAT1 to hallmarks of cancer. FUNDING: SERB Start-Up Grant (DST, India); DHR Young Scientist Scheme (ICMR, India) Citation Format: Khushboo Irshad, Chitrangda Srivastava, Nargis Malik, Yakhlesh Gupta, Vaishali Suri, Swati Mahajan, Deepak Gupta, Ashish Suri, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1 and the immunosuppressive milieu in glioblastoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3175.

Heat Shock Protein Inhibitor 17-Allyamino-17-Demethoxygeldanamycin, a Potent Inductor of Apoptosis in Human Glioma Tumor Cell Lines, Is a Weak Substrate for ABCB1 and ABCG2 Transporters.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and has a poor prognosis. Complex genetic alterations and the protective effect of the blood–brain barrier (BBB) have so far hampered effective treatment. Here, we investigated the cytotoxic effects of heat shock protein 90 (HSP90) inhibitors, geldanamycin (GDN) and 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), in a panel of glioma tumor cell lines with various genetic alterations. We also assessed the ability of the main drug transporters, ABCB1 and ABCG2, to efflux GDN and 17-AAG. We found that GDN and 17-AAG induced extensive cell death with the morphological and biochemical hallmarks of apoptosis in all studied glioma cell lines at sub-micro-molar and nanomolar concentrations. Moderate efflux efficacy of GDN and 17-AAG mediated by ABCB1 was observed. There was an insignificant and low efflux efficacy of GDN and 17-AAG mediated by ABCG2. Conclusion: GDN and 17-AAG, in particular, exhibited strong proapoptotic effects in glioma tumor cell lines irrespective of genetic alterations. GDN and 17-AAG appeared to be weak substrates of ABCB1 and ABCG2. Therefore, the BBB would compromise their cytotoxic effects only partially. We hypothesize that GBM patients may benefit from 17-AAG either as a single agent or in combination with other drugs.

Research on HOXA11-AS in Malignant Tumors

Long-chain non-coding RNA HOXA11 antisense RNA (HOXA11-AS) is a kind of lncRNA discovered in recent years. Long-chain non-coding RNA (LncRNA) is an important regulatory factor of protein-coding genes, especially the disorder of LncRNA in more and more diseases which are found, including cancer. HOXA11-AS was first discovered in mouse embryo cDNA library using probes, and then it was discovered by scholars and played an important role in human cervical cancer, gastric cancer, glioma and other malignant tumor cells. Overexpression of HOXA11-AS has been found to promote cell proliferation, migration and tumor invasion, and has a carcinogenic effect. HOXA11-AS can promote tumor proliferation, metastasis and other malignant biological behaviors by interacting with miRNA and EZH2 protein, and is considered to be carcinogenic lncRNA. The discovery of HOXA11-AS provides new ideas for tumor prevention and treatment.

RNA Interference-Mediated Aurora Kinase A Gene Silencing Inhibits Human Glioma Cells Proliferation and Tumor Growth in Mice.

Objective: This study aims to explore the roles of Aurora Kinase A (Aurora A) in human glioma progression and relevant molecular mechanisms involved.Methods: RNA interference (RNAi) technology was performed to silence the Aurora A gene in human glioma cell line U251 and U87. Western blot and real-time PCR were used to determine the protein and mRNA expression levels of Aurora A. Flow cytometry was performed to analyze the cell cycle distribution and MTT was used to examine the cell viability. Annexin V/FITC double staining and Hoechst 33258 staining were carried out to examine cell apoptosis. Xenograft tumor model was established to examine the effect of Aurora A siRNA on tumor growth in vivo.Results: RNAi-mediated Aurora A gene silencing with specific short interfering RNA (siRNA) significantly decreased Aurora A protein and mRNA expression levels in human glioma cell line U251 and U87. Aurora A knockdown in glioma cells with siRNA strongly inhibited cell proliferation, along with the accumulation of cells in the G1, G2/M phase and decrease in S phase. Furthermore, the enhancement of cell apoptosis in vitro and the suppression of xenograft tumor growth in vivo were also observed after Aurora A silencing in U251 cell. In addition, Aurora A knockdown resulted in decreased expression of anti-apoptotic protein Bcl-2 and cell cycle protein Cyclin D1, while increased expression of pro-apoptotic factor caspase-3.Conclusion: Aurora A can be used as a candidate targeting gene and inhibition of Aurora A is a potentially promising therapy for glioblastoma.

The Effect of Gold Nanoparticles on Chemo and Radiotherapy of Brain Tumor Cells

To test the hypothesis that gold nanoparticles (GNPs) can improve the sensitivity of human glioma tumors, to radiotherapy (RT) and chemotherapy. Major objectives were to isolate and culture the human glioma tumor cells in vitro, to evaluate the chemo- & radiosensitizing effects of the GNPs, and to extrapolate this sensitization effect to the isocitrate dehydrogenase (IDH) mutation status of the tumor. Fresh post-operative glioma tumor samples were obtained (n = 43) and cultured in-vitro. Morphology of the cultured cells was evaluated using Giemsa staining and the proliferation index was calculated based on their population doubling time (PDT). Western blot assay was performed to evaluate the expression of apoptotic proteins, viz. Bad, Bax, Caspase-3 and Bcl2, involved in the tumor cell death. Each of such cultured patient samples were then divided into 8 therapeutic interventional groups, i.e. control, GNP alone, Temozolomide (TMZ) alone, TMZ+GNP, RT alone, RT+GNP, RT+TMZ, RT+TMZ+GNP groups. Based on this group assignment, the cultured cells were exposed to the following interventions: carboxylic acid functionalized GNPs of diameter 30 nm at a concentration of 100ng/mL, 6MV energy X-rays to an absorbed dose of 10 Grays (Gy) using Varian linear accelerator and/or to the drug TMZ at the concentration of 500nM. Cell viability and surviving fraction of cells (SF) were assessed 24 hours after the interventions using MTT assay & optical density (OD) value comparisons with the control group (mean SF 100). Study results showed that 36 patients’ samples, cultured successfully in vitro, were evaluable. The RT+TMZ+GNP group was found to have the least mean SF {76.8, (range 50.4 - 92.9)}. The mean (+/- SD) SF in the RT vs RT+GNP group was 88.3 (+/- 11.4) vs 84.5 (+/- 13.3); TMZ vs TMZ+GNP group was 85.1 (+/- 7.2) vs 83.6 (+/- 7.9); and RT+TMZ vs RT+TMZ+GNP group was 77.9 (+/- 12.2) vs 76.8 (+/- 12.4), respectively. Thus, the mean difference in the SF achieved due to addition of GNPs to RT, TMZ, RT+TMZ was 3.79 (p-value 0.001), 1.47 (p-value 0.001), and 1.06 (p-value 0.011), respectively, with statistical significance. More so, this beneficial effect was pronounced in the samples with high proliferation index (HPI) (PDT <72 hrs, n = 13) than in those with low proliferation index (LPI) (PDT >72 hrs, n = 23), with statistical significance. Although the GNPs were found to be advantageous in the IDH positive samples (n = 11) when compared to the IDH negative samples (n = 14), the results were not statistically significant. Apoptosis, a mechanism of tumor cell death, has been confirmed using western blotting technique in our study. Our study is the first to demonstrate that GNPs could potentially be used to enhance the sensitivity of the human glioma tumors to radiotherapy and chemotherapy. Future larger, in vivo studies are needed to confirm our study findings.

Long non-coding RNA NEAT1 promotes human glioma tumor progression via miR-152-3p/CCT6A pathway

BackgroundLong non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) has been documented to implicate in diverse tumor progression. However, the mechanism of NEAT1 in glioma was rarely reported. MethodsThe levels of NEAT1, microRNA-152-3p (miR-152-3p) and chaperonin containing TCP1 subunit 6A (CCT6A) in glioma tissues and cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability, apoptotic rate, the migrated and invaded abilities of A172 and U251 cells were evaluated via cell counting kit-8 (CCK-8), flow cytometry and Transwell assay, respectively. The mice xenograft model was constructed to further verify the effect of NEAT1. The interactions between miR-152-3p and NEAT1 or CCT6A were predicted by starBase v2.0 or TargetScan, then luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull-down assay were performed to validate the interaction. The protein level of CCT6A was detected by Western blot assay. ResultsThe levels of NEAT1, CCT6A were highly expressed, but miR-152-3p was decreased in glioma tissues and cells. NEAT1 depletion or miR-152-3p mimics suppressed cell viability, migrated and invaded abilities but induced apoptotic rate in A172 and U251 cells, while the introduction of CCT6A partly counteracted these impacts. In addition, NEAT1 silencing impeded xenograft tumor growth in vivo. MiR-152-3p was verified as a direct target of NEAT1 and directly targeted CCT6A. CCT6A expression was upregulated by NEAT1 and reversed by miR-152-3p. ConclusionNEAT1 enhanced glioma progression, partially through miR-152-3p/CCT6A pathway. The novel regulatory network might contribute to the diagnosis and treatment of glioma.

Modeling human pediatric and adult gliomas in immunocompetent mice through costimulatory blockade

ABSTRACT Currently, human glioma tumors are mostly modeled in immunodeficient recipients; however, lack of interactions with adaptive immune system is a serious flaw, particularly in the era when immunotherapies dominate treatment strategies. Our group was the first to successfully establish the orthotopic transplantation of human glioblastoma (GBM) in immunocompetent mice by inducing immunological tolerance using a short-term, systemic costimulation blockade strategy (CTLA-4-Ig and MR1). In this study, we further validated the feasibility of this method by modeling pediatric diffuse intrinsic pontine glioma (DIPG) and two types of adult GBM (GBM1, GBM551), in mice with intact immune systems and immunodeficient mice. We found that all three glioma models were successfully established, with distinct difference in tumor growth patterns and morphologies, after orthotopic xenotransplantation in tolerance-induced immunocompetent mice. Long-lasting tolerance that is maintained for up to nearly 200 d in GBM551 confirmed the robustness of this model. Moreover, we found that tumors in immunocompetent mice displayed features more similar to the clinical pathophysiology found in glioma patients, characterized by inflammatory infiltration and strong neovascularization, as compared with tumors in immunodeficient mice. In summary, we have validated the robustness of the costimulatory blockade strategy for tumor modeling and successfully established three human glioma models including the pediatric DIPG whose preclinical study is particularly thwarted by the lack of proper animal models.

Characterization of G protein β subunit expression of human brain Glioma tumor.

Proteome indicates the protein content of a genome. Proteome analysis is effective in a new system formulation and prediction, prevention, and treatment based on protein. One of the purposes of proteomics researches is to know and understand the cancer mechanism. In this study, we separated the proteins by the Two-Dimensional Electrophorese method and analyzed and compared protein spots by statistical and software data. The spots were separated and identified by the proteins' Isoelectric PH differences, molecular weights, and data bank. In continuation, the protein profiles were clustered by MALDI-TOF-TOF and the main element was identified and confirmed. We have used site PhosphoSitePlus® to review post-translational modifications. The findings indicated that the G protein Beta subunit rate increased in the astrocytoma, oligodendroglia, and glioblastoma cerebral malignant tumors. The βγ complex formation may prevent and activates many paths of cellular growth. The βγ complex activity control of the changes after the conversion parallel to GTPase activity of this α subunit may be a formulation mechanism for the G signal path (Tab. 5, Fig. 4, Diagram 2, Ref. 29). Keywords: glioma, G protein β subunit, proteomics.

Abstract 2441: Association between NRF1 regulatory gene networks and cancer and aging

Abstract There is a growing convergence between molecular aspects of aging and cancer. Cellular senescence acts as a barrier through preventing the proliferation of cells under oncogenic stress. Therefore, we decided to examine the role of nuclear respiratory factor 1 (NRF1) transcription factor in human brain cancer and brain aging. In contrast to higher expression of NRF1 in human glioma tumors, lower NRF1 levels are observed in Alzheimer’s disease (AD) brain tissue. Using the glioma and AD transcriptomic data of NRF1 target genes, we conducted Transcription Factor Target Enrichment Analysis that detects transcription factor (TF) activity based on quantification of the collective transcriptional activation of their target genes. NRF1 TF activity was significantly lower in AD brain tissues from four independent cohorts compared to non-AD brain tissues. In contrast, we observed higher NRF1 transcription factor activity in glioma. NRF1 activity as well as expression of several cell cycle pathway genes including CDK1,2,6,9 and NRF1 target genes such as NR2E1, APOE and MAPT are significantly altered in these two population cohorts. We also discovered that expression of NRF1, RB1 and E2F influenced survival in glioma. Specifically, the mean overall survival for patients with a high NRF1, E2F1,2,6,7,8 and RB1 gene signature was 20.91 months (95% CI = 9.37-32.46), compared to 101.99 months (95% CI = 74.28-129.70), in high NRF1 expressing patients that lacked the specific gene signature. NRF1 based gene signatures combined with IDH1 mutation status are necessary and sufficient for explaining the observed differences in survival curves. We examined the association of NRF1, the E2Fs and RB1 through Bayesian graphical modeling and observed gender differences in gene networks between NRF1 and E2F family members in the glioma patient sub-populations as compared to normal aging human brain samples. Furthermore, the differentially expressed NRF1 regulated genes are part of the KEGG pathways associated with spliceosome, ribosome and lysosome function as well as a diverse array of other functions are enriched, indicating ways in which NRF1 may be involved in glioma biology. In summary these findings support our postulate that NRF1 is an important transcription factor in the human brain, where it plays an active role in both aging and cancer. Citation Format: Kaumudi Bhawe, Quentin Felty, Changwon Yoo, Deodutta Roy. Association between NRF1 regulatory gene networks and cancer and aging [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 2441.