Glioma Cell Lines Research Articles

Expression of brain-derived neurotrophic factor and formation of migrasome increases in the glioma cells induced by the adipokinetic hormone.

It has been previously shown that brain-derived neurotrophic factor is linked with various types of cancer. Brain-derived neurotrophic factor is found to be highly expressed in multiple human cancers and associated with tumor growth, invasion, and metastasis. Adipokinetic hormones are functionally related to the vertebrate glucagon, as they have similar functionalities that manage the nutrient-dependent secretion of these two hormones. Migrasomes are new organelles that contain numerous small vesicles, which aid in transmitting signals between the migrating cells. Therefore, the aim of this study was to investigate the effects of Anax imperator adipokinetic hormone on brain-derived neurotrophic factor expression and ultrastructure of cells in the C6 glioma cell line. The rat C6 glioma cells were treated with concentrations of 5 and 10 Anax imperator adipokinetic hormone for 24 h. The effects of the Anax imperator adipokinetic hormone on the migrasome formation and brain-derived neurotrophic factor expression were analyzed using immunocytochemistry and transmission electron microscope. The rat C6 glioma cells of the 5 and 10 μM Anax imperator adipokinetic hormone groups showed significantly high expressions of brain-derived neurotrophic factor and migrasomes numbers, compared with the control group. A positive correlation was found between the brain-derived neurotrophic factor expression level and the formation of migrasome, which indicates that the increased expression of brain-derived neurotrophic factor and the number of migrasomes may be involved to metastasis of the rat C6 glioma cell line induced by the Anax imperator adipokinetic hormone. Therefore, the expression of brain-derived neurotrophic factor and migrasome formation may be promising targets for preventing tumor proliferation, invasion, and metastasis in glioma.

Elevated SH3 and Multiple Ankyrin Repeat Domains 2 Expression Correlates With Improved Glioma Prognosis.

This study investigates the prognostic significance of SH3 and multiple ankyrin repeat domains 2 (SHANK2) gene expression in glioma patients, using data from The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, and the Gene Expression Omnibus (GEO). Through comprehensive analysis, we found a significant association between higher SHANK2 expression and improved survival outcomes across various glioma subtypes. To further validate the clinical relevance of SHANK2, we conducted cellular experiments involving siRNA-mediated knockdown of SHANK2 in U87 and A172 glioma cell lines. Quantitative real-time PCR (qPCR) and Western blot analyses confirmed the successful knockdown of SHANK2, and subsequent MTT assays revealed that silencing SHANK2 significantly promoted glioma cell proliferation. These findings underscore the potential role of SHANK2 as a tumor suppressor in glioma. The study also introduces a multivariate prognostic model incorporating SHANK2, providing a novel perspective on glioma prognosis. While the retrospective nature of the study presents limitations, our results suggest that SHANK2 expression could serve as a valuable biomarker for glioma prognosis and inform future therapeutic strategies.

Investigation of the Effect of Indatraline on Oxidative Damage Induced by Hydrogen Peroxide in C6 Glioma Cell Line

Oxidative stress is defined as an imbalance between the generation of reactive oxygen species (ROS) and their scavenging. Indatralin, which has serotonin reuptake inhibitory activity, has not yet been studied for its ability to prevent oxidative damage. Our research's objective was to find out how indatraline defends against oxidative damage. C6 cells were used in the study and four different cell groups were created. The control group received no therapy at all. For 24 hours, cells in the H2O2 group were exposed to 0.5 mM H2O2. The indatraline group received indatraline treatments for 24 hours at various doses (0.5, 1, 2.5, 5 and 10 μM). For one hour, indatraline was administered to the indatraline + H2O2 group at various concentrations (0.5, 1, 2.5, 5 and 10 μM) before the group was subjected to 0.5 mM H2O2 for 24 hours. Following the occurrence of oxidative damage, total antioxidant status (TAS) and total oxidant status (TOS) levels were determined. Cell viability was also evaluated using the XTT assay. As a result, after hydrogen peroxide-induced oxidative damage, indatraline at doses of 10, 5, and 2.5 μM showed a protective effect by significantly enhanced cell survival in C6 cells(p < 0.001). Additionally, indatraline boosted the lowered TAS level while decreasing the elevated TOS levels following hydrogen peroxide-induced oxidative damage (p<0.001).

Transmembrane and Ubiquitin-Like Domain-Containing 1 Promotes Glioma Growth and Indicates Unfavorable Prognosis.

Ubiquitin-related proteins have garnered increasing attention for their roles in tumorigenesis. Transmembrane and ubiquitin-like domain-containing 1 (TMUB1) is a recently discovered protein in the ubiquitin-like domain family, yet its involvement in glioma remains poorly understood. This study is aimed at investigating the functional significance and clinical relevance of TMUB1 in glioma. We conducted a comprehensive analysis using two cohorts: a retrospective glioma cohort from our hospital and The Cancer Genome Atlas (TCGA) cohort. The mRNA levels of TMUB1 were assessed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Clinical associations of TMUB1 in these cohorts were evaluated using correlation tests, chi-square tests, and survival analyses. Additionally, we performed TMUB1 knockdown in U87 and LN-229 human glioma cell lines, and cellular growth was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Our results revealed that TMUB1 expression was elevated in glioma tissues compared to normal brain tissues. Notably, lower TMUB1 expression correlated with favorable characteristics such as lower World Health Organization (WHO) grade and 1p/19q codeletion. Moreover, patients with higher TMUB1 levels in glioma tissues exhibited worse prognosis in both TCGA cohort and our retrospective cohort, underscoring its prognostic significance in gliomas. Cellular experiments demonstrated that TMUB1 silencing suppressed the growth of glioma cells. TMUB1 emerges as a novel and clinically relevant prognostic biomarker for gliomas. Targeting TMUB1 holds promise as a potential strategy for glioma treatment. This study contributes valuable insights into the multifaceted role of TMUB1 in glioma pathogenesis and its potential as a diagnostic and therapeutic target.

KLC1-ROS1 Fusion Exerts Oncogenic Properties of Glioma Cells via Specific Activation of JAK-STAT Pathway.

Here, we investigated the detailed molecular oncogenic mechanisms of a novel receptor tyrosine kinase (RTK) fusion, KLC1-ROS1, with an adapter molecule, KLC1, and an RTK, ROS1, discovered in pediatric glioma, and we explored a novel therapeutic target for glioma that possesses oncogenic RTK fusion. When wild-type ROS1 and KLC1-ROS1 fusions were stably expressed in the human glioma cell lines A172 and U343MG, immunoblotting revealed that KLC1-ROS1 fusion specifically activated the JAK2-STAT3 pathway, a major RTK downstream signaling pathway, when compared with wild-type ROS1. Immunoprecipitation of the fractionated cell lysates revealed a more abundant association of the KLC1-ROS1 fusion with JAK2 than that observed for wild-type ROS1 in the cytosolic fraction. A mutagenesis study of the KLC1-ROS1 fusion protein demonstrated the fundamental roles of both the KLC1 and ROS1 domains in the constitutive activation of KLC1-ROS1 fusion. Additionally, in vitro assays demonstrated that KLC1-ROS1 fusion upregulated cell proliferation, invasion, and chemoresistance when compared to wild-type ROS1. Combination treatment with the chemotherapeutic agent temozolomide and an inhibitor of ROS1, JAK2, or a downstream target of STAT3, demonstrated antitumor effects against KLC1-ROS1 fusion-expressing glioma cells. Our results demonstrate that KLC1-ROS1 fusion exerts oncogenic activity through serum-independent constitutive activation, resulting in specific activation of the JAK-STAT pathway. Our data suggested that molecules other than RTKs may serve as novel therapeutic targets for RTK fusion in gliomas.

Efficacy of antitumor vaccines based on photoinduced GL261 glioma cells using photosensitizers from the group of tetra(aryl)tetracyanoporphyrases with different aryl substituents

Glioblastomas are solid tumors in the brain that pose a challenge for traditional treatments like surgery, radiation therapy, and chemotherapy. Complete cures are not guaranteed, and these treatments cause numerous side effects. First-line treatment approval has only been granted to Temozolomide (TMZ), the sole chemotherapy drug available. Using TMZ increases the median overall survival from 15 to 17 months. In general clinical practice, innovative interventions have not demonstrated efficacy due to glioma heterogeneity and their immunosuppressive microenvironment. Immunogenic cell death (ICD) activates an immune response against cancer cells by emitting damage-associated molecular patterns (DAMPs) upon cell death/dying. The DAMPs activate an anti-tumor immune response. Photodynamic therapy (PDT) can induce ICD. Integrating the principles of immunogenic cell death into glioma immunotherapy could elicit a targeted immune response against the diverse tumor. Vaccination with dendritic cells represents a promising approach for immunotherapy. The goals of this research are to evaluate the efficacy of a prophylactic vaccine using immunogenically-photoinduced glioma GL261 cells and a dendritic cell vaccine in an orthotopic in vivo model. The glioma cell line (GL261) is cultured in RPMI medium supplemented with 10% serum, L-glutamine, 1% penicillin, and 1% streptomycin in a CO2 incubator. Photodynamic treatment involves using photosensitizers from the tetra(aryl)tetracyanoporphyrazine group with 9-phenanthrenyl (pz I) or 4-(4-fluorobenzyloxy)phenyl (pz III) as side substituents. The GL261 cell line is incubated in a serum-free solution containing a selected porphyrazine for four hours. Subsequently, the solution is exchanged with complete medium and the cells are activated through photodynamic treatment at a dose of 20 J/cm2. The cells are then further incubated for 24 hours in a CO2 incubator. Immunizing mice includes subcutaneously injecting photoinduced GL261 glioma cell lysates twice with a 7-day interval. One week after the final immunization, viable GL261 glioma cells are injected into the brain using a stereotaxis frame. Measuring the neurological status of the animals occurs for 25 days, and tumor localization and volume are detected on day 23 by MRI. The survival rate of the experimental groups was 100% (pz III) or significantly higher (pz I) than that of the control groups. Additionally, neurological symptoms were either not identified (pz III) or were significantly lower (pz I) compared to the control animal groups experienced. The dendritic cell vaccine was based on photoinduced GL261 glioma cells. The femur and tibia bones of C57BL/6J mice were used and differentiated for 9 days in RPMI medium to isolate bone marrow stem cells. The medium was supplemented with 5% fetal bovine serum, 20 ng/ml GM-CSF, 1% L-glutamine, 1mM sodium pyruvate, 50 µM β-mercaptoethanol, 100 units/ml penicillin, and 100 µg/L streptomycin. The culture medium was renewed on days 3 and 6 to maintain stability. Protein levels are quantified in cell lysates collected. 2 mg of protein is added to a dendritic cell suspension, and after 90 minutes, 0.5 µg/ml lipopolysaccharide is added for 24 hours. Intraperitoneal dendritic cell vaccine immunization on animals is conducted, with injections 7 days apart. One week post-last immunization, viable GL261 cells are intracranially injected via a stereotactic frame. The neurological status of the animals was monitored for 25 days, and an MRI was conducted on day 23 to evaluate the brain tumor. The survival rate of animals in the experimental groups did not significantly differ from those in the control groups. However, the experimental groups exhibited significantly lower neurological symptoms, and the tumor was visualized without any areas of necrosis, with a smaller volume.

Gastrodin Induces Ferroptosis of Glioma Cells via Upregulation of Homeobox D10.

Gastrodin, the primary bioactive compound found in Gastrodia elata, has been shown to exhibit neuroprotective properties in a range of neurological disorders. However, the precise mechanisms through which gastrodin influences glioma cells remain unclear, and there is a scarcity of data regarding its specific effects. To ascertain the viability of glioma cell lines LN229, U251, and T98, the CCK-8 assay, a colony formation assay, and a 3D culture model were employed, utilizing varying concentrations of gastrodin (0, 5, 10, and 20 μM). Gastrodin exhibited a notable inhibitory effect on the growth of glioma cells, as evidenced by its ability to suppress colony formation and spheroid formation. Additionally, gastrodin induced ferroptosis in glioma cells, as it can increase the levels of reactive oxygen species (ROS) and peroxidized lipids, and reduced the levels of glutathione. Using a subcutaneous tumor model, gastrodin was found to significantly inhibit the growth of the T98 glioma cell line in vivo. Using high-throughput sequencing, PPI analysis, and RT-qPCR, we successfully identified Homeobox D10 (HOXD10) as the principal target of gastrodin. Gastrodin administration significantly enhanced the expression of HOXD10 in glioma cells. Furthermore, treatment with gastrodin facilitated the transcription of ACSL4 via HOXD10. Notably, the inhibition of HOXD10 expression impeded ferroptosis in the cells, which was subsequently restored upon rescue with gastrodin treatment. Overall, our findings suggest that gastrodin acts as an anti-cancer agent by inducing ferroptosis and inhibiting cell proliferation in HOXD10/ACSL4-dependent pathways. As a prospective treatment for gliomas, gastrodin will hopefully be effective.

Synthesis of Some New 1,3,4-Oxadiazole Derivatives and Evaluation of Their Anticancer Activity.

In this work, some new 2-[(5-((2-acetamidophenoxy)methyl)-1,3,4-oxadiazol-2-yl)thio]acetamide derivatives (4a-4l) were synthesized and studied for their anticancer activity. Twelve new compounds were tested on the A549 human lung cancer cell line, C6 rat glioma cell line, and L929 murine fibroblast cell line. Compounds 4f, 4i, 4k, and 4l (IC50: 1.59-7.48 μM), and especially 4h (IC50: <0.14 μM), exhibited excellent cytotoxic profile on A549 with selectivity. Compounds 4g and 4h showed remarkable antiproliferative activity on the C6 cell line with IC50 values of 8.16 and 13.04 μM, respectively. The compounds with the lowest IC50 value on the A549 cell line (4f, 4h, 4i, 4k, and 4l) were further studied to determine the mechanism of action. These compounds were found to induce apoptosis with a higher ratio (16.10-21.54%) than that of the standard drug cisplatin (10.07%). Compound 4f displayed mitochondrial membrane depolarization and caspase-3 activation at most, whereas compounds 4h (89.66%) and 4i (78.78%) had outstanding retention rates in the G0/G1phase of the cell cycle (cisplatin 74.75%). Compounds 4f, 4g, 4h, and 4l exhibited matrix metalloproteinase-9 (MMP-9) inhibition higher than 75% at 100 μg/mL; even IC50 values were found to be 1.65 and 2.55 μM for 4h and 4l. In addition, in silico physicochemical properties of the compounds and molecular docking interaction of compound 4h on the MMP-9 enzyme were evaluated; the desired and expected results were obtained.

Construction and validation of a risk model based on the key SNARE proteins to predict the prognosis and immune microenvironment of gliomas.

Synaptic transmission between neurons and glioma cells can promote glioma progression. The soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors (SNARE) play a key role in synaptic functions. We aimed to construct and validate a novel model based on the SNARE proteins to predict the prognosis and immune microenvironment of glioma. Differential expression analysis and COX regression analysis were used to identify key SRGs in glioma datasets, and we constructed a prognostic risk model based on the key SRGs. The prognostic value and predictive performance of the model were assessed in The Cancer Genome Atlas (TCGA) and Chinese glioma Genome Atlas (CGGA) datasets. Functional enrichment analysis and immune-related evaluation were employed to reveal the association of risk scores with tumor progression and microenvironment. A prognostic nomogram containing the risk score was established and assessed by calibration curves and time-dependent receiver operating characteristic curves. We verified the changes of the key SRGs in glioma specimens and cells by real-time quantitative PCR and Western blot analyses. Vesicle-associated membrane protein 2 (VAMP2) and vesicle-associated membrane protein 5 (VAMP5) were identified as two SRGs affecting the prognoses of glioma patients. High-risk patients characterized by higher VAMP5 and lower VAMP2 expression had a worse prognosis. Higher risk scores were associated with older age, higher tumor grades, IDH wild-type, and 1p19q non-codeletion. The SRGs risk model showed an excellent predictive performance in predicting the prognosis in TCGA and CGGA datasets. Differentially expressed genes between low- and high-risk groups were mainly enriched in the pathways related to immune infiltration, tumor metastasis, and neuronal activity. Immune score, stromal score, estimate score, tumor mutational burden, and expression of checkpoint genes were positively correlated with risk scores. The nomogram containing the risk score showed good performance in predicting the prognosis of glioma. Low VAMP2 and high VAMP5 were found in different grades of glioma specimens and cell lines. We constructed and validated a novel risk model based on the expression of VAMP2 and VAMP5 by bioinformatics analysis and experimental confirmation. This model might be helpful for clinically predicting the prognosis and response to immunotherapy of glioma patients.

Glycation Interferes with the Expression of Sialyltransferases and Leads to Increased Polysialylation in Glioblastoma Cells.

Glioblastoma (GBM) is a highly aggressive brain tumor that often utilizes aerobic glycolysis for energy production (Warburg effect), resulting in increased methylglyoxal (MGO) production. MGO, a reactive dicarbonyl compound, causes protein alterations and cellular dysfunction via glycation. In this study, we investigated the effect of glycation on sialylation, a common post-translational modification implicated in cancer. Our experiments using glioma cell lines, human astrocytes (hA), and primary glioma samples revealed different gene expressions of sialyltransferases among cells, highlighting the complexity of the system. Glycation has a differential effect on sialyltransferase expression, upregulating ST8SIA4 in the LN229 and U251 cell lines and decreasing the expression in normal hA. Subsequently, polysialylation increased in the LN229 and U251 cell lines and decreased in hA. This increase in polysialylation could lead to a more aggressive phenotype due to its involvement in cancer hallmark processes such as immune evasion, resistance to apoptosis, and enhancing invasion. Our findings provide insights into the mechanisms underlying GBM aggressiveness and suggest that targeting glycation and sialylation could be a potential therapeutic strategy.

Abstract C141: Targeting SHP2-dependent adaptive resistance to BRAF and MEK inhibition in gliomas

Abstract Introduction: BRAF-mutant gliomas, common in children, are sensitive to MEK and/or BRAF inhibitors. However, responses are incomplete and can be short-lived, due to poorly understood mechanisms of resistance that are likely to involve kinome re-programming, treatment-emergent resistance mutations, and adaptive resistance, by which upstream signaling components become activated due to release of negative feedback. Based on the critical role of SHP2 upstream of RAS pathway, we postulated that the combination of SHP2i and BRAFi/MEKi would maintain superior ERK pathway inhibition in gliomas. Here, we demonstrate that combined SHP2i+BRAFi can be efficacious in BRAFV600E mutant gliomas, potentially without the toxicity posed by SHP2i+MEKi in human participants. Methods: Using a panel of BRAFV600E mutant and wild-type glioma cell lines, we examined cell growth with Incucyte live-cell imaging, RAS activity with pull-down assay, and immunoblots of ERK signaling activity in response to MEKi (Trametinib), BRAFi (Dabrafenib), and/or SHP2i (TNO155). NSG immune-deficient mice were implanted subcutaneously with human-derived glioma lines and patient-derived xenografts (PDXs), then treated via oral gavage for ≤28 days while recording tumor volume and weight. Results: In BRAFV600E glioma cells, we observed ERK reactivation and increased RAS activity in response to BRAFi or MEKi. SHP2 knockdown using PTPN11 pooled siRNA partially inhibited cell growth, while the addition of BRAFi/MEKi enhanced growth inhibition compared with either intervention alone. BRAFV600E cells showed insensitivity to small molecule SHP2i monotherapy, but augmented growth inhibition upon the combination with either BRAFi or MEKi due to prolonged ERK pathway suppression. BRAF wt glioma cell lines exhibit sensitivity to TNO155 monotherapy, and combination with MEKi demonstrates sustained pERK suppression in NF1-mut and NF1 wt cell lines. Additionally, we evaluated the effect of combined BRAFi+SHP2i in heterotopic xenografts using human-derived glioma lines and PDXs. In both models, tumor growth was significantly slower in the combined treatment group than either monotherapy. Moreover, mice tolerated the combination well with no weight loss or lethargy. Conclusion. Combined MEKi and SHP2i attenuate RAS reactivation in response to MEKi and demonstrates additive effects in glioma cell lines that are resistant to MEKi monotherapy. Moreover, SHP2i in combination with BRAFi in BRAFV600E mutant glioma cells synergistically attenuated tumor growth in vitro and shows superior preclinical activity to BRAFi or SHP2i alone in BRAFV600E models of gliomas. Citation Format: Abiola Abdulrahman Ayanlaja, Kriti Lalwani, Maria Ioannou, Christine Pratilas, Karisa Schreck. Targeting SHP2-dependent adaptive resistance to BRAF and MEK inhibition in gliomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C141.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Evaluation of in vitro intrinsic radiosensitivity and characterization of five canine high-grade glioma cell lines

Glioma is the most common primary brain tumor in dogs and predominantly affects brachycephalic breeds. Diagnosis relies on CT or MRI imaging, and the proposed treatments include surgical resection, chemotherapy, and radiotherapy depending on the tumor’s location. Canine glioma from domestic dogs could be used as a more powerful model to study radiotherapy for human glioma than the murine model. Indeed, (i) contrary to mice, immunocompetent dogs develop spontaneous glioma, (ii) the canine brain structure is closer to human than mice, and (iii) domestic dogs are exposed to the same environmental factors than humans. Moreover, imaging techniques and radiation therapy used in human medicine can be applied to dogs, facilitating the direct transposition of results. The objective of this study is to fully characterize 5 canine glioma cell lines and to evaluate their intrinsic radiosensitivity. Canine cell lines present numerous analogies between the data obtained during this study on different glioma cell lines in dogs. Cell morphology is identical, such as doubling time, clonality test and karyotype. Immunohistochemical study of surface proteins, directly on cell lines and after stereotaxic injection in mice also reveals close similarity. Radiosensitivity profile of canine glial cells present high profile of radioresistance.

METTL3-mediated HOTAIRM1 promotes vasculogenic mimicry icontributionsn glioma via regulating IGFBP2 expression

BackgroundHOTAIRM1 is revealed to facilitate the malignant progression of glioma. Vasculogenic mimicry (VM) is critically involved in glioma progression. Nevertheless, the molecular mechanism of HOTAIRM1 in regulating glioma VM formation remains elusive. Thus, we attempted to clarify the role and mechanism of HOTAIRM1 in VM formation in glioma.MethodsqRT-PCR and western blot assays were used to evaluate the gene and protein expression levels of HOTAIRM1 in glioma patient tissue samples and cell lines. The role of HOTAIRM1 in glioma cell progression and VM formation was explored using a series of function gain-and-loss experiments. RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and mechanism experiments were conducted to assess the interaction between HOTAIRM1/METTL3/IGFBP2 axis. Furthermore, rescue assays were conducted to explore the regulatory function of HOTAIRM1/METTL3/IGFBP2 in glioma cell cellular processes and VM formation.ResultsWe found that HOTAIRM1 presented up-regulation in glioma tissues and cells and overexpression of HOTAIRM1 facilitated glioma cell proliferation, migration, invasion, and VM formation. Furthermore, overexpression of HOTAIRM1 promoted glioma tumor growth and VM formation capacity in tumor xenograft mouse model. Moreover, HOTAIRM1 was demonstrated to interact with IGFBP2 and positively regulated IGFBP2 expression. IGFBP2 was found to promote glioma cell malignancy and VM formation. Mechanistically, METTL3 was highly expressed in glioma tissues and cells and was bound with HOTAIRM1 which stabilized HOTAIRM1 expression. Rescue assays demonstrated that METTL3 silencing counteracted the impact of HOTAIRM1 on glioma cell malignancy and VM formation capacity.ConclusionHOTAIRM1, post-transcriptionally stabilized by METTL3, promotes VM formation in glioma via up-regulating IGFBP2 expression, which provides a new direction for glioma therapy.Graphical

Retrovirus-like gag protein Arc/Arg3.1 is involved in extracellular-vesicle-mediated mRNA transfer between glioma cells

BackgroundActivity-regulated cytoskeleton-associated (Arc) protein is predominantly expressed in excitatory glutamatergic neurons of vertebrates, where it plays a pivotal role in regulation of synaptic plasticity. Arc protein forms capsid-like particles, which can encapsulate and transfer mRNA in extracellular vesicles (EVs) between hippocampal neurons. Once glioma cell networks actively interact with neurons via paracrine signaling and formation of neurogliomal glutamatergic synapses, we predicted the involvement of Arc in a process of EV-mediated mRNA transfer between glioma cells. Materials and methodsArc expression in three human glioma cell lines was evaluated by WB and immunocytochemistry. The properties of Arc protein/mRNA-containing EVs produced by glioma cells were analyzed by RT-PCR, TEM, and WB. Flow cytometry, RT-PCR, and fluorescent microscopy were used to show the involvement of Arc in EV-mediated mRNA transfer between glioma cells. ResultsIt was found that human glioma cells can produce EVs containing Arc/Arg3.1 protein and Arc mRNA (or “Arc EVs”). Arc EVs from U87 glioma cells internalize and deliver Arc mRNA to recipient U87 cells, where it is translated into a protein. Arc overexpression significantly increases EV production, alters EV morphology, and enhances intercellular transfer of highly expressed mRNA in glioma cell culture. ConclusionThese findings indicate involvement of Arc EVs into mRNA transfer between glioma cells that could contribute to tumor progression and affect synaptic plasticity in cancer patients.

EXTH-55. ALL-IN-ONE GENETIC CONSTRUCT TO KNOCKDOWN MIR-21 REDUCES GLIOMA PROLIFERATION IN VITRO

Abstract Glioma, the most aggressive tumor of the CNS has poor patient outcome with limited effective treatments. CRISPR-CAS technology recently opened a new avenue for gene therapy and has been previously used to target non-coding RNAs, including microRNA-21, an oncogene associated with glioma progression. Here we show that an all-in-one construct containing both Staphylococcus Aureus Cas9 (SaCas9) guided by a single-guide RNA (sgRNA) specifically targeting miR-21 resulted in a deletion causing a downregulation of miR-21 expression levels and reduced proliferation in glioma cell lines. Here, we aim to design a single adeno-associated vector (rAAV) vector platform that express both saCas9 and sgRNA to target miR-21 in a glioma syngeneic mouse model. With the future goal to engineer a single AAV that can achieve therapeutic genome editing outcomes.

CSIG-16. PTEN LOSS IN GLIOMA CELL LINES FACILITATES IMMUNE EVASION BY UPREGULATING EXTRACELLULAR VESICLE BIOGENESIS AND PD-L1 CARGO

Abstract Phosphatase and tensin homologue (PTEN) is deleted or mutated in over 50% of all glioblastoma (GBM) cases. Recently, PTEN loss has been implicated as a prognostic biomarker for patients likely to be non-responders treated with immune checkpoint inhibitor (ICI) therapy. Indeed, a growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1) and the development of an immunosuppressive tumor immune microenvironment (TIME), likely the result of an altered secretome. This raised the question of whether the loss of PTEN impacts the biogenesis and function of extracellular vesicles (EVs), given their functional contributions to immunosuppression and tumor growth. Here we show that glioma cell lines with no or reduced PTEN expression produce elevated numbers of EVs, as measured through nanoparticle tracking analysis. Pharmacological inhibition of PI3K reversed this observation, indicating that this phenomenon occurs in a PI3K-dependent manner. Moreover, we show through flow cytometry and immunoblot analysis that the ability for the inflammatory cytokine interferon-γ (IFN-γ) to upregulate PD-L1 at the cell surface and as EV-associated cargo is markedly increased in cells lacking PTEN. Lastly, we show that in response to IFN-γ, EVs derived from glioma cells lacking PTEN exhibit an enhanced ability to inhibit TCR signaling in a Jurkat reporter system. Taken together, these findings shed new light on how the loss of a major tumor suppressor, PTEN, can alter EV biogenesis and function and contribute to an immunosuppressive extracellular milieu. Future studies will be directed toward exploring whether loss of PTEN function alters additional EV-associated immuno-modulatory proteins relevant to other immune cell populations, toward gaining a further understanding of how EVs shape the “cold” TIME of GBM and the potential therapeutic benefit of combining PI3K inhibitors with ICIs.

TMIC-30. HYPOXIA-INDUCED COMPLEMENT COMPONENT 3 PROMOTES AGGRESSIVE TUMOR GROWTH IN THE GLIOBLASTOMA MICROENVIRONMENT

Abstract Glioblastoma is the most aggressive primary adult brain tumor. Treatment includes surgery, irradiation and chemotherapy. Still, virtually all patients suffer a treatment resistant recurrent tumor. In previous studies from our lab, we have investigated the tumor microenvironment contribution to tumor progression, showing that stromal astrocytes become reactive upon irradiation and hypoxia and generate tumor-supportive conditions for glioblastoma cells. Common features of astrocyte reactivity in several neurological diseases, include upregulation of complement proteins including complement component 3 (C3). However, it remains unexplored if these complement proteins are expressed in stromal astrocytes in glioblastoma. In a genetically engineered glioma mouse model, C3 was expressed in the invasive front, hypoxic, and perivascular tumor areas. TCGA data analysis revealed an overall strong correlation between hypoxia and complement gene expression signatures in glioblastoma patients. Astrocytes and glioma cells cultured in hypoxic conditions upregulated C3, as well as other genes associated with an infiltrative phenotype of GBM. In hypoxia, glioma cell lines, but not astrocytes, upregulated the C3a Receptor. GBM tumor single-cell sequencing data confirmed a subpopulation of highly C3-expressing astrocytes, enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, hypoxia, and IFN-signaling. In vitro, C3 enhanced proliferation of glioma cells specifically under hypoxic conditions. Blocking C3a-C3aR signaling in vivo prolonged survival of glioma-bearing mice both alone and in combination with radiotherapy. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment of glioblastoma, where local expression of complement proteins lead to tumor promoting signaling. Inhibition of C3a-C3aR enhanced survival of glioma-bearing mice and could represent a promising avenue for further investigations.

CNSC-07. USING CRISPR-CAS9 SCREENING TO IDENTIFY BIOMARKERS FOR MMF SENSITIVITY IN GLIOBLASTOMA MULTIFORME

Abstract Glioblastoma (GBM) the most common malignant brain tumor with a median survival of 21 months. Recurrent GBM remains resistant to available therapies. Our phase I clinical trial investigates combining mycophenolate mofetil (MMF) and temozolomide (TMZ) to target de novo purine biosynthesis in GBM. Previously, our lab revealed that GBM cells upregulate purine biosynthesis and inhibit purine salvage through the ARL13B-IMPDH2 axis during TMZ therapy. Targeting IMPDH2 with MMF/TMZ extended survival in GBM patient-derived xenograft models. In preparation for Phase II of our clinical trial, we aim to identify a predictive biomarker for MMF+ TMZ therapy. In a CRISPR-Cas9-mediated knockout screen, we identified ten genes, including SLC1A5, showing enrichment (differential β score mean values &amp;gt; 60), indicating sensitivity to MMF/TMZ. SLC1A5 displays a wide distribution of expression in Glioviz patient datasets and encodes the glutamine transporter ASCT2, which is crucial for de novo purine biosynthesis. RNA-seq and protein expression analysis in six glioma cell lines confirmed that high SLC1A5 expression appears to correlate with sensitivity to MMF/TMZ (IC50 0.58-21.30 μM), while low expression led to resistance (IC50 73.43-1148 μM). Knockdown of SLC1A5 in a high-expression cell line demonstrated reduced sensitivity to MMF/TMZ (IC50 2.099 vs IC50 1007 μM). To further validate our findings, we plan to perform SLC1A5 overexpression in low-expression glioma cell lines. We hypothesize that GBM patients with higher SLC1A5 expression may respond favorably to MMF/TMZ therapy due to the reliance of their tumors on de novo purine biosynthesis. We aim to combine this data with analysis of our Phase I clinical samples to retrospectively establish SLC1A5 as a biomarker for MMF/TMZ sensitivity and investigate its role in tumorigenic purine metabolism. Identifying predictive markers will enhance personalized treatment strategies for GBM, offering hope for improved outcomes.

CTNI-23. SINGLE CELL TRANSCRIPTOMICS, PHARMACOKINETICS, AND PHARMACODYNAMICS OF COMBINED CDK4/6 AND MTOR INHIBITION IN A PHASE 0 TRIAL OF RECURRENT HIGH-GRADE GLIOMA

Abstract Adult High-grade gliomas (HGGs) often exhibit deregulation of RB-CDK4/6 and mTOR signaling pathways. This study investigated tumor single cell transcriptomics, pharmacokinetics, and pharmacodynamics immediately following combined CDK4/6 (ribociclib) and mTOR (everolimus) inhibition in recurrent HGG. 24 patients with recurrent gliomas harboring RB+, CDKN2A/B deletion or CDK4/6 amplification, and PTEN loss or PIK3CA mutations were enrolled in a phase 0 trial. Patients received ribociclib treatment for 5 days before surgery, including six who received ribociclib (400mg QD) plus everolimus (2.5mg QD), and six dose-escalation cohorts (n = 3 each) reaching ribociclib 600mg QD plus everolimus 70mg QW. No dose-limiting toxicities were observed. Validated LC-MS/MS methods determined total and unbound drug concentrations. Preoperative MRI revealed a marked decrease in gadolinium-enhancement in all patients. In gadolinium-nonenhancing tumor regions, the median unbound concentration of ribociclib was 561 nM, while no unbound everolimus concentrations were detected. Ribociclib’s pharmacodynamic effects in surgical specimens were compared with matched archival tissue using IHC. Across IDH-wild-type tumors, there was significant decrease in MIB1 levels between the matched archival and surgical specimens (p &amp;lt; 0.05) suggesting drug-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) analyses were performed on 17 IDH-wild-type recurrent samples treated with ribociclib plus everolimus and 29 IDH-wild-type recurrences treated with standard therapies. Cellular state analysis based on snRNAseq data demonstrated a decreased neural progenitor-like (NPC-like) malignant cell state compared with standard therapies (p &amp;lt; 0.01), indicating inhibition of the CDK4-driven neural progenitor-like cell state. This observation was validated using snRNA and patient-derived glioma cell lines exposed to ribociclib, showing a consistent decrease in NPC-like proportions and cycling cells (p &amp;lt; 0.05). This study highlights the value of integrating pharmacokinetics/pharmacodynamics and snRNA data to assess treatment effects in phase 0 surgical tissues. These findings support further development of ribociclib, but not everolimus, for the treatment of glioma patients.